CA2183461A1 - Antiviral transgenic plants, vectors, cells and methods - Google Patents

Abstract

Isolated 2-5A-dependent RNases, an inteferon-induced enzyme which is activated by 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A) and implicated in both the molecular mechanisms of interferon action and in the fundamental control of RNA stability in mammalian cells, and encoding sequences therefor are disclosed. The expression cloning and analysis of murine and human 2-5A-dependent RNases is also disclosed. In addidon, recombinant nucleotide sequences, recombinant vectors, recombinant cells and antiviral plants which express, for example, 2-5A-dependent RNase, 2-5A synthetase and/or double-stranded RNA dependent protein kinase (PKR), or other amino acid sequences which have activity that interferes with or inhibits viral replication are disclosed.

Description

2 1 ~3461 Vl1~2.T. .~ PLaNTS. ~3~:TORS.
~T.T~C ,~ F~nn~
Related ~ tions This application for ~.s. patent is a continuation-in-part of U.S. patent application, which was a~signed Serial No. 08/028,086 and filed on March 8, 1993.
Field of the Invention The present invention relates to isolated 2-5A-~lP~ t RNases having the ability to bind 2-5A
and/or cleave single stranded RNA when bound to 2-5A, ~-nro-l;nq S~Tl~nrr~c therefor, recombinant nucleotide molecules, recombinant vectors, recombinant cells, and antiviral ~Lal~Syl:niC plants which express, for example, antiviral animal amino acid seq~lr-nr~C which have activity similar or identical to 2-5A-d~r~nrl~nt RNase, 2-5A synthetase and~or PKR.

Control of RNA degradation is a critical cell function, and gene expression is often regulated WO 9~l~5 2 1 8 3 4 6 1 r~~ ~ A
at the level of RNA stability. See, e.g., Sh~w, G.
and Ramen, R., Ç~ll, 46:659-667 (1986). Neverthe-less, relatively little is known about the bio-rh~Tnir~l p~ ya that mediate RNA degradation in 1 l~n or plant 6ystems. For instance, most if not all of the r;hnn~rl-~Acn~ r~qr~nc;hle for mRNA
~UL~ L in l; An or plant cells remain lln;AF~ntif;OA. This was reviewed in Brawerman, G., 5~Ll, 57:9-10 (1989).
Presently, the 2-5A system i5 believed to be the only well-charac ~ oA RNA degradation pathway from higher animals inrl~ ;ng man. see FIG.
1. See also, e.g., Rerr, I.M. and Brown, R.E., Prod.
Natl. Acad. sci. U.S.A., 75:256-260 (1978) and Cayley, P.J. et al., BioDhYs Res~ Commun..
108:1243-1250 (1982); reviewed in Sen, G.C. and Lengyel , P., J . Biol . Chem., 267 : 5017-5020 (1992) .
The activity of the 2-5A system is believed to be mediated by an endoribonuclease known as 2-5A-~1 l nn~l~nt RNase. See Clemens, M.J. and Williams, B.R.G., Cell, 13:565-572 (1978). 2-5A A~ A~"L
RNase is a unique enzyme in that it requires 2-5A, unusual oligoadenylates with 2',5' ~ A;e~ter 1 ;nkA~c~c~ pn(A2'p)nA, for ribonuclease activity. See Rerr, I.M. and Brown, R.E., Prod. Natl. Acad. S~i.
U.S.A., 75:256-260 (1978). 2-5A is ~L~du~ d from ATP
by a family of - y~Ll e~Ac-~c in reactions requiring . . _ _ _ _ _ . _ _ _ _ _ _ _ _ wo95n224s - 21 83461 E~
doubl~ OLL~.ded RNA (dsRNA). See FIG. 1. See also r ~ ;An~ A.G. et al., ~3~3~, 268:537--539 (1977);

Marie, I. and ll~V~ iAn, A.G., J. Biol. Chem..
267:9933-993g (1992). 2-5A is unstable in cells and in cell-free systems due to the combined action of 2',5' r~h~ Ast I and 5' pl~oO~h~L~Oe~ See Williams, B.R.G. et al.; F~lr. J. Biochem., 92:455-562 (1978); and Johnson, M.I. and Hearl, W.G., J. Biol.
~Chem., 262:8377-8382 (1987). The interaction of 2-SA-cl~rDn~lDnt RNase and 2-5A(Kd = 4 X 10 11 N), Silverman, R.H. et al., Biol. Chem., 263:7336-7341 (1988), is hiqhly speclfi~. See Knight, ~. et al., Nature, 288:189-192 (1980). 2-5A-~DrDn~lDnt RNase is believed to have no detectable RNase activity until it--is converted to its active state by binding to 2-5A. See Silverman, R.H., ~nAl. Biochem..
144:450-460 (1985). Activated 2-5A-rlDrDn~lDnt RNa~Oe cleaves single sLL~ ed regions of RNA 3' of UpNp, with preference for W and UA sDqnDnr Dc. See WL~s. ll-.el-, D.H. et al., Nature, 289:414-417 (1981a);
and Floyd-Smith, G. et al., Science, 212:1020-1032 (1981). Analysis of inactive 2-5A-dc~rDn~Dnt RNase from mouse liver revealed it to be a single polypeptide of approximately 80 kDa. See Silverman, R.H. et al., Biol. Chem., 263:7336-7341 (1988).
Although the full scope and bi olo~j cAl significance of the 2-5A system remains unknown, W095/22245 2 1 8346 1 P~
studies on the ~ e~ Ar - ' ~n; Of interferon action have provided at least some of the fllnrt j nn~.
Interferons a, ~ or Y are believed to induce the ~ 1 Ation of both 2-SA ~ 4 L RNase, Jacobsen, H. et al., Virolocv, 125:496-501 (1983A) and Floyd-S~ith, G., J. A~ lAr Biochem., 38:12-21 (1988), and 2-5A ay~Ll~:L~ses~ HovAn~c~i~n~ A.G. et al., l~a~, 268:537-539 (1977), reviewed in Sen, G.C. and Lengy-el~ P., J. Bi~l. Chem., 267:5017-5020 (1992)- Furth- ~, several investigations have implicated the 2-5A system in the - - AhAn i F~ by which interferon inhibits the replication of picornaviruses. Indeed, 2-5A per se and highly specif ic 2-5A mediated rRNA cleavage products were induced in interferon-treated, ~nA~rhAl ~ rditis virus (EMCV)-infected cells. See Williams, B.R.G., Nature, 282:582-586 (1979); Wreschner, D.H. et al., Nucleic A. ids Res. . 9:1571-1581 (1981b); and Silverman, R.~. et al., Eur. J. Biochem.. 124:131-138 (1982a). In addition, expression of 2-5A synthetase cDNA inhibited the replication of picornaviruses, Chebath, J., Nature, 330:587-588 (1987) and Rysiecki, E.F. et al., J. Interferon Res., 9:649-657 (1989), and the i.-L~u- Lion of a 2-5A analogue inhibitor of 2-5A-cl~r~n~nt RNase into cells reduced the interf~r~ Ated inhibition of E.~CV replication.
~ee Watling, D. et al., EMRr~ J.. 4:431-436 (1985).

W0 9512224~ ~ 2 1 8 3 4 6 1 P~

Further, 2-5A ~lo~ e l RNase levels were correlated with the anti-El~CV activity of interferon, Kumar, R.
et al., J. Virol., 62:3175-3181 (1988), and EMCV-derived dsRNA both bound to and activated 2-5A
ay~lU~ 5~ in i--~el~r~.. treated, infected cells.
See Gribaudo, G. et al., J. Virol., 65:1948-1757 ( 1991) .
The 2-5A E;ystem, however, almost certainly provides fllnrt;nnG beyond the antipicornavirus activity of inteL r~ ,..5 . For instance, introduction of 2-5A into cells, H.,v~ GlFn, A.G. and Wood, J.N., Viroloqv, 101:81-90 tl980), or expression of 2-5A
synthetase cDNA, Rysiecki, G. et al., J. Interferon Res., 9:649-657 (1989), inhibits cell growth rates.
~IGL~,V~:r, 2-5A ~ 1_ ,L RNase levels are elevated in growth arrested cells , Jacobsen , H . et al ., P~oc .
Natl. Acad. sci. U.S.A., 80:4954-4958 (1983b), and 2-5A synthetase, Stark, G. et al., Nature, 278:471-473 (1979), and 2-5A ~ RNase levels are induced during cell differentiation. See, e.g., Krause, D. et al., Eur. J. Biochem., 146:611-618 (1985). Therefore, interesting correlations exist between 2-5A-d~ l,o .lQ ~ RNase and the flln~l tal control of cell growth and differentiation suggesting that the 2-5A system may function in general RNA
me~hol i ~- The ubi~uitous presence of the 2-5A
system in reptiles, avians and 1 j ?nF certainly W095/22245 ~ 2 1 8 3 4 6 1 .

DU~J~J' L:j a wider role ~or the pathway. See, for example, Cayley, P.J. et al., Biochem. BioDhv. Res.
commun., 108:1243-1250 (1982).
While it is presently believed that the 2-5A system is the only well-characterized RNA
d~yL..d~tion pathway from higher ~nimals, the dsRNA-~lo~ t protein kinase enzyme, known as PRR, is also thought to have antiviral effects in higher animals. Like the 2-5A synthetase enzyme, it is believed that PRR is stimulated by dsRNA. It is believed that activated PRR rhnsrhnrylates the alpha subunit of translation factor eIF2, known as elF2-alpha, which indirectly inhibits protein synthesis initiation. It is believed that interferons , ,~, and y induce the A~ l~tion of PKR. See HoavAnocQi~n et al.: J. Interferon Res..
9:641-647 (1989).
Like the 2-5A system, the PKR system is also likely to provide fllnr tinne beyond the antipicornavirus activity of interf erons . See Meurs, E.F. et al.: J. Virolocnr, 66:5805-5814 (1992). For example, expression of mutant forms of PRR in NIH 3T3 cells resulted in tumor formation when injected into nude mice. See Meurs , E . F . et al .: Proc. Natl .
Ac:ad. sci U.S.A., 90:232-236 (1993).
In short, the 2-5A system and the PKR
system inhibit viral protein synthesis. This is _ _ _ _ _ _ _ _ _ _ _ . , . , . . _ . _ w0 95n2245 2 1 8 3 4 6 1 , believed to be ~ 1 i Chc~d by the 2-5A system by degrading mRNA and rRNA whereas the PKR system i8 believed to : 1 i Ch this by indirectly inhibiting protein synthesis initiation.
Viral plant tlic~J-coc are r~n~mic and their severity varies from mild Dy i to plant death.
The majority of plant viruses are believed to have single stranded RNA genomes. ~OL~uvo:I, it is currently believed that plants are void of the three enzymes llicc--c~P~l above, i.e., PKR, 2-5A :~y~U~t~lse and 2-5A ~el~Dl~ t RNase. See cayley, P.J. et al.:
Biochem. biol~hvs Res. Commun., 108:1243-12S0 (1982) and Devash, Y. et al.: Bioc:hemistrv. 24: 593-599 (1985): but see Crum, C. et al.: J. Biol. Chem. .
263-13440-13443 (1988); T~ in~, H.J. et al.:
Science, 241:451-453 (1988); Sela, I.: TIBS, pp.
31-33 (Feb 1981); and Devash, Y. et al.: Science, 216: 1415-1416.
Notwithstanding the il~ly~L Lallce of 2-5A-rlorPn~t RNase to the 2-5A system, 2-5A-~ t RNase enzymes having ribonuclease function have not been isolated, purified or 5~qu~n~d heretofore.
C ~ce~ ly, there is a demand for isolated, active 2-5A-r~ - L RNases and their complete amino ~cid seS~uences, as well as a demand for ~nl~o~in~ seqn~n~-~c for active 2-5A ~ c L RNases. There is also a W0 9~r2224s ` - 2 1 8 3 4 6 1 P~

demand for plants which are resistant to viruses ~uch a~ the picornaviruses.
~E9~ ~ of th~ TnventiOn In brief, the present invention alleviateF
and .-L~ ~~ certain of the a~u~_ - Lioned problems and ~I~UL L n~s of the present state of the art through the discovery of novel, isolated 2-5A-d~r~n~ nt RNases and r nro~l i ng sr~ C therefor.
Broadly srr Ak;n~r the novel 2-5A d~r~n~ nt R13ases of the instant invention are involved in the flln~l 1 control of single stranded RIJA decay in animal cells, such as mammals, and are also present in animal cells, such as avian and reptilian cells.
More particularly, the novel 2-5A ~ L RNases of the present invention have the ability to degrade single stranded RNA, mainly 3 ' of UpUp or UpAp Fe~ c ~ after they are activated by binding to 5' 1h~ ylated,2',5'-linked n~;goafl~ylates (hereinafter "2-5A"). As a result, it is believed that the novel 2-5A ~ RNases are useful in cnnnPr~; nn with inhibition of cell growth rates, viral replication and in connection with interferon treatment of viral infection and cancer. As used herein, the term "2-5A-~ rDn~l~nt RNase(s) " is used in a broad sense and is meant to include any amino acid r~ nre which includes a 2-SA binding domain and/or ~ W0 9~2224~ 2 1 ~ 3 4 6 1 r ~
g ri homlrl ease function when the 2-5A-d~l - 1PI ~ L RNase i8 activated by 2-5A.
The novel 2-5A dr~ \ t RNases of the present invention are protein enzymes having molecular weights on the order of between about 74 KDa (murine) and about 84 KDa (human), as detorm; no~l by gel el~.LL~ L~is migration and/or prediction from their respective c~nro~l i n~ nucleotide 50q~l0n0_ .
For example, a human 2-5A-d~ t RNase of the instant invention has a lorl~ r weight of about 83,539 Da as ~lotprminod from the amino acid soTlonre predicted from the onro~in~ soquonre therefor, whereas the murine 2-5A-AorPn~30nt RNase has a molecular weight of about 74 KDa as detprm;notl by gel ele~.L u~h.~sis migration and from prediction of the amino acid 5~ lv-l~ e from the onrorlirtr soqnonro While an about 74 KDa molecular weight is reported herein for a murine 2-5A-d~ ~ lP L RNase, it should nevertheless be appreciated that the reported molecular weight is for an incomplete murine 2-5A-doren~ont RNase. It is nevertheless believed that once completely se~l~-, ed, i.e., when an about 84 amino acid end region is identified, the 1 ec~ r weight of a complete murine 2-5A d~l,o.~lPIIL RNase will be similar to that of human, i.e., about 84 KDa.
It should also be readily apparent to those versed in this art, however, that since gel electro-=

W095/22245 21 83461 r~

phoresis migration has been employed to antorm~ n-~Ar weight of a murine 2-5A A~ L RNase, the 74 KDa - ~ 1 e~ r weight is only an estimate based upon relative migration.
The amino acid ~ r. ~ for human 2-'A-~ RNase protein is depicted in FIG. 3 and T~tole 1. The onro~in~ EC~ e for the human 2-5A tlr~ RNase protein i5 also set forth in Table 1. The mRNA f or human 2 -5A ~ RNase is about S. 0 ~b in size. The virtually complete amino acid sequence for the murine 2-5A ~ l_r,L RNase protein and the ~nro~ seq~l~noe therefore i5 depicted in Table 2. The mRNA for murine 2-5A-~Qp~-n~ont RNase is about 5 . 7 Rb in sizQ.
~ Analysis of the amino acid 60~-~n, ~c of the 2-5A-~l~ron~nt RNases of the present invention have revealed several characteristics unique to the 2-5A-d~ RNases. For example, it has been discuv~Led that the novel 2-SA ~lpp~ndont RNases of the instant invention include the following unique domains which span between the amino tormi n-lc and the carboxy tormi m-c. For instance, it has been disc-lvel d that there are at least four and possibly as many as nine or more ankyrin repeats, of which three lie closest to the amino torm;n~. However, while four ankyrin repeats have been di~cuveLed, it is believed that there may be additional ankyrin _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ W095/2224~ 21 ~3461 r repeats that may total, for instance, about eight or more when the amino acid 3~ 5 of the 2 'A f~ _ t RNases of the present invention are further analyzed. It is believed that these ankyrin repeats may possibly function in protein-protein interaction. Anlcyrin repeat 1 generally lies between amino acids designated as 58-90 in Tables 1 and 2 .
Ankyrin repeat 2 generally lies between amino acids desigDated as 91-123 in Tables l and 2 . Ankyrin repeat 3 generally lies between amino acids designated as 124-156 in Tables 1 and 2. Ankyrin repeat 4 f~nnGrAl 1 y lies between amino acids designated as 238 and 270 in Tables 1 and 2. See also FIGS. lOA and lOB.
- ~ It has also been discuv~Le d that the novel 2-5A f~ ases include a cysteine rich region (which has homology to zinc ~ingers~ that lies closer to the carboxy t~rm;n~q than the amino tf~rm;nllc which may possibly function in RNA recognition or in ~ormation of protein dimers. The cystQine rich region is believed to include about 5 or 6 cysteine residues which generally lie between amino acids designated as 395-444 in the human sef~ Dnf e as reported in Table 1 ~nd FIG. 4, or between amino acids designated as 401-436 in the murine ~frl~nre as reported in Table 2 and FIG. 4.

wo g5n224s 2 18 3 4 6 1 P .,~

Still further, it has been dis.uv~:Led that the novel 2-5A A~ ,L RNases include a duplicated phosphate binding (2 P-loops) motif which lies generally within the ankyrin repeat motifs. It is believed that the two P-loops ~re in the same orientation ~nd constitute the binding do~ain n~ y for binding 2-5A. It is further believed that each P-loop motif i nrll~A~ a lysine residue which is essential for maximum 2-5A binding activity. The lysine residues are designated as 240 and 274 in Tables 1 and 2.
It has been further discovered that the 2-5A-d~r~nA~nt RNase proteins contain an amino acid region which follows the cysteine rich region that is be~ieved to be homologous to protein kinases. Within this region, there is believed to be separate domains designated as domains VI and VII which generally lie between amino acid residues designated as 470-504 in Tables 1 and 2 . More particularly, as to the human seqUpnre of 2-5A-A~ RNase, domain VI generally lies between amino acid residues designated as 471-491 and domain VII generally lies between amino acid residures designated as 501-504, as reported in Table 1 and FIG. 4. As to the murine S~ nre of the 2-5A-Aer- A~ - RNase, domain VI generally lies between amino acids designated as 470-489 and domain wo gsn224s 2 1 ~ 3 4 6 1 PCTrUS95rO2058 VII generally lies between amino acid residues desig-nated aæ 499-502, a8 reported in Tabie 2 and FIG. 4.
It ha5 al50 been dis.;uvc Led that there is limited homoloqy between the amino acid seqll~-nr~ f or the 2-~r~ .L ~Wases Or the present invention and RNase E, encoded by the altered mRllA stability (ams)/rne qene of E. Coli. Uniquely, the limited homoloqy i5 generally cu..~el ved between the murine and human amino acid 5~ c for 2-5A-~ r~n~lont RNases and qenerally lies between a 200 amino acid region. More particularly, for the human se~ e, the amino acid region spans amino acid residues designated as 160-349 in Table 1 and FIGS. 9A and 9B. With respect to the murine seqn~n~ e, the amino acid region spans amino acid residues designated as 160-348 in ~able 2 and FIGS. 9A and gB.
It has been further disuuv~:r. ~ and is believed that almost the entire, if not complete, amino acid SG~ of the novel 2-5A-d-~r~ - L
RNase proteins of the instant invention are n~r~ccs~ry for r;h~n~ lease function. For example, it is believed that, when an about 84 amino acid region at the carboxy t~--m;m~C is present in the human 2-5A-~ 1P.,L RNase, the human 2-5A ~ RNase has ribonuclease function in the ~L~ e of 2-5A.
I~ contrast, when th.e murine 2-5A d~ t RNase ~o 95~2245 - 2 ~ o lacks the about 84 amino acid region at the carboxy to--min~, it lacks r;hnnllr~lf--- funnt;~n, With respect to the binding activity of a murine 2-~A de~ RNase protein to 2-SA, it has been dis~ .,v.:-O-l that, when one P-loop is deleted from the repeated P-loop motif of a murine 2-5A ~ r~nt RNase protein, nearly all 2-5A binding activity is lost, ~nd that when both P-loops are deleted, virtually ~ leto activity is lost. HowevQr, it has been found that, even though the carboxy tr~n~l~;mlc portion of the amino acid CQ~ nre of a murine 2-5A Ael l_.L R~7ase protein following the repeated P-loop motif has been deleted, partial 2-5A binding activity is ~- i ntA; nr~
It has been further discvvere d that whQn lysine residues 240 and 274 are replaced with asparagine residues in both P-loop motifs, cign;firAnt 2-5A binding activity of a ~urine 2-5A-~ o t RNase protein is lost. It has been further di~ ,veL~, however, that when either lysine residue 240 or 274 is replaced in either P-loop motif, only partial 2-5A binding activity is lost.
It is therefore believed that the ~L~:s~ .e of both P-loop motifs in the amino acid seq~ n~ es for the 2-5A d~ l-\ t RNases of the present invention plays an; cl-L rolO in 2-5A binding activity. It is further believed that the pLe:3e~ e of lysine residues _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ , . _ _ ~ wo g5n224s 2 1 8 3 4 6 1 ~ o 240 and 274 in each P-loop motif plays An; La~lL
role for ~ 2-5A binding activity. It is also believed that the ~r.g_l.ce of virtually the entire amino acid ~ e of the 2-;A dc~ .t RNases of the present invention proYides f or even further ~-nh~nr~d 2-5A binding activity, as well as provides for r;hr~m~ ce function.
In addition, the present invention relates to the cloning of murinQ and human 2-5A-cl~r~n~nt RNases and novel murine and human clones.
~^ ~ ;nAnt and naturally occurring forms of 2-5A-~r~ L RNase displayed virtually identical 2-5A binding properties and ribonuclease sp~c; f i c ities.
- The present invention further contemplates the use of the novel isolated, 2-5A-d~r~n~ont R~ases and ~n~o~l;ng s~ c therefor, as well as analogs and active f _ ~ Ls thereof, for use, for instance, 1. ) in gene therapy for human and animal lli CPAC~C
including viral disease and cancer, 2. ) as genetic markers f or human disease due to perhaps cancer or viral infection, 3 . ) to develop plants and animals resistant to certain viruses, and 4 . ) as enzymes in connection with research and development, such as for studying the ~ u~LuL~ of R~A. In one manner to r , lich the above, and as ~ nt~ l~ted by the present invention, the ~nr~o~lin~ c of the Wossl22245 2 1 8 34 6 1 P~ CQ

instant invention may be l~t i ~ ' in ex vivo therapy, i.e., to develop L~ ` inAnt cells using the ~nrn~7;n~
s , . of the present invention using tr~rhn; q-l~c known to those versed in this art. In another manner which may be employed to ~ h the above, the enro~;ng ~ CC of the present invention may be _ ~ ;ncd with ~n al.~L~},riate ~LI ~ to form a reco~binant 1 ecl~l c and inserted into a suitable vector for i--t-~ I ;rn into an animal, plant, or other lower lie fsrms also using techniques known to those skilled in this art. Of course, other suitable methods or means known to those versed in this art may be selected to accomplish the abuv~ sLated objectives or other objectives for which the novel 2--~A ~ c ~ RNases and -nrn~; n~ 8C~ cnrcc: of the present invention are suited.
The present invention also contemplates novel tr~n~cn;r- plants, as indicated above, which are resistant to viruses such as the picornaviruses.
Generally spe~k;n~, the transgenic plants of the present invention include any inserted nucleotide sequence cnro~; ng any type of antiviral amino acid Scrlllcnre, including proteins. Preferably, the antiviral nucleotide scq--cnrC~ L~lu~.~d into plants in accordance with the present invention are animal antiviral genes, such as those genes which are stimulated in L. s~ul.se to interferon production `` 21 ~3461 and/or LLe3~ . These include, for example, those animal antiviral genes that encode 2-5A-synthetase, 2 _A~ RNase, and PRR. These interferon-regulated proteins, 2-5A-synth~t~ce, 2--A d ~ _ L RNase and PE~ tthe dsRNA ~ L
protein kinase) have r~cogn~ 7~`~ antiviral effects in higher animals and are believed to have antiviral effects in the tr~n~g~nir plants of the present ~invention. PKR is stimulated by dsRNA to ,Lylate translation factor eIF2 which indirectly inhibits protein synthesis intiation. On the other hand, 2-5A synthetase is activated by dsRNA
resulting in the protll~rt;orl of "2-5A," pXA(2'p5'A)y wherein X = about l to ~bout 3 and Y 2 about 2, from ATP. The 2-5A then activates an endoribonuclease entitled 2-SA cler- ~- l RNase (also known as RNase L
or nuclease F). The activated ribonuclease deqrades mRNA and rRNA thus inhibiting protein synthesis.
These above-described pathways are particularly e$~ective at inhibiting viruses in animals with single stranded RNA genomes that replicate through dsRNA intermediates, such as the picornaviruses, and are believed to be effective at inhibiting similar types of viruses that infect plants. This belief is premised upon the understanding that most single stranded RNA plant viruses produce double ~L..nded ~L~u~Lu~:s during wo ssl2n4s F~~ n~#

replication by their viral rorlicA~sc~ see Dawson, WØ et al.: Acad. Press, 38: 307-342 (1990~, and that plant viruses are similar to animal viruses in ~L~....LUL., composition and i F~ of repliciltion in cells. In addition, even viral so-called single .,LL,~ ed RNA may contain Sorrnr3:~ry structures which could activate Pl~ and 2-5A synthetase leading to widespread plant protection against plant viruses. It is believed that co ~ La~ion of 2 -A " L _ . lont RNase and 2-5A-synthetase, will lead to the destruction of viral mRNA and viral genomic RNA thereby protecting the LLAj~C~ plants of the present invention from viruses. ~So- t:vvaI, it i8 believed that expression of P~ by the trAn~: ~rnir~
plants of the present invention will inhibit viral protein synthesis leading to inhibition of virus replication and protection of the LLar. ,y~..ic plants.
The present invention is therefore premised in part upon the belief that plant virus RNAs activate 2-5A-synthetase and PKR in the transgenic plants of the instant invention leading to immunity against virus infection. Fur'~h~ ., expression of 2-5A
Ly"l ~ .~'r~ce alone or 2-5A d~lJ- l~ L RNase alone or PKR
alone may protect plants against viruses, perhaps by binding to viral RNA, such as viral replicative into ';Ates thereby blo~in'J viral replication.
~oL~vvar, eYpression of only the dsRNA binding ~ wo gsn224~ 2 1 ~ 3 4 6 1 P~

domains of PRR and/or of 2-5A Ly.,U.eLase may ~m11 Arly protect the ~ J :.i r plants of the present invention against viral infection.
It should Ulæ~ef.,L~ be appreciated by those versed in this art that novel ~lAr J~ r plants which are resistant to viral infection can now be produced in a~-,L~ ;e with the present invention. It is believed that the effectiveness of the anti-viral protection can be Dnh~nred or even r-Yi~i 7e~ when at least the three-above animal antiviral genes are inserted into plants to form ~ lArY trAnCAjen;C
plants of the present invention, since the animal antiviral proteins encoded by these three animal antiviral genes interfere with different stages of t~e viral life cycles. ~S~ r, these animal antiviral proteins or amino acid seyuences are believed likely to be safe to give or i-lLl-~d~ ~ into animals, including humans, since these antiviral proteins or amino acid q~DA~u~nrc~q are naturally occurring in humans as well as in other mammals, avians and reptiles.
While the present invention is described herein with reference to the particular sO~AluDnrec disclosed, it should nevertheless be understood by those skilled in this art that the present invention contemplates variations to the amino acid and/or n=cleotide ~ "q wh~ch do not destroy 2-5A

-Wogs/22245 ` 21 83461 r~

~yllLII-t -e ~ctivity, PKR activity ~nd/or 2 'A ~ ribonuclease activity. I~I~L~fULe:r the present invention , 1 Ates any analogs, parts or rL ~ f 2 SA~ L RNase, 2--SA syntht~tAce~
and P~R which are active, such as any active part, and any -nro~l~n~ 5~ " e- therefor. In other words, the present invention inr~t1~Dc, among other thingfi, any amino acid 5~ re, any nucleotide 5e~U~ and any LLA"-~J'~;r plant which have the ahility to r 1 ~ Ch the ob; ectives of the instant invention .
For example, the instant invention includes any amino acid se~l re which has antiviral activity and any nucleotide s~ which encodes therefor and those LLAI.-~J~'.;r plants that express such nucleotide 8~, -. More ~Cr~cifir~lly, the present invention ;nrlllA~C, for instance: 1. ) any animal amino acid s~ nre which has the ability to inhibit or interfere with viral replication such as those amino acid se~U~nr~c that have activity similar or identical to PKR activity, 2-5A synthetase activity and/or 2-5A r;hrn~lrl~Ace activity, and ~ny nucleotide s~r~ nre which encodes f or an amino acid se~ nr~
having any such activity; and 2 . ) any trAncgeni r.
plant having any animal antiviral nucleotide s~Tl~nre which encodes any such amino acid ~ which has any such antiviral activity.

W0 95l22245 !~ 2 1 8 3 4 6 I F~ c The above features and adv~ s of the present invention will be better ul.d_~ DLa~d with r 6~ Le:l~Ce to the a~: , 2 ing FIGS ., ~et~ i 1 ed Description and r ~1AC~ It should also be understood that the particular methods, amino acid se.~ =, Anro~;nj Se~ IrAC~ C;UII~- u~;LC:, vectors, recombinant cells, and antiviral ~ J i c plants illustrating the invention are , lAry only and not to be regarded as limitations of the invention.
Brief Descril~tion of the FTCC.
Ref erence is now made to the ~ - y ing FIGS . in which is shown illustrative ~ ' _ ' i ts of the present invention from which its novel feaLu~s and advantages will be apparent.
-~ FIG. l is the 2-5A system: a ribonuclease pathway which is believed to function in the molecular - Ani r-~ of interferon action.
5'-phosphatase, p'tase; 2'-5'-phns~hn~l;esterase, 2 '--PDE .
FIGS. 2A and 2B is a comparison of 2-5A
binding activity of re~ ' ;nAnt and naturally occurring forms of murine 2-5A-dc l, ~ L RNase.
FIG. 2A is a spDc; f i c affinity of truncated murine 2-5A-~ .L RNase for 2-5A. W covalent cros~l inkinj of the 32P-2-5A probe (lanes 1-7) to protein is performed after translation reactions in wheat germ extract (5 t~l) with murine 2-5A d~ L

Wo 95/2224s : 2 1 8 3 4 6 1 1 "~ ~

RNase mRNA (from clone ZBl) (lanes 1-3) or without aaded RNA (lane 4) or in extr~ct of interferon treated mouse L cells (100 llg of protein) (lanes 5-7), moArt; ~na are without added competitor (lanes 1, 4, and 5) or in the ~L~ of either trimer core. (A2'p)2A, (100 nM) (lanes 2 nnd 6) or trimer 2-5A, p3(A2'p)2A (100 nN~ (lanes 3 and 7). Lanes 8 and 9 are ~JL u.lu~=d by incubating the wheat germ extract with 35S - inn;n~o in the absence or presence of 2-5A ~ L RNase mRNA, respectively.
FIG. 2B are iAontit~l ul~y ~Ly~ain cleavage ~Lud~La and are obtained from recomhinant and naturally occurring form of 2-5A-~r~nA~nt RNase.
Partial ul~y ~Lyyain digests (arrows) are p~
on ~ truncated 2-5A d~ G I RNase (c:lone ZBl) produced in wheat germ extract ( "Recombinant" ) and murine L cell 2-5A A I-~ --A_ -~ RNase ("Naturally ûccurring") after crosslinking to the 2-5A prohe and purification from gels.
FIGS. 3A and 3B are clonings of the complete coding s~lu-~r~ for human 2-5A-doronAPnt RNasQ .
FIG. 3A is the ~ol.a-Luu~ion of a human' 2-5A-~A~ o~ o.l- RNase clone. The initial human 2-5A-d-~ o ~ RNase cDNA clone, HZBl, is isolated from an adult human kidney cDNA library in ~gtlO
using r~A i f~lAho~ f.A murine 2-5A-~ronAont RNase cDNA

_ _ _ _ _ _ _ _ _ _ _ . _ _ . _ _ _ _ WO ~5l22~45 21 8 3 4 6 1 ~ . ;n (clone ZBl) as probe. Se~ Example. P~i nli~hP~ ed ~ZB1 DNA is used to isolate a partially overlapping cDNA clone, ~ZB22, which is fused to HZBl DNA at the NcoI site to form clone ZCl. The 5 ' -region of the coding se~ Q~e is obtained from a genomic SacI
LL _ isolated using a ra~linl~hPled HZB22 DNA
.CL, L as probe. Fusion of the genomic SACI
~L _ ' with ZCl at the indicated SacI site pL~ ~uces clone ZC3. The coding EequPrl~re with some fl~nking Eeqt~AnrQe: is then s~hrlnnPd as a E~indIII rL, - L
into pBl I~Qc~ript KS t+) (Stratagene) resulting in clone ZC5. The restriction map for the composite clone, ZC5, is shown. Clone HZBl includes nucleotides designated as 658-2223 in Table I which Rncode for amino acids designated as 220-741 in Table I. Clone ~ZB22 includes a nucleotide s~QT~nce which encodes for amino acids designated as 62-397 in Table I . Clone ZCl i nrl ~ q a nucleotide sequence which encodes for amino acids designated as 62-741 in Table I. Clones ZC3 and ZC5 both include nucleotide sequences which encode for amino acids designated as 1-741 in Table I.
FIG. 3B is a nucleotide s~uuel~e and predicted amino acid se~ , e of human 2-sA ~Ql~nrlt...
RNase with fl~nkinq nucleotide se~ . The numbers to the right indicate the positions of nucleotides and amino acid res ~ dues .

Woss/2224s I S ~ 2 1 834 6 1 FIG. 4 is Al; j ' of the predicted amino acid 5~ c for murine nnd human formS of 2 5A Ar~ "L RNase. The positions of the repeated P-loop motifs, the cysteine (Cys)-rich regions with homology to zinc finqers, and the regions of homology to protein kinase domains VI and VII are indicated.
Amino acids residues which are; L~ L , ~s of the indicated domains are LC:~L~5_1~Led in bold type and are italicized. T~ nt; r~l amino acid residues in murine and human 2-5A-~ L RNase are indicated with colon (: ) symbols adjacent th~L b~L~_en.
FIGS. 5A and 5B are 2-5A binding properties and ri hrrll~rle~ce activity of recombinant human 2-5A-A- ,L RNase ~Ludu~ d in vitro.
-~ FIG. 5A is specific affinity of recombinant human 2-5~ A~ l L RNase for 2-5A. Crosslinking of the 2-5A probe (lanes 1-7) to protein is p~Lr~ ' after translation r~r~t;nnC in wheat germ extract (5 1) with human 2-CA~ L RNase mRNA (lanes 1-3) or without added RNA (lane 4) or in extract of hum2n interferon treated (lOOO units per ml for 16 h) human HeLa cells t350 tlg of protein) (lanes 5-7).
pc~rt;onc were without added competitor (lanes 1, 4, and 5) or in the ~L-_...e of either trimer core, (A2 'p) 2A, (100 nM) (lanes 2 and 6) or trimer 2-5A, p3(A2'p)2A ~100 nM) (lanes 3 and 7). Incubations with 355-me~ h;t~n;n~ are shown in lanes 8 to 12. Lane W0 95/21245 2 1 ~ 3 4 6 1 r~1,u 8 is with wheat germ extract and human 2-5A-d~
RNase mRNA. 7~ati~10cyte lysate p}~A- L~d to 2-SA-rDlll-loFe is ir~,ub~ed with human 2-5A ~7~
~lase mRNA in the absence (lane 9) or ~L~ se~.~ e (lane 10) of cy~ ha~ im~r7e, or in the absence of addeo m7~A
(l~ne ll). Lane 12 shows human 2-5A d~r-~7~L RNase which i.5 ~L~,-l.-.;ed in the r... 7r~1.,,7, crude reticulocyte lysate. The positions and relative molecular masses ( in kDa) of the marker proteins are indicated .
FIG. 5B is reticulocyte lysate ~LeLLe~ted to remove ~ 7~ o~- 2-5A-~-1,a~ l 7~Nase and is ine~at~d in the absence of added mRNA 0 ), in the e ~ ~ ~ of human 2-5A-~7-1~ L rNase mrNA without inhibitor ( ~, O ) or in the pLeSe~ ê of both 2--5A--daran~7ant RNase m7~NA and cycl~heY;mi~7a (50 ~Ig per ml (-). See Example I. S~hsa~antly, the recombinant 2-5A d~ . t rNase (or controls) is adsorbed to 2-5A-cellulose and r~h~m~rle~ce assays are performed after extensive washing of the matrix to reduce general nuclease activity. 7~AA;t77Ahaled substrate ?NA was either poly(U) (0, ,~ ) or poly(C) (O) -FIGS. 6A, 6B and 6C show levels of 2-5A ~7 ~ lr7_1lL r~Nase mRNA which are induced by interferon tLe~ L of murine L929 cells even in the p~ ..ce of cycl~lhaYimi,7~a.

W0 95/2~245 ` ~ 2 1 8 3 4 6 1 r~l" - ~

FIG. 6A is a northern blot ~ ~aL_d with poly(A)+RNA (4 llg per lane) that is isolated from murine L929 cells treated with murine interferon (a +
,~) (1000 units per ml) and/or cyclnhoYimi-lo (50 ~g per ml) for different durations (indicated) which is probed with r2~; ol 5~1hol orl murine 2-SA ~o~ nt RN2se cDNA. InLe Lt:L~I~, IFN; cyclnh~Yim;~lor CHI.
FIG. 6B shows levels of 2-5A-~ 5 L
RNase which are estimated rom the autoradiogram shown in panel ~a) with a video camera and Qlli ckC~rture and Image computer pL~yLal..C..
FIG. 6C shows levels of glyceraldehyde-3-phosphate d~lydL ~y~ S~ (GAPDH) mRNA
as detorm;n~ in the same blot shown in panel tA).
FIGS. 7A and 7B are the truncated, recombinant murine 2-5A-~ L RNase, clone ZBl, and murine L cell 2-5A-doron~lont RNase having identical 2-5A binding activities localized to a repeated P-loop motif.
FIG. 7A shows incubations of truncated 2-5A-t~ L RNase, clone ZB1, ("Recombinant") which is ~L~Iuced in wheat germ extract (upper panel) or of murine L cell 2-5A-~oron~lont RNase ( labeled "Naturally Occurring, " lower panel) with the 32P-2-5A
probe, (2.4 n~), are in the absence of pL~S~ of unlabeled 2',5' F~ n~;ester linked oligonucleo-tides (as indicated) followed by uv covalent W0 95/22245 2 1 8 3 4 6 1 r~

crnCsl inkinq. Autoradiograms of the dried SDS/10~
polyacrylamide gels are 8hown. Cl~..c~llL- ~tions of the ol; qnn~ ntide competitors are indicatea. I is inosine .
EIG. 7B shoWS a truncated series of murine 2-~A-d~ l RNase mutants (ZBl to Z815) which is U~ e d in wheat germ extract which are assayed for 2-5A binding activity by a f ilter binding method.
See Example and Knight et al. 1980). The positions of the P-loop motifs and the lengths of the translation products are indicated. Clone ZBl encodes for amino acids designated as 1-656 in Table 2, except for the last 5 amino acid residues which are Lys, Pro, Leu, ser, and Gly. Clone ZB2 encodes for amino acids designated as 1-619 in Table 2 .
Clone ZB3 encodes for amino acids designated as 1-515 in Table 2 . Clone ZB5 encodes for amino acids designated as 1-474 in Table 2 . Clone ZB9 encodes for a~ino acids designated as 1-403 in Table 2 .
Clone ZB10 encodes for amino acids designated as 1-365 in Table 2 . Clone ZB13 encodes for amino acids designated as 1-294 in Table 2 . Clone ZB14 encodes for amino acids designated as 1-265 in Table 2 . Clone ZB15 encodes for amino acids designated as 1-218 in Table 2 ~v0 9s~22245 ~ ; 2 1 8 3 4 6 1 ~ n~

FIGS. 8A and 8B are suhstitution mutations of the lysine residues in the P-loop motifs of 2 'A ~ Ar l RNase.
FIG. 8A shows the truncated murine 2-5b A~ RNase, clone ZBl, and ly~iine to asparagine substitution mutants of clone ZBl, which are synth~ci 7 ,d in wheat germ extract. In (A) translation pl~du~,~5 are covalently crr~ecl ink~ to the bromin~ _,LaLituted, 32P-labeled 2-5A probe, Br-2-5A-[32P]pCp. See Nolan-Sorden et al., 1990.
FIG. 8B shows the mRNA species which are translated in the ~Lt:s~lue of 35-S -- t ;~n;nF- in separate rGi~ ~rf; cmc. Autoradiograms of the dried, SDS7polyacrylamide gels are shown. The order and positions of the translation products (labelled "RNasen) and the relative molecular Dasses (in kDa) of the protein markers are indicated.
FIGS. 9A and 9B are a comparison of the amino acid Seq~l~nr~C of RNase E and 2-5A-cl~r~n~l~nt RNase.
FIG. 9A shows identical and ccl.seLv-tive matches which are shown between E. coli RNase E and the murine and human forms of 2DR.
FIG. 9B is a model for the ~LU~LuL~ and function of 2DR. Abbreviations: P-loop motifs, a repeated s~ e with homology to P-loops: Cysx, a Wo 95/22245 2 1 8 3 4 6 1 P~ S/~7llc~

cysteine-rich region with homology to certain zinc fingQrs; PK, homology to protein kinase domains VI
and VII.
FIGS. lOA and lOB are ~ comparison of the amino acid s~ P~ c of the ankyrin repeats in the human and murine 2-5A-~Dp~nAPnt RNase proteins.
FIG . lOA æhows murine and human f orms of 2-5A A~ A_ l RNases containing four ankyrin repeats. Homology between the ankyrin ~- ~c~ c 8e~ and the murine and human forms of 2-5A A~ L RNase are indicated. ~, hydrophobic amino acids.
FIG. lOB is a model showing the relative positions of the four ankyrin repeats in 2-5A-ADr~n~ nt RNase in comparison to the position of the ~lvyvz;ed 2-5A binding domain (t) (the repeated P-loop motif): Cysx, the cysteine-rich region; PK, the protein kinase homology region, and the carboxy-~ nAl region required for RNase activity.
FIG. 11 shows the role of 2-5A-d~rPn~Pnt RNase in the anti-viral le~.~vnse of cells to interferon treatment. Interferon binds to specific cell surface receptors resulting in the generation of a signal which activates a set of genes in the cell nucleus. The genes for 2-5A synthetase are thus activated producing inactive, native 2-5A
synthetase. Interferon treatment of the cell also _ _ W0 95/22245 ~ 218 3 4 61 r~

~ctivates the 2-5A cl~r~ A-~ L RNa6e gene (not shown in the FIGure) . S~ -"Lly, the interfeLu~- tL. ~ed cells i8 infectPd by a virus. Tlhe virus ~Lu-l...es double ~iLLC..Ided RNA tdsRNA~ during its replicative cycle. The viral dsRNA then activates the 2-5A
Dy..U.~L..se resulting in the production of 2-5A. The 2-5A then activates the 2--5A-~ t RNase to degrade the viral RNA thus de:.LLvying the virus itself .
FIG. 12 depicts a physical map of T: based binary vector pAM943 which is about 12 Rbp.
Abbreviations: BL, left border; ~3R~ right border;
Kanr, kanamycin resistance; AMT, promoter of adenyl methyl transferase gene from Chlorella virus; 355, promoter for 355 RNA from Cauliflower mosaic virus;
TER, RNA termination signal; Ovi V and ûri K origins of DNA replication.
FIG. 13 depicts physical maps of portions of certain recombinant plasmid constructs containing cDNAs Pnro~ling 1 i~n antiviral proteins and showing the;, Li~nL DNA elements in between right border and left border of T-DNAs that are transferred to plant genomes. ~IG. 13A depicts a certain portion of plasmid pAM943: PR68; FIG. 13B depicts a certain portion of plasmid pAM943 :muPR68; FIG. 13C depicts a certain portion of plasmid pAM943:5ynthetase; FIG.
13D depict6 a certain portion of plasmid ~ W0 95/22245 2 1 ~ 3 4 6 1 r~ o pAM943:2-5A-dep. RNase (sense); FIG. 13D/a depicts a certain portion of plasmid pAM943:2-5A-dep. RNare and FIG. 13B depicts pAM822:2-SA dep. RNase (antisense~.
Ab~reviations: BL, left border; BR, right border;
R~nr, ~ in r~ t~nce; Hygror, L~YL~ ~-;in resistance; AMT, E~L~ LeI of adenyl methyl tr~nsferAse gene from Chlorella virus; 355, promoter for 35S RNA from ~ liflower mosaic virus; PXR, cDNA
to human P~R; muPRR, cDNA to a lysine (amino acid #
296) to arginine mutant form of PKR; Sy~L~,e Lase, cDNA
to a low molecular weight form of human 2-5A-s-ynthetase; 2-5Adep. RNase, cDNA to human 2-5A d~l ,Ar~"t RNase; TER, RNA termination signal.
FIG. 14 shows a physical map of Ti based binary vector pAM822 which is about 14 . 6 Kbp.
Abbreviations: BL, le~t border; BR, right border;
Kanr, kanamycin resistance; Hygror, IIYYL~ ~in resistance; Tetr, tetracycline resistance; AMT, promoter o~ adenyl methyl transferase gene from Chlorella virus; 35S, promoter for 35S RNA from Cauliflower mosaic virus; TER, RNA termination signal; ovi V, origin of DNA replication.
FIG. 15 shows expression of human 2-5A-synthetase cDNA intr~n~n; c tobacco plants as ,lDt,~in,~,~ by measuring mRNA levels in a Northern blot. Cu.-,LLucL C (pAM943:Synthetase~ was i--LLv~uced into the plants . Total RNA was prepared f rom the .

W095/2_245 2 1 8 3 4 6 1 F~~ 8 leaves of control tlabeled "C") ~nd LLAr~ c pl~nts using RNASTAT-60 (Tel-Te5t B., Inc- ) Thirty ilg of ~'.NA was treated with glyoxal and separated in a 1. 5%
agarose gel. After el~.LLUU~oLC:Sis RNA was LLallDr~:L~ to 11~ La~l, (MSI) Nylon ' anc and probed with human 2-5A ~yllLlle~ -- cDNA labeled with t-32P]dcTp by random priming. Autoradiograms were made from the dried blots.
FIG. 16 shows expression of mutant and wild type forms of human PKR cDNA in LLa~s~e"ic tobacco plants as ~Pt~rm; n~d by measuring mRNA levels in a Northern blot. CUIIDLLUL.LS A (pAM943:PK68) and B
(pAM943:muPK68) ~nl~o~lin~ wild type and mutant (lysine at position 296 to arginine) forms of PKR, respectively, were introduced into the plants. Total RNA was prepared from the leaves of control (labeled "C") and LLA~ ;C plants using RNASTAT-60 (Tel-Test B., Inc. ) . Thirty ~g of RNA was treated with glyoxal and separated in a 1. 5% agarose gel . After elecLLu~lloLt:sis RNA was transferred to Mayl~&yLc~ (MSI) Nylon al,e and probed with human PKR cDNA labeled with [-32P]dCTP by random priming.
Autoradiograms were made from the dried blots.
FIG. 17 shows a pL.~ e of 2-5A d-~ L
RNase cDNA in LLar.s~ellic plants as det~rm;n~ on a Southern blot. Genomic DNA wa6 isolated from leaves of LLA~- J--I~;C plants ~nnt~inin~ cull~LLu~L D/a W0 9~/22245 2 1 8 3 4 6 1 r~ 205X
(pAM943:2-5A-dep.RNase, Ant;p e) using CTAB
(cetyltrimethy~ bromide) following the method of Rogers and Bendich ( 1988, Plant Molecular Biology Manual, A6, pp. 1-10, Rluwar ~ Dm;C Pulbisher, DULdLt d~L) . Ten ~Ig of genomic DNA was digested with HindIII for 5 h at 37-C and fr~ti~n~ted in a 19c agarose gel followed by LL~nsfe~ to Mayl~ayL~h ~nylon transfer membrane, Micron Separations, Inc. ) using a CAril1Ary transfer method. The cDNA for 2-5A-c~opPn~P~t RNase (from plasmid pZC5) was labeled by random priming with [a-32P]dCTP (3,000 Ci/mmole) using a Pri- 3 ~I,e kit from (Promega) according to the protocol s~r~liPd by the company. The labeled 2-5A-tiPr~n~lpnt RNase cDNA (Sper;fi- activity of 1.0 X
109 c.p.m. per ~g DNA) was washed and an autoradiogram was made from the dried memhrane. The sizes (in lcil~lhsl-c~-_) and the positions of the DNA
markers are indicated. The band indicated as "2-5A-dep. RNase cDNA" tsee arrow) was absent in Southern blots of control plants tdata not shown).
FIG . 18 depicts a coding seq~ nre f or human p68 kinase DRNA tPRR).
FIG. 19 depicts a translation product of the complete coding ~, ~ for human p68 kinase mRNA tP~R) of FIG. 18.
FIG. 20 depicts a coding se~ e for human 2-5A synthPtr a cDNA.

wo g5n2245 2 18 3 4 6 1 r~"~ ~

lPIG. 21 depicts a translation product of t_e coding 5~ for human 2-5~ ~y~ e of FIG.

20 .
r~PtA i ~ iu~iorl ~ 3y way of illustrating and providing a more lete appreciation of the present invention and ~any of the attendant Ld~ll.L~ges thereof, the following Det~; l ed Description and Examples are given cnn-~rnin~ the novel 2--5A--~r~-n~l~nt RNases, ~nrn~;
se~ c therefor, recombinant nucleotide molecules, ~;UII~LU-_~S~ vectors, recombinant cells, antiviral ~LA . y`~ ic plants and methods.
Because 2-5A~ r~n~ t RNase is very low in Ahlln~lAnre (one fi-~e ~.u-,dLe~ thn~ An~th of the total .protein in mouse liver, Silverman, R.H. et al., J.
Biol. Chem. . 263:7336-7341 (1988~ ), its cloning requires the development of a sensitive screening method. ~urine L929 cells are selected as the source of mRNA due to high basal levels o~ 2-5A d~ L~
RNase. A protocol to enhance 2-5A ~ RNase mRNA levels is developed based on the OLs-L V~ltiOn that optimal ;nt91- -t j nn of 2--5A dr ~ ont RNase is obtained by treating cells with both interferon and cyclnh~Yi~n;cle, then with medium alone. See Example.
The cDNA library is s~Leelled by an adaptation of t~ hniq~ developed for cloning DNA binding proteins, Singh, H. et al., Ç~, 52:415-423 (1988);

WO 95~22245 ~ i 2 1 ~ 3 4 6 ~ PcrluS95JO20S8 Singh H. et ~1., BioTe~-,htni~t~c, 7:252-261 (1989), in whic~t a bromin~ ~DLituted 32P-labeled 2-5A analogue ("2-5A probe"), Example and Nolan-Sorden, N.L. et al., An~l. 8iochem.. 184:298-304 (1990), replaced a r~ hDlec~ nl ;go~ yLibonucleotide. A single clone (ZB1) is thus isolated from about three million plaques. The protein ~ L~c~s~d from the Z81 clone, transfQrred from plaques to filter-lifts, showfi reactivity to both the 2-5A probe ~nd to a highly purif ied polyclonal antibody directed against 2-5A d~ ~lr~.L RNase.
To obtain recombinant protein for characterization, the cDNA is transcribed and translated in cell-free systems. See Example. 2-5A
binaing activity i8 then detr~--m;n~d by covalently crnC5l inkin~ the 2-5A probe to the protein with uv light, for e~ample, Nolan-Sorden, N.L. et al-, ~
Bioch~m., 184:298-304 (1990). The recombinant 74 kDa protein produced in a wheat germ extract shows specific affinity for the 2-5A probe. See FIG. 2A, lanes 1 to 3. A core derivative of 2-5A lacking s~-rhncrhnryl groups, (A2~p)2A, ~ails to interfere with binding of the protein to the 2-5A probe whereas trimer 205A, p3(A2'p)2A, completely ~Lev~ ; probe binding. See FIG. 2A, lanes 2 and 3, respectively.
~here is no detectable 2-5A binding proteins in the wheat germ extract as shown in the incubation without W0 95l22245 2 1 8 3 4 6 ~

added RNA, FIG. 2A, lane 4. For comparison, a simil2r profile of 2-5A binding activity i5 obtained for the 80 kDa 2-5A-~ A- ~ RNase from murine L929 cells, ir.~ uL.~ted without added ol; ~n~ eotide or with (A2'p)2A or p3(A2'p)2A as competitors. See FIG.
2A, lanes 5 to 7. The 35S-labeled translation product is shown in FIG. 2A, lane 9. In a further comparison, covalent linkage of the 2-5A probe to the about 74 kDa protein and to murine L929 cell 2-5A-~ Qr L RNase followed by partial digestion with ~1~y LLy~sin ~ lu~es an identical pattern of six labeled peptides. See FIG. 2B. Similarly, partial digestion of the two labeled proteins with 5.
aureus V8 protease also ~uluces identical paLLeL,.s of- labeled cleavage ~Lu-lu- Ls. These results and the apparent molecular weight of about 74 kDa for the recombinant protein, as compared to about 80 kDa for 2-5A-APr~nA~nt RNase, see FIG. 2A, ~uyye::~Ls that the about 74 kDa protein is a truncated, or partial clone f or 2 -5A~ L RNase .
To obtain the entire coding sequence for human 2-5A-~ r~ "L RNase, a composite DNA
containing genomic and cDNA is ~ UII~LL u~Led. See FIG .
3A. The initial cDNA portion of the human 2-5A-A-l- l- L RNase clone (HZBl) is obtained by screening a human kidney cDNA library with raA;ol~h~-led murine 2-5A A~ P L RNase cDNA. See ~ . -~ wogs/2224s 21~346t P~llu~ o Example. A genomic clone, ~ nl-~;n;nq the 5'-part of the coding ~ e _ ~ is ; Col~t~ with rP~l i ol~h~
hum~n 2 _A d~ - L RNase cDNA. The nucleotide and predicted amino acid ~ of human 2-5A ~ Rlla_e are ~ rmirlp~ FIG. 3B, resulting an open reading frame ~n~ o~l;nq a protein of 83, 539 Da.
A comparison is made between the predicted amino acid S~G~ * 0~ the human and murine forms of 2-5A ~ L RNase in order to identify and evaluate the C~ ael v~d regions of the proteins. See FIG. 4. The murine cDNA, clone ZBl, cont~;nC about 88% of the coding 5~ '- re for 2-5A d~ RNase to which an additional L-._,.L~ eight 3 '-codons are addea from a murine genomic clone. Al i~, L of the murine and human forms of 2-5A A~ L RNase indicates about 65% identity between the overlapping regions. See FIG. 4. In addition, there is 73%
identity between the ~uLL__l,.. .l;nq nucleotide Gr~ ~c for murine and human 2-5A-~ L RNase.
The apparent translation start codons for both the murine and human 2-5A-~r~n~nt RNases, are in an ~p~ru~L iate context f or tran51ational initiation, namely ACCATGG and GTC~ÇG, respectively. See FIG.
3B. See also, for example, Kozak, M., 5~gll, 44:283-292 (1986). In addition, both the human and ~rine 2-5A-d~L F ~ RNase 5~ _R contain W0 9sl2224s 2 1 8 3 4 6 1 in-frame stop codons ~ LLe:~u of the translation ~tart sites. See FIG. 3B.
The 2-5A binding properties of the , ~,c ; nAnt and naturally occurring forms of human 2-5A ~-L- ,-1_--~ Rl~ase are _ e l by uv covAlent crn~l;nl~in~ to the 2--5A probe. The ~ inAnt human 2-5A A-~ t. RNase produces in wheat germ extract shows -rac~fic affinity for 2-5A. See FIG.
5A, lanes 1 to 3. l~AA;ol~hol in~ of the cloned human 2-5A d~r- l ~ L RNase with the 2-5A probe i5 not prevented by (A2'p)2A. See FIG. 5A, lanes 1 and 2.
In contrast, addition of trimer 2-SA, p3(A2'p)2A, effectively ~ l~~ with the 2-5A probe for binding to the . ~ l nAnt 2-5A-~'Ioron~lont RNase. See lane

3.~ The same pattern of 2-5A binding activity is obtained with 2-5A do~ l&~ t RNase in an extract of interferon-treated human HeLa cells. See FIG. 5A, lanes 5 to 7 . The Ys,al- -.L lo~ r weights of HeLa cell 2-5A-~ all~ RNase and 355-labeled recombinant human 2-5A-~rn~nt Rllase ~udu~ in reticulocyte lysate are believed to be exactly the same (about 80 kDa). See PIG. 5A, lanes 5 and 9. The recombinant human 2-5A-~ L RNase ~l u-luc~d in wheat germ extract migrates slightly faster probably due to post-translational i f i cations . See FIG . 5A, lanes 1, 2 and 8.

w095~22245 2 1 8 3 4 6 1 r~
.

To '- L,ate - and characterize the rihnn~ leArAi activity of the cloned 2-5A ~F~ '3C ~t RNase, translation is p~Lr- -' in a reticulocyte lysate instead of a wheat germ extract due to the ;A11Y greater efficiency of protein æynthesis in the former cystem. See FIG. 5A, compare lanes 9 ana 8. Prior to translation, _ l..,J,-.n,-c reticulocyte 2-5A d~ Fl~ RNa_e is removed by A~lC~bin~ the lysate to the affinity matrix, 2-5A-cellulo6e. See Example. See also, Silverman, R.H., Anal. Riochesl..
144:450-460 (1985). The LL~al ~ with 2-5A-cellulose effectively removes all measurable g-- ~ c 2-5A A~ - L RNase activity f rom the lysate, as detP~m;n~d by 2-5A-d~ rihnm~-lP~ce .assays, and FIG. 58. In addition, the adsorption-depletion protocol did not reduce translational ~fficiFnry. FIG. 5A, lanes g and 12 show the 35S_translation ~JLUd~ S produced in the 2-5A-cellulose-E ~e ~L-ated and untreated lysates, respectively .
Rihnnl~lease assays with recombinant 2-5A-~lPrPn~lPmt RNase are p~L r( - ~ after immobilizing and purifying the translation product on the activating affinity matrix, 2-5A-cellulose. It was previously shown that murine L cell 2-5A-d-l r~
RNase bound to 2-5A-cellulose, re-culting in ri ho~ Ace activity against poly (U~ but not -W0 95/2224~ 2 1 ~ 3 4 6 1 r~ 0 poly(C). See Silverman, R.H., anpl. BiQchem., 144:450-460 (1985). F~h~ ~, by washing 2 e~_~3~ L RNase:2-5A--coll~lnce prior to adding the .uL,,L.e~te the lovel of general, non-2-5A ~ lr-l~ ~Wase, is greatly reduced. See Silverman, R.EI., An~ 1nrhom.. 144:450-460 (1985).
Incubations of lysate in the absence of added ~NA or in the presence of both human 2-5A-'.-~ L RNase mRNA and cyclnhoYim; 'o resulted in only low levels of poly(U) breakdown. See PIG. 5B. In addition, it is shown that CyclnhoY;m;,-O completely prevented 2-5A-'oprn'ont RNase synthesis. See FIG. 5A, lane 10. In CullLLcl5L~ translation of the human 2-5A-fl~lJ~ lr L RNase m~?NA~ in the absence of inhibitor, results in substantial ribonuclease activity against poly(U) but not against poly(C).
See FIG . SB. The poly (U) is degraded with a half-life of about 10 minutes whereas only 20% of the poly(C) is degraded after one hour of incubation.
Binding of recombinant 2-5A-deron'r~nt RNase to the affinity matrix was also shown by monitoring the pLesenc~ o~ the 35S-labeled translation product.
These results are believed to ' LLate that the recombinant human 2-5A-d~- lo L RNase E~ lu~d in vitro is a f~lnl-tinn~l and potent ribonuclease.
Fu~ both - ; nJ~nt and naturally occurring forms of 2-5A fl~ "L RNase are capa~le of cleaving _ = = = . = = = = =, =, , = = = _ . = = = = = = = = _ WO95/22245 218346 ~ r~.111J_,.I~

poly(U) but not poly(C). See FIG. 5B. See also Silverman, R.H., AnAl. Biochem., 144:450-460 (1985) and Floyd-Smith, G. et al., Science, 212 :1020-1032 1981) .
To ~ Prm;no if 2 ~A d~l-- l- l L RNase mRNA
level6 are regulated by i--~e~ re~ , a northern blot ~rom murine Lg29 cells treated with interferon and cyc~nhPY~m~-le is probed with the radiolabeled murine 2-5A ~e~ RNase cDNA. See FIG . 6 .
2-5A ,~ "l RNase mRNA levels are orlh:~nced three-fold by interferon (~ + ~B) LL-_~t. L even in the ~ ence of cyclnhPY;mi-le. See FIGS. 6A and B, compare lanes 1 and 2 ) . Regulation of 2-5A-depon~lont RNase mRNA levels by interferon as a function of time iS--~ LL-~t~d tFIGS. 6A and B, lanes 3 to 6.
Maximum 2-5A-dep~rulont RNase mRNA levels are obs~r~_d after 14 hours of interferon tre~tment. See FIGS. 6A
and B, lane 6. A similar in~rease in levelsD of 2-5A-~opPn~lont RNase per se is observed after interferon treatment of the cells. Relatively invariant levels of GAPDH _RNA indicates that equivalent levels of RNA are present in every lane of the blot. See FIG. 6C. These results are believed to show that the in~ t;Qn of 2-5A-~ QnL RNase eYpression is a primary l.:D~,..se to interferon treatment. The murine and human 2-5A-A~ L RNase mRNAs are ~otPrm~nP~ from r.~LU.e-.. blots to be 5.7 kb _ _ W09512224~ 2 ~ ~ 3 4 6 1 r~~ A
and 5. 0 kb in length, respectively. See FIG. 6A.
The 2-5A d ~ --L RNase coding ~ 6~ U-el~fuL-comprlse only about 40% the nucleotide 5 C~ntA; n~-A in the mRNAs.
The 2-5A binding f~lnr~t; rnlc Or the L-__ ' ;n:l~nt and naturally occurring forms of murine 2 rA ~ RNase are characterized by covalent crossllnking to the 2-5A probe in the yl~Ea~ e of nl ~h~l e~l 2-sA or 2-sA analogues as competitors . See FIG. ~A. Interestingly, although the about 74 kDa truncated 2-5A A~ A_- ~ RNase i5 missing about 84 ~mino acids from its ~aLL~ f~m;n.lc, see FIG. 4, it nonetheless p ~ s ~?- a 2-5A binding activity indistinguishable from that of naturally occurring 2-SA A~ RNace. See FIG. 7A. Trimer 2--5A[p3(A2'p)2A], at about 20 nM effectively ~Lt:v~llL:i the 2-5A probe- rrOm binding to either protein. See FIG. 7A, lane 8. In comparison, a 500-fold higher . ol~c~,-LLation of (A2'p)2A (10 IIM) is required to prevent probe binding to both proteins. See lane 13. The dimer species, p3A2'pA, is unable to prevent the 2-5A probe from binding to the proteins even at a ~h~ LL~-t.iOn of lO~M (lane 18) . However, the inosine analogue, p3I2'pA2'pA, Imai, J. et al., J.
Biol. Chem., 260:1390-1393 (1985), is able to prevent probe binding to both proteins but only when added at a ~ Lration of about 1.0 I~M (lane 22).

wo 95/22245 2 1 8 3 4 6 1 r~

To further define 8F~.~.. -r~ ~ involved in 2-5A binding, nested 3'-dPl~tinn of the murine 2-5A ~P~ L RNase cDNA, clone ZBl, are ol-D~Lu~ed, Llc-l-svLibed in vitro, And e~L~..D~d in a wheat germ extract. See FIG. 7B. The different APle~irn clones ~.Vlu~.eD comparable amounts of polypeptide as monitored by incvL~uL~tion of 35S_methirninP. The levels of 2-5A binding activity are detP~inn~l with the 2-5A probe in both a filter binding assay, Knight, M. et al., ~a, 288:189-192 (1980), and the uv crosslinking ~ssay, Nolan-Sorden, N.L. et al., An~l. Biochem.. 184:298-304 (lg90), with similar results. See FIG. 7B. Expression of clone ZBll, Pnror3in~ amino acid residues 1 to 342, results in- a 10s5 of only about 26% of the 2-5A binding activity as _ ~1 to clone ZBl (amino zlcids 1 to 656). See FIG. 7B. Clones intr- -iate in length between ZBl and ZBll ~11 result in si~ni fir~nt levels of 2-5A binding activity. In ~ v--LL~DL, protein pLv~uced from ZB13 (amino acids 1 to 294) results in only about 38.3% of the 2-5A binding activity of clone ZBl, suggesting that a region ; L~ f or the 2-5A binding function is affected. Indeed, clone ZB14 ~Lv-lu~æd a protein ~nrorlin J amino acids 1 to 265 which is nearly inactive in the 2-5A ~inding assay (only 1.9% of th activity of clone ZBl).
Interestingly, the significant decrease in 2-5A

W09~/2224~ 21 8346 I r~ 30 binding activlty OLa~v-:-l with ZB14 occurs with the d le~inn of one of two P-loop motifs; nucleotide blnding domains in many proteins. See FIGS. 4 and 7B. See also Saraste, M. et al., TIBS, 14:430-434 (1990). Deletion of both P-loop motifs in clone ZB15 results ln protein (amino acids 1 to 218) which is 1 ef p~ y lacking in 2-5A binding activity . See FIG. 7B.
To probe the involvement of the COncpnc~
lysine residues in the P-loop motifs in 2-5A binding activity, site-directed , --ic is performed on the LLu~ ted form of 31urine 2-51;~ L RNase encoded by clone ZBl. Previously, it is l~u- Led that substitution mutations of the Cu~ el ved lysine residues in P-loop motifs o~ eucaryotic initi~tion factor 4A and for R~ c anthracis adenylyl cyclase results in a 1085 of ATP binding and catalytic activities, respectively. See Rozen et al., ~
rPl l . Biol., 9:4061-4063 tl989) and Xia, Z. and Storm, D.R., J. Biol. Chem., 265:6517-6520 (1990).
In the ~ormer study the invariant lysine residue is mutated to a5paragine. See Rozen et al., Mol. Cell.
Biol., 9:4061-4063 (1989). We substituted, individually and t~J~ , the ~....CC-I.F..C lysines with asparagines at positions 240 and 274 in the two P-loop motifs of 2-5A-d~ RNase. See FIG. 8 and the ~ . Analysis of the effects of these _ _ . _ _ _ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ WO 951~2245 2 1 8 3 4 6 1 r~

mutations on 2-5A binding activity is ~ rmi n-~d by covalently ,;~, -1 ;nlr;n~ the 32P-2-5A probe to the in vitro translation ~L~d~ L:i under uv light. See FIG.
8A. See also Nolan-Sorden, N.L. et al., Anal.
Bi~rhP"., 184:298-304 ~1990). Simil~r levels of proteins are synth~s; 7~ from the different mRNA
species as shown in separate reactions containing 35~ - '' ;nnin~. See FIG. 8B. The three mutant forms of 2-5A A~ " ~ RNase Eihows reduced binding to the 2-5A probe. See FIG. 8A, lanes 2 to 4. Clone ZBl~Lys240-~Asn), FIG. 8A, lane 2, expresses a mutant 2-5A-~ r~n~ t RNase with a ~ Lially reduced affinity for 2-5A; about 48.4% of the activity of clone ZBl as det~rmin~d by rhrrrhorimager analysis .(MoIecular Dynamics) of the dried gel. A more modest reduction in 2-5A binding activity, to 79% of the control value, is obtained from clone ZBl (Lys274-)Asn) . See FIG. 8A, lane 3 . In cu--~Las~, 2-5A binding activity from clone ZBl(Lys240~274-~Asn), FIG. 8A, lane 4, in which both conserved lysine residues are replaced with asparagine residues, is reduced to only 12.2% of the activity of clone ZBl (avt:l ed from three separate experiments). These results suggest that the lysine residues at positions 240 and 274 function within the context of a repeated P-loop motif in the binding of 2-5A to 2-5A d~ -, L RNase.

WO 95/2~245 ;~ 1 8 3 4 6 1 r~
The ~ l~-rt~ cloning and expression of 2-'' d-,_ rl~ l RNase, the t~min~7 factor in the 2-5A
~ystem and a key enzyme in the - 1 ,D-ll A~ -ni of interferon action is described. See FIG. 1. The r~_ ' in~nt proteins ~-du~ in vitro are d Ll a L~.~ to possess 2 -5A binding properties identical to naturally occurring forms of murine and human 2-5A A_~ 4D L RNase. See FIGS. 2, 5A, and 7.
In addition, lin~cage of a 32P-2-5A analogue to a truncated murine 2-5A ~ , L RNase and to murine L
cell 2-5A r~ lc t RNase followed by partial proteolysis reveals i~Dnt i~ll patterns of labeled peptides. See FIG. 2B. Fur~h- ~, the full-length recombinant human 2-SA-~ . L RNase isolated on the ~ activating, af f inity matrix, 2 -5A-c~ e, shows potent ribonuclease activity towards poly (U) but none against poly(C). See FIG. 5B. Similarly, it is p~eviously '- L- ated that murine L cell 2-5A-~Pr~nADnt RNase was activated by 2-5A-cellulose resulting in the cleavage of poly(U), but not of poly(C). See Silverman, R.H., An~l. Biochem..
144:450-460 (1985). The full-length human 2-5A-d~DrDnADnt RNase, which is ~L.~ul ~d in reticulocyte lysate, had the same apparent molecular weight as did naturally occurring 2-5A ~
RNase. See FIG. 5A. E~owever, the actual molecular m~ss of human 2-5A-~ _ l RNase is APtPnminD~3 from W0 95/222~5 2 1 ~ 3 4 6 1 the predicted amino acid c~ , FIG. 3B, to be about 83,539 Da.
Previously, it was L~ L ~d that interferon r~nhAnr ~s levels of 2-5A A~ l_ L RNase by between two- to twenty-fold r~r~r~ { nq on the cell type. See Silverm~n, R.H. et al., F~lr, J. Biochem.. 126:333-341 (1982b~ and Jacobsen, H. et al., Viroloqy, 125:496-501 (1983a). Results presented herein suggest that the gene for 2-5A del~enA~ t RNase may be an int~Lr_Lul. ..Limulated gene. See FIG. 6. Levels of 2-5~ c~ ,A,~nt RN~e mRNA in murine L929 cells are elevated as a function of time of interferon (a + 3) LL~a; L by a factor of about three. FUL UleL `e, the induction a~ al- ~ to be a primary Le:~OI~:~e to interferon LL~a; ~ because it is obseLv- d in the ylesl=llce of cyc~r~h~Y;mid~. Therefore, interferon is believed to regulate the 2-5A pathway by elevating levels of both 2-5A synfh~t~ces, llv~r~ siAnr A.G. et al., Nature, 268:537-539 (1977), and 2-5A-d~ A~ L
RNase, Jacobsen, H. et al., Viroloav. 125:496-501 tl983a)- See. FIGS. 1, 6 ~nd 11.
The cloning of 2-5A-~ n~l~ t RNase reveals several features of the protein. The 2-5A binding domain i5 of particular interest because it is the ability of 2-5A-A-I~ l'A_"L RNase to be activated by 2-5A that sets it apart from other nucleases. By e~pressing nested 3 ' -d~letions of murine Wo 9sl2224~ P

2-5A c~ - L RNase, a region between amino acids refiidues 218 and 294 which is ~elieved to be critical for 2-5A binding activity is l~ t~ . See FIG.
7B. Interestingly, the i~t~nt;fied region cnt~t~in~ a repeated P-loop motif, one from residues 229 to 241 and another from residues 253 to 275. See FIG. 4 and Table 2 . ~lhen t~te latter P-loop motif (antino acids 253-275) is partially deleted, there is a precipitous decline in 2-5A binding activity. See clone ZB14 in FIG. 7B.
The homology with P-loops is believed to be highly CUIIS~LV~1 between the hu~tan and murine forms of 2-5A-ci~ L RNase; thus ~.. d6~-cuLing the belief of the i ~hce of this region for 2-5A binding activity. See FIG. 4. The similarity to P-loops consists of the tripeptides, glycine-lysine-threonine, ~L Lceded by glycine-rich s~qu-~nr ~c . In this regard, the unusual feature of 2-5A-~ .L
RNase is that the P-loop motif is repeated and are in the same orientation. Adenylyl cyclase from g~ril l,-c anthracis also contains a duplicated P-loop motif, however, the two se~ c are in opposite orientation and are overlapping. See Xia, Z. and Storm, D.R., J. Biol. Chem., 265:6517-6520 (1990~.
The relative i _ ~nce of the _ulla~L v~
P-loop lysines (at positions 240 and 274) are evaluated by site-directed ~ ic of the murine ~ W0 95/22245 2 1 8 3 4 6 1 2-5A~ L RNase, clone ZBl. Although individual substitution mutations of the two lysines ~tgn~f1~J-ntly reducea 2-5A binding ~ctivity, r~rl ~; n~J both of the lysines with as~e.L ~ine residues in the same mutant RN~se severely L~ 5e6 2-5A binding. See FIG. 8. Perhaps the trimer 2-5A
requirement for activation of most forms of 2-5A ~ t RNase could be r~Ypl ~ i nr~d if the f irst and third adenylyl residues of 2-5A interact with the separ~te P-loop s ~ s ;nrlllr ;h~ o.,f~ a1Lional changes in 2-5~ d 1~ L RNase. In this regard, dimer 2-5A neither binds 2-S~ r ~ RNase effici~ntly nor does it activate 2-5A de~
RNase , FIG . 7A ; Kerr , I . N. and Brown , R. E ., Prod .
~atl. Acad. Sci. U.S.A., 75:265-260 (1978) and Knight, M. et al., I~at~e, 288:189-192 (1980), perhaps because it is too short to span the two P-loop motifs. Alternately, the residual 2-5A
binding activity ~bseL~._d in the point mutants, ZBl (Lys240-)Asn) and ZBl (Lys274-)Asn), and the very low af f inity of the double mutant, ZBl(Lys240~274-)Asn) for 2-5A, could indicate that the two P-loop motifs are parts of separate 2-5A
binding domains.
~ omology with protein kinase domains VI and VII is also identified in 2-5A-~l~p~n~^nt RNase. See FIG . 4 . See also Hanks , S . K. et al ., Science , Wo 9512224S _ 2 1 8 3 4 6 1 ~ o .

241:42-~2 (1988). Although domain VI i8 believed to be involved in ATP bindinq, this region in 2-5A ~ ' L RN~se i8 believed not to be i _ L~
for 2-5A binding because its ~le] et~ n-~ caused only a ~ninimal r~ t~nr~ in affinity for 2-5A. See FIG.
7B. However, a modest ttwo-fold) stimulatory effect of ATP on 2-5A ~ RNase activity has bQen L~JUL ~ed. See ~- ~=a~ L..er, D.H. et al., Fl-r. J.
Bior:hPm.. 124:261-268 (1982) and Rrause, D. et al., J. Biol. ~'h~-~., 261:6836-6839 (1986). ThQ latter report ~ n~ Ate~l that ATP was not required for 2-5A tle~ RNase activity but may act to 81 :~hi 11 7e the enzyme. lh I ~ru ~, the region of homology with protein kinases could perhaps bind ATP
resulting in stimulation of rihnn~ Ace activity through stabilization of the enzyme.
A ~ c zinc finger domain, reviewed in Evans, R.M. and Rnllf~nh~rg~ S.~l., ~, 52:1-3 (1988), consisting of six cysteine residues with the ~LLU-.LULe CX4CX3CX17CX3CX3C (amino acid residues 401-436 in Table 2 ) is i~ntifi~d in the murine form of 2-5A ~ RNa6e . See ~IG. 4 . The ~ - lO~o -c region in the human for~ of 2-5A-depenent RNase is CXl1CX25CX3CX6C (amino acid numbers 395 to 444 in Table 1 ). Because zinc fingers are nucleic acid binding domains, the cysteine-rich region in 2-5A d~ RNase could be involved in binding to W09~i/22245 2 1 ~ 3 4 6 ~

the RNA .uL~,L,ate. Alternatively, the cysteine-rich domain in 2-5A~ r- l~ ~L RNâse could mediate formation of 2-_A A ~ ,t RNase dimers. Analysis of crude pL~Le-L~OnS of 2-5A d~ RNase suggest that 2-5A A~ RNase may form dimers in a~e~ but not in dilute ~ L- CL~i. See Slattery, E. et al., Proc. Natl. Acad. Sci. U.S.A..
~6:4778-4782 (1979) and Wreschner, D.}~. et al-, J. Biochem., 124:261-268 tl982).
Comparison between the amino acid 2.~ c of other ribonucleases with 2-5A-~lDron~ont RNase identif ies some limited homology with RNase E, an endor;h~r~ P~e from E. coli. See FIG. 9A. See also Apirion D. and Lassar, A.B., J. Biol. rhO~..
25~-1738-1742 (1978) and Claverie-Martin, F. et al., J. Biol. Chem. 266:2843-2851 tl991). The homology with RNase E is relatively l_VIID~::' v~d between the human and murine forms of 2-5~ d~ RNase and spans a region of about 200 amino acid residues.
Within these regions there are 24 and 32% ;r~ontiCAl plu5 ~v..se~vative matches, with some gaps, between RNase E and the human and murine f orms of 2-5A-~lop~ n~lont RNase, respectively. See FIG. 9A.
The rne gene which encodes RNase E and the altered mRNA stability tams) gene, Ono, N. and Kumano, N., J.
Mol. Biol.. 129:343-357 (1979), map to the same genetic locus. See Mudd E.A. et al-, l~P~

4~ 2 1 8 3 4 6 1 ~"1/l ~ r Mi~robiol., 4:2127-2135 (1990); Babitzke, P. and Rushner, S.R., Proc. Natl. Acad. Sci. U.S.A.. 88:1-5 (1991) and l.,L--_~iciene, L. et al., Mol. Mi~robiol.,

5:851-855 (1991). RNase E is required for both eff~ri~nt mRNA tUL~ and rRNA procQ~;n~ in E.
coli . s~e Mudd E.A. et al., Nol . Mi rrobiol ., 4:2127-2135 (1990) and Babitzke, P. and Rushner, S.R., Proc. Natl. Acad. SCi. U.S.A., 88:1-5 (1991).
The cleavage ~:r~r;fi~ties of 2-5A-d~r~nr7Dnt RNase and Rllase E are similar in that 2-5A-d~ _\ L RNase cleaves mainly a~ter W or UA, Wreschner, D.H. et al., Nature, 289:414-417 ~1981a) and Floyd-Smith, G.
et al., Science. 212 :1020-1032 (1981), and RNase E
usually cleaves within the central AW s~ n~e Or tG
Dr- A)AUU(A or U), Ehretsmann, C.P. et al., Genes &
Develol~mer;t. 6:149-159 (1992). The location of the RNase E ho~ology and other identiried features in 2-5A ~_IJ~ ,L RNase are shown. See FIG. 9B. These f;n-lin~ raise the pnce:ihil ity that RNase E may be the ancestral ~LI:C~ L~oI of 2-5A-~prDnrl~nt RNase. In this regard, there are indications Or 2' ,5'-~1 jgnl~lPnylates in E. coli. See Brown, R.E.
and Kerr, I.M., Process ,in Clinical and Biolo~ical RP~ rCh, 202:3-10 (1985) and Trujillo, N.A. et al., ~llr. J. Biochem.. 169:167-173 (1987~. However, the evolutionary distribution Or a complete 2-5A system (i.e. 2-5A ay--U._klse and 2-5A ~ L RNase) is _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ W0 9512Z245 ~ 2 1 ~ 3 4 6 1 Y .~ e~ c8 I~ ~UL ~ed to begin only with reptiles or possibly ` ;hj~. See Cayley, P.J. et al., Biochem. Bio~hvs.
Res. Commun., 108:1243-1250 (1982).
Enaorihnn~ leA--F play a controlling role in RNA ~ by cat~lyzing the rate-limiting steps in RNA decay. See BL __ , G ., Cell , 57 : 9-10 (1989). 2-5A ~PI~ RNase is a uniquely regulated endor; h~n~ which mediates ef rects of interf eron against picornaviruses. It functions by binding 2-5A
and ~ y d_~L d~s both viral and cellular RNA. See WLes.l~er, D.H. et al., Nucleic l~r;~e Res., 9:15?1-1581 (1981b). In addition, the 2-5A system may be involved in the antiproliferative effects of interferon and in the fllnrl Lal control of RNA
stability. Cellular levels of 2-5A~ .t RNase and/or 2-5A-synthetase are regul~ted during int~lrer~,.. treatment, HovAn~cc;~n, A.G. et al., ~, 268:537-539 (1977) and Jacobsen, H. et al., Viroloc~v, 125:496-501 (1983a), cell growth arrest, Stark, G. et al., Natu~e, 278:471-473 (1979) and Jacobsen, H. et al., Proc. Natl. Acad. Sci. U.S.A., 80:4954-4958 (1983b), cell differentiation, Krause, D. et al., Fllr. J. Biochem., 146: 611-618 (1985), rh~n~;n~ hormone status, e.g., Stark, G. et al., Nature, 278:471-473 (1979), and liver Leyt:~u=ration, Etienne-Smekens , N. et al ., Proc . Natl . Acad . sci .
IJ.S.A., 80:4609-4613 (1983). However, basal levels w095/22245 2 1 834 6 1 ~lr~ ~

of 2 _A r~ _ L RNase and 2-5A synth~tr-- ~re present in most if not nll 1; An cells. The existence of multiple forms of 2-5A ayllU.. Ldse with different intr~ lAr locAtions, H~ vA~ ;An, A.G.
et al., ~MRn -J., 6:1273-1280 (1987), could indicate diverse fl~nrt;nn~ for the 2-SA system. Similarly, the ubiquitous ~l e sen ~: of the 2 -5A system in higher aniDals :~u~yc:~S an i La..L function for 2-5A ~ L RNase, Cayley, P.J. et al., Biochem.
BioDhYs. Res. comm~, 108:1243-1250 tl982). For in-:t~ln-e, 2-5A-d~ "1 RNase cleaves rRNA at spec;f~c sites in lntact ri - --, Wreschner, D.H.
et al., Nucleic Acids Res., 9:1571-1581 (1981b) and Silverman, R.H. et al., J. Virol., 46:1051-1055 (1983), possibly affecting translation rates. The transient nature of 2-5A, Williams, B.R.G. et al., J. Biochem.. 92:455-562 (1978), and its growth inhibitory effect after illLr ,l I inn into cells, Hovanessian, A.G. ~nd Wood, J.N., ViroloaY, 101:81-89 (1980), indicate that the 2-5A system i5 a tightly regulated pathway.

~y~T.lZ I
The source of mRNA for preparing the cDNA
library is murine L929 cells grown in EMEM
(Whittaker, Inc. ) and supple~ented with about 10% FBS
(Gibco-8RL), and antibiotics. The cells are treated with about 50 llg per ml of cyclnh~Yi~ and 1000 W0 95/2224S 2 1 8 3 4 6 1 r~ 7n~x units per ml of murine i.lL~aLL~u,. ( + ,1~) (1. 3 X 107 units per mg protein: Lee Ril lec~ r) for about 2 . 5 hours to increase levels of 2 '~-dG~ .,L RNase mRNA. Total RNA was then ;~ 1At--r7, e.g. ~ y~ ki~
P. and Sacchi, N., An~l. B$ochem., 162:156-159 (1987), from which poly(A)+ RNA is ~Le~L.d by oligo(dT)-c~ l o~:e ~ lIL~ U~L~ ly as described. see Sambrook, J. et al., Cold sl~rin~l Harbor Lab-,-ato~v Press tl989). Synthesis of the first strand of cDNA
is done by using reverse ~Lan~Liptase as described (SU~è~- Lipt; BRL) except that 5-mêthyl-dCTP is substituted for dCTP and an XhoI-oligo-dT
adapter-primer tSLLata~el.e) is usQd. Synthesis of the econd strand of cDNA and ligation of EcoRI
linker was as described (Stratagene). The cDNA is digested with EcoRI and XhoI and unidirectionally cloned into predigested ~ZAPII vector (sLrc~b~e~e).
The library- is p~k~gecl by using Giagpack Gold extract and titered on P.R-F bacteria.
The cDNA library is screened directly without prior amplification at a density of about 25,000 phage per 150 mm plate. Phage are grown for 3.5 hours at about 42C until plaques arê visiblê.
Nitrocellulose filters saturated in IPTG ~10 ml~) and then dried, are overlaid on the plates and growth was continued for an additional 4 to 6 hours at 37C.
The ~ilters are pL~ ed by a ';f~-~Ation of the wo g~ 2 1 8 3 4 6 1 ~ T. c methods of Singh, ~I. et al., Cell, 52:415-423 (1988) and Singh, R. et al., BioTprlln~ 7:252-261 (1989). Filters are washed in ice-cold binding buffer (about 20 ~M Tris-}~Cl, about pH 7.5, about 20 oM -i acetate, about 50 mM potassium chloride, about 1 ni!! EDTA, about 50 mM B ~ ~.a~oethanol, ~bout O .1 mlS PNSF, ~bout 5% glycerol ) containing about 6 M
;rl;nC~-~Cl for about 20 min. The solution containing the filters is then diluted two-fold with binding buffer and washing on ice is continued for about an additional 5 minutes; seri~l two-fold dilutions were cc~Tlt;n~-ed until the gll~n;~;nP
..,.,- ~..~L~tion was about 187 mM. The filters are then washed twice with binding buffer, and incubated with binaing buffer containing about 5% nonfat milk for one hour at about room t~ ~ ~LULI:. The filters are then washed twice with binding buf fer and incubated in binding buffer (supplemented with about 0.25%
nonfat dry milk and about 0 . 02~ sodiu~ azide) containing p(A2'p)2(br8A2'p)2A3'-[32P]Cp (the "2-5A
probe"), Nolan-Sorden, N.L. et al., Anal. ginf-hF~m., 184:298-304 (1990), at about 2 X 105 counts per minute per ml (about 3, OOo Ci per mmole) at about 4C
with shaking for about 24 hours. The filters are washed twice with binding buffer and then twice with water before air drying and PYpoS;n~ to film.

WO 95l22245 2 1 ~3 3 4 6 1 1~ n~
5~--~ lurine L929 cells are treated with about 1000 units per ml illLeLLeL.~I (a + 13) with or without about 50 llg per ml of cyrl nh~-Y~m~ and the total RNA
is then isolated as described. See ~'hl _y~lski, P.
~md Sacchi, N., AnAl. Biochem., 162:156-159 (1987).
Poly(A)~ RNA i~ prepared by oligo(dT)-c~ lose ~ r, I,CyLc~ y~ as described in Sambrook, J. et al., Cold SDrin~ ~:~rhnr T~Ah~ ~Lat~r v Press (1989), and is separated on glyoxal ~garosQ gels and transferred to Nytran membranes. RNA is immobilized on the membrane by uv croq~l inkin~ (Stra1 Al ;nkDrr Stratagene) . The murine 2-5A ~~ RNase cDNA is 32P-labeled by random priming and then hybridized to the f ilter [about 50% forr~mi~le, about 10% dextran sulphate, Denfiardt's solution about 1% SDS, 6X SSPE, Sambrook, J. et al., Cold SDrinq Harbor Laboratorv Press (1989), about 250 llg per ml salmon sperm DNA] at about 42 C .
The Human 2-5A A ".~1"1_.,1 RNase cDNA clone, HZBl, is isolated f rom an adult human kidney cDNA
library in ~gtlO with rA~;nlAh~ed (random primed) murine 2-5A~ r~-n~ t RNase cDNA (clone ZBl) as probe, S _ook, J. et al., Cold sDrinq E7:~lrbor T ~hnratory Press (1989~ . Clone HBZ22 is isolated using ra~l i nl ;~h--l e~ HZB1 DNA as probe . The genomic human 2-5A r~ RNase clone is isolated from a human p~ ACF~nt~ cosmid library- in vector pVE15 Woss/2224s 2183461 p (SL.c.t~ge,,~) with a r~A;n~Ah~ed ~L of HZB22 DNA as probe. The murine genomic 2-sA A ~ Ar . l RNa6e clone is isolated from a mouse 129SV genomic library in vector ~FIXII (Stratagene) with a r~;clAh~led LL_, of 2-5A-BP cDNA (clone ZBl) as probe . S~h~ n i n~ of DNA is in Bluescript vectors (SLLaL g_..~) .
~ rLar__~iption of rla~ q with phage RNA
poly ~3e6 is in the ~L~sence of mGppppG as described (Promega) except that reaction mixtures are s~rpl Led with 15~ dimethyl sulfoxide and incubations are at about 37C for about 90 minutes.
RNA is p--ri f ied through ~-rh~A~-Y G50 spun-columns and ethanol precipitated prior to translation. Protein syn~hesis was p~lL~ -', as described (Promega), at about 30C for about one hour in mi~;L.~uc~;al nucleas.e pLt ~L~=ated rabbit reticulocyte lysate or in an extract of wheat germ at about room t~ ~LUL~
for about one hour and then at about 40C for about 12 hours. Translation reactions contain about 50 ~M
zinc sulfate. F ~ uc 2-5A-d-~ o L RNase in the reticulocyte lysated is removed by adsorption to about 30 ~ of p2(A2'p)3A covalently attached to cellulose (2-5A-C~ 1 oqe), prepared as described in Wells, J.A. et al., J. Biol. Chem.. 259:1363-1370 (1984) and Silverman, R.H. and Krause, D., ~.~
Press. Oxford. Enaland. pp. 149-193 (1987), for about WO 9512224s 2 1 8 3 4 6 1 ~ ~
one hour on ice afi described. See Silverman, R.H., An~l. Biochem., 144:450-460 (1985). The 2 A~ RNase:2-5A-cellulose complex is removed by twice centrifuging at about 400 x g for about 5 minutes at about 2C. The ~iU~L~at~
completely lacking in measurable levels of 2-5A ~ RNase. See FIG. 5.
The set of nested 3 ' -deletions of the truncated murine 2-5A ~ lr~ L RNase cDNA, ZBl, is generated with exonuclease III/Sl nuclease digestion followed by filling-in with Klenow DNA Polymerase using the "Erase-A-Base" system (Promega).
The synthesis of the 2-5A probe, p(A2'p)2(br8A2'p)2A[32P]Cp, and its cro~link;nrJ to 2-5A-d-~ L RNase is performed exactly as described. See Nolan-Sorden, N.L. et al., Anal.
Biochem.. 184:298-304 (1990). Briefly, the 2-5A
probe, about 0.7 to 2.5 nM at 3,0009 Ci/mmole, is incubated for about one hour on ice with cell extract partd as described, Silverman, R.H. and Krause, D., I.R.L. Press Oxford. Enqland. pp. 149-193 (1987), in the absence or p~ .c~, of llnlAhr~led oligonucleotide competitors. Covalent cr~ l ;nk;n~
is done under a uv lamp (308 nm) for one hour on ice and the proteins are s~aLated on SDS/10%
polyacrylamide gels. Filter assays for 2-5A binding activity using the 2-5A probe for about one hour on w09s/2224~ 2 1 8346 1 ice, as described in Knight, M. et al-, ~a~, 288: 189-192 (1980) .
Protease ~ c are p~L L 1 on gel-purified proteins in a gel, as described by Cleveland, D.W. et al., J. Biol. Chem.. 252:1102-1106 (1977) .
The r~h^nl~rl~~Q~ assay with 2-5A-cellulose is pe:LL~ ', as described by Silverman, R.H., Anal.
h~ n., 144 450-460 (1985) . sriefly~ lysates are ,~Ac~ .1 to about 30 ~M of 2-sA-cellulose on ice for about two hours. The matrix is then washed three times by centrifuging and L~ in~ in buffer A.
See Silverman, R.H., An:~l. Biochem., 144:450-460 (1985). The matrix is then in~ubated with po~y(U)-~32P]Cp or poly(C)-[32P]Cp (both at about 16 ~IN in nucleotide equivalents) at about 30C and the levels of zlcid-precipitable radioactive RNA are det~ n~fl by ~iltration on glass-~iber filters.
The Sanger dideoxy SDTl~nri ng method is used to detersline the DNA se~lucS ~s (sDq~l~nA
United States ~i i- ~l ) .
The lysines in the truncated murine 2-5A-d~ L RNase, clone ZBl, at positions 240 and 274 are mutated, individually and together, to asparagine residues . Nutants ZBl (Lys274-)Asn) and the double mutant, ZBl(Lys240~274-)Asn), are obtained with mutant ~ q^r~ leotides after sl~h~loninq ZBl _ _, _ _ _ _ ., _, WO9~/222~ 21 ~3461 IJo~ o cDNA into pALTER-l as described (Promega~. Mutant ZBl ~Lys240-~Asn) is obtained after polymerase chain reaction ; f ~ tion of a segment of ZBl with an U~DLr.~u primer ~nn~;nin~ a unique Hinc~I site L~ d to the mutant fi~ re and a second primer .u of a unique 8glII site. The HincII- and BGlII-digested polymerase chain reaction product and similarly-digested clone ZBl are then ligated. The specific mutations are: for codon 240, AAA-~AAC and for codon 274, AAG-)AAC. Mutants are cnnf i ~ ' by DNA s~ l~ - . i n~.

F~Y~MPT.F~ II
Seeds of tobacco (Nicotiana tabacum cv.
Wi~nn~;n) and Ti based binary vectors pAM943 and pA~822 were obtained from Dr. Amit Mitra, Department ~f Plant Pathology, University of Nebraska, Lincoln, Nebraska. The Arqobacterium l f~-iens LBA4404 and the E. coli strains K802 and MM294 were purchased from Clonetech, Palo Alto, California and SLL~ e, LaJolla, California. The plant tissue culture medium Murashige and Skoog s ready mix (MS media ~ was purchased from Sigma `h~m;c~l company, St. Louis, Missouri. The human cDNAs for PRR, the lysine ~
arginine mutant PKR, and 2-5A synthetase were obtained from Dr. B.R.G. Williams, Department of Cancer Biology, The Cleveland Clinic Foundation.
See, for example, Meurs, E. et al .: ~1, 62: 379-390 .

w0 95n2245 2 1 ~ 3 4 6 1 ~ 7~8 (1990); Chong, K.L. et al. ~ MR- J., 11:1553-1562 (1992); Rysieki, G. et al.: J. Interferon Res 9:649-657 (1989); 8enech, P. et al.: ~;~MRt- J,, 4:2249-2256 (1985); and S~ , M.E. et al.: ~B0 J., 4:1761-1768 (1985). me human cDNA for 2-5A
d~ RNase, as shown in FIG. 3A, was cloned in Dr. R.H. Silverman's laboratory in the Department of Cancer Biology and i8 the ~ y of The Cleveland Clinic Foundation. See, Zhou, A. et al.: ~, 72:753-765 (1993).
The expression vector pAM943 is used to obtain ArgnhAr~riul 'iAted transfer of T DNA
containing the cDl~As and kanamycin resistance marker gene. The physical map of the plasmid vector pAM943 shows its elements. See FIG. 12. The plasmid pA~943 C~nnt~ i nc a dual promoter consisting of the adenyl methyl transferase (ANT) gene promoter of Chlorella virus and the wild type 35S promoter of Cauliflower mosaic virus. The vector also contains the gene for kanamycin resistance to select the transformed plants. Initially, the cDNAs are sl~hclnnF~l in pAN943 and ~ ~ i f iF~d in E. coli strains K802 or N~5294 using tetracycline resistance as the selectable marker.
The Arg~h~ct~rium cells are L~ .ar~ ' with the LL _ i nJ~nt pAM943 pl A~ c and selected by growth in medium containing about 5 llg/ml of tetracycline, W0 95122245 ~ ` 2 1 8 3 4 6 1 P~

about 10 71g/ml of kanamycin and about 25 llg/ml of 1~L~ ; r U in To subclone cDNAs for P~ (PR68), a lysine ~ arginine mutant PRR (muPk68: the mutant PRR protein binds to dsRNA but has no kinase activity and will thus function as a control~, and a low molecular weight form of 2-5A ~y..Ll,eLase (synthetase), the pl i~ C pRS (+ ) PRR, pRS (+) muPRR, and pRS (+) synthetase are digested first with XbaI and than with ClaI
restriction ~nAr~n~ AC~c, the cDNA rL are purified from low melting point agarose gels and subcloned in sense orientation at XbaI and ClaI sites of pAM943. See FIG. 13. The recombinant r~ c, e.g., ~ullaLLucL A, pAM943:PR68, CUlla~LUU~ B, pAMg43:muPR68, and c;ol.LLu~ ~ C, pAM943:synthetase, which cu- L._~ond to th~ cu..a~LuuLs depicted in FIG.
13A-C, respectively, are used to transf orm ~raobacterium tumefaciens LBA4404. The resultant bacteria, identified as AG68, AGmu68 and AGsyn, respectively, are used for tobacco leaf disc transformations. Pro~ ion of the 1-- in~nt c~ c, i e , o ~ a ~L u~ L A, pAM9 4 3: PR6 8, construct B, pAM943:muPR68, and ~ U~LLUCL C pAM943:~yl~ se~
is described in greater detail hereinafter.
To subclone cDNA for 2-5A-d~r~-n~ nt RNase, the plasmid pRS(+)2C5 DNA is digested with HindIII
enzyme and s~h~ n~d in the HindIII site of pAM943 in W095/22245 2 1 8346 1 P~

both orientations, see FIG. 13, and the L. ln~nt plA~AC, v...-L-~L D, pAM943:2-5A-dep. RNase sense and cu.-DLLu~;L D/a, pAM943:2-5A-dep. RNase antisense, both of which ~.uLL~ u~d to _u-.,LL..uLs D and D/a, respectively, in FIG. 13D and D/a, are used to LL.,I.DruL.u AL~ t A ~Pr ium to obtain the bacteria called AG2DR sense _nd AG2DR Ant~n~:e, respectively.
Production of the recombinant pl P~ c ~ i . e., u ul~DLL~ L D, pAM943:2-5A-dep. RNase sense, ~u~ ~LLucL
D/a, pAM943:2-5A-dep. RNase ~nti~once, and ~U~DLLU-:L
E, pAM822:2-5A dep. RNase antisense, is also described in greater detail hereinafter.
The competent Argobacterium cells are prepared and L.~-nDruL-u~tion follows the method of, for example, An, G. et al.: Plant Molecular Bioloav , AD:l-l9 tl988) The pIese~ e of 1~ ~ in~nt plasmids in the transformed Argobacterium cells is confirmed by preparing plasmid DNA and by performing PCR using specific complementary oligonucleotides and by observing restriction enzyme digests.
The physical map of plasmid pAM822, one of the vectors used to deliver the reverse orientation cDNA for 2-5A ~ C~ c,,L RNase into plant cells by elecL.u~u,-tion, is also shown. See FIGS. 13E and 14. To subclone cDNA for 2-5A A~ n~lc ~L RNase into pAM822 the entire coding region of 2-5A A~
RNase was PCR 1 i f i c-l using two ol i ~n~ leotide wo gst2~s ~` 2 1 8 3 4 6 1 primers cm~t~;n;n~ BamHI res~iction sites before ATG
(~tart codon) and after TGA (stop codon). The product was digested with BamHI 2nd subcloned at BglII site of pAM822 vector. The cDNA used for 2 5A-d~ L RNase is in plasmid pZC5 referenced in 2hou et al. Cell ~2, 753-765 (1994), the human form of the cDNA. The E~ ? is also disclosed herein.
The plasmid pAM822 contains a second selectable marker gene, the ~ LI ~:in resistance gene, permitting the ~_...,,LLuuLion of plants containing both 2-5A-synthetase and 2-5A-d~ L RNase cDNAs.
Insertion of pAM822:2-5Adep. RNase (Fig. 13E), containing 2-5A cl_~ RNase cl)NA, into kanamycin-resistant, LL..1.~ tobacco leaf discs rnn~:~inin~ 2-sA-synthetase cDNA is thus pe, ruL ~.
Tobacco plants are grown aseptically in Murashige and Skoog's medium, known as MS medium, containing about 3% sucrose (MSû medium) and about 0.8% agar in plastic boxes (l.~yLaLray) at about 28-C
under cycles consisting of about 16 hr of light and about 8 hr of dark in a growth chamber. Leaves bigger than about 2" long are cut into about 2 to 3 cm2 pieces under the MSû medium and 6-8 leaf pieces are placed in a 6 cm Petri dish containing about 2 ml of MSO medium and holes are made in the leaf pieces with a sterile pointed forcep. Overnight cultures of AG68, AGmu68, AGSyn, AG2DR sense and AG2DR :-lnt ;c: n~e WO 95n224s 21~ 3 4 61 ~ 5/;J;v ~d are grown in LB (L broth) onnt~;n;nq about 50 ~M of r yLingone and a~,v~Liate antibiotics at about 28-C in a waterbath. One hundred microliter of overnight culture is added to e~ch of the Petri dishes containing leaf pieces. Incubation is at about 28-C under diffuse light in the growth chamber for about 2 days. Lea~ pieces are washed extensively with MSO medium and LL~ ,relL.d to ~;olid agar for selection in shoot Ley- I.tLation medium [MSO; sbout 0.5 mg/l BAP (benzylF~m;nopl~rine); about 200 pg/ml kana_ycin; about 200 pg/ml r-Arh~n;~illin; and about 100 pg/ml of cefotaxine], under diffuse light at about 28 C in the growth chamber . Within about 3 weeks, ~e~e~ Le.tion of plantlets is observed. When the~ plantlets are about 2-3cm long they are transferred to root-ina-lrin~, t ~ free MSO solid agar medium rnnt~inin~ about 200 pg/ml kanamycin and about 200 pg/ml carb~tnirillin. The trAn~:g~nir- plants expressing 2-5A synthetase are substantially transformed to i..LLu.luce the cDNA for 2-5A-d~rc~n~nt RNase (with pA~q943:2-5Adep.RNase sense, construct D;
FIG. 13D). Alternatively, the vector pAM822 (FIG.
14 ) containing the 2-5A ~ L RNase cDNA in sense orientation and the lly~LI y~in resistance gene is used to tLallarUL-u 2-5A-synthetase cnnt~;ninrJ plants.
This allows selection in 1IYYL~ ~ in rnnt~ inin~ MSO
media. Tissue culture and l- ~el.~=L~tion of plants are W09S/22245 2 1 8 3 4 6 1 I~.~ VA?AC~

done as described above. T ~ i c plants are grown to produce flowers ~nd seeds to ' ~L..-e the transfer of the antiviral genes or nucleotide - to E~ ,y~ t generations. Although ~re~if;c plaDmid ~u~ LLu-;L:i are described herein, the present invention is intended to include any plant vector ; nrl~A i n~ those with ; n~h-r~ hle promoters .
Expression of P~, mutant PRR, 2-5A-syn~hDt~e, and 2-5~ d~ RNase in plants that are 4" to 5" tall are tested in protein extracts of leaves (,-u~t:LI.ata..L of 10, 000 x g centrifugation). Results of Northern and Southern blot assays and fllnrtinnAl binding assays for 2-5A-deL~ 3D~ RNase are L_r~UL ~ed in Tables I-V. See also FIG. 15 wherein expression of human 2-5A
synthetase cDNA in LLCI1~5Ye~liC tobacco plants as ~l~t~; nDd by measuring the mRNA levels in a Northern blot is shown. FIG. 16, on the other hand, shows expression of mutant and wild type forms of human P~R
cDNA in LL~ .;r tobacco plants as dDtD~;n~ by measuring mRNA levels in a 1I L1- L11 blot. FIG. 17 depicts yLc ~ ce of 2-5A-~ L RNase cDNA in LLA~ J_I~;r. tobacco plants as detDnm;nDd on a Southern blot .

wo gs/ ~s 2 1 8 3 4 6 1 ~ '07~8 TABIE I
TL ~, ; c Tobacco Plants E xpressing ~ild Type and Mutant Forms of Human PKR cDNA
(pl~smid pA~943:PR68) FIG. 13A
(plasmid pAM943:muPK68) FIG 13B
Tr~n^~n;c- Plant: Southern 810t: Nr- LlleL~I Blot:
(clone #) (~- s~l.ce of DNA) (expression of mRNA) Mutant PRR: 1 + N . T .
(plasmid 2 ++ +
pAM943:PK68) 4 N.T. N.T.
FIG. 13A 6 N . T . +
7 N.T. +
N.T. +
11 N.T. +
12 N.T. +
17 N.T. +
Wild Type 1 N . T . +
PKR: 2 N.T. N.T.
(plasmid 5 N . T . +
pAM943:muPK68) 6 N.T. N.T.
FIG. 13B 7 N.T. N.T.
-~ 8 N.T. +
N.T. +
N.T. N.T.
22 N.T. N.T.
N . T., Not Tested wo gs/zz24~ ~ 2 1 ~ 3 4 6 1 1 ~IIL ~
--6g--TABLE II
J :~; C Tobacco Plants ~xpressing Hum~n 2--5A--Sy-ntho~ ? cDNA
(Plasmid pAN943: ~yl~Lh~ L - FIG. 13C~
Plant: Southorn Blot: Northern Blot:
(clone#) t~. sel~ce of DNA) (expression of mRNA) ++ +
3 s N.T.
4 + ++
5 ~ N . T .

6 ~ N.T.
N . T .
8 +++ +
9 + N.T.
+
12 + N.T.
13 + N.T.
14 ++
+
16 +
17 N. T . ++
18 N.T. ++
a N.T. N.T.
b N.T. N.T.
c N.T. N.T.
d N.T. N.T.
N. T., Not Tested .
.

W0 95122245 ~-- 2 1 8 3 4 6 1 r ~

TABIE III
IL A ~ ~ ~- "; C Tobacco Plants Containin Sense or ~nti~an~e Orientntion ~uman 2-5A-D~ L RNase cDNA
(plasmid pAM943:2-SA-dep. RNase sense - FIG. 13D) tPlasmid pAM943:2-5A-dep. i~Nase :~lnti~Pn~e - FIG. 13D/a) TL A ~ - . . i r Southern Northern 2-5A-Binding Plant: (pl~3el~Ce (expression Assay: (pro-rclone #) of DNA) o~ ~NA) tP;n activitv ~nt icPne:e: 1 + N.T. N.T.
2 + N.T. N.T.
3 + N.T. N.T.
4 + N.T. N.T.
5 + N.T. N.T.
a N.T. N.T. N.T.
b N.T. N.T. N.T.
c N.T. N.T. N.T.
sense: Zl + - +
22 ++
Z3 ++ N.T. ++
Z4 + N.T. N.T.
z5 N.T. N.T. +++
Z6 N.T. N.T. ++
z7 N.T. N.T. +/--N . T ., Not Tested .

W095122245 ' 2 1 8 3 4 6 1 PCrlUS95~02058 TABIiE IV
TI ~ Tobacco Plants Containing Both Human 2-5A-Sy~ t, ~~ and Human 2-5A-D~L,~ t RNase cDNA
(plasmid pAM943:- yl"~ A~e - PIG. 13C) (plasmid pAM943:2 'A de~. RNase sense - FIG. 13D) Plant: Southern Blots: i Northern Blot:
(clone #) (2-5A-Syn (2-5A-Dep. (2-5A Syn. (2-5A-dep.
rlN~ ' DNA) InRNA) RNase mRNA
14tl N.T. - +
14/2 N.T. - +
14/3 N.T. N.T. N.T. N.T.
14/4 N.T. N.T. N.T. N.T.
14/5 N.T. N.T. N.T. N.T.
14/6 N.T. N.T. N.T. N.T.
15/1 N.T. - +
15/2 N . T . - +
15/3 N.T. - +
15/4 N.T. N.T. +
15/5 N.T. N.T. N.T. N.T.
15/6 N.T. -- +
15/7 N.T. - N.T. N.T.
N . T ., Not Te~ted .

WO9S122245 2 1 834 6 1 r~ ~r J~.v.Jo Assays of dsRN~ d~L ~ Vl h~7l 1~ Lyl-ation of P~, 2-5A syAthP~A~e ~ctivated with dsRNA, and 2-5A ~ l RNase by IJV-crnssl ;nkln~ to rA~ rtive 2-5A, ~ee Nolan-Sorden et al.: AnAlytica Bio~h~ ts. (184) :298-304 (l990), m~y be E~ ~L.
on the leaf extracts. The levels of the proteins may also be d~t.~rminr~rl by Western blot analysis using the Ant;ho~ against PRR, 2-5A-synthetase and 2-5A~ cr~n~nt RNase.
To ~' LL ,~e the expression of 2-5A-d~ L RNase in LLAn~ i c plants containing aL~ u~.L D, pAM943:2-5A-dep. RNase sense, as depicted in FIG. 13D, functional assays that measure binding of r~tl;r~lAho~ed 2-5A analog to 2-5A ~
RNase are perf ormed . See Tables III and V . Results show the ~lt sence of 2-5A-tl~r~nrl~nt rwaSe in tran6genic plants Zl, Z2, Z3, Z5 and Z6. It is believed that the highest levels of human, recombinant 2-5A ~r~n~l~nt RNase are in plant Z5.
S~e ~able V.

W0 95/2224s ~ 2 1 8 3 4 6 1 TABLE V
Functional Expression of 2-5A-D~1 ~ n~ RNase in T~ ~ ~c Tobacco Plants ad Detormin }~y a 2-5A Binding Assay (plasmid pAM943: 2-5A-dep. RN~se sense - FIG. 13D) , Plant; 2-5A Binding Activitya:

Z2 1, 618 Z3 1,545 Z5 2,575 Z6 1, 547 aTobacco plants contain U~I~LLU- ~ D, pAM943:2-5Adep. RNase (sense). 2-5A binding assays are performed by the filter binding method of Knight, M. et al. Nature (288) :189-192 (1~80) with '; fications. A 32P-labeled and b~omine substituted 2-5A analog, p(A2'p)2(br~A2'p)2A3'-3~p]Cp, about 15,000 counts per min per assay, at about 3,000 Ci per mmole , Nolan-Sorden , N . L., et al . Anal . Biochem . .
(184) :298-304 (1990), is incubated with plant extracts, c~ntAinin~ about 100 miuLu~L~ of protein per assay, on ice f or about 4 h . The reaction mixtures are then transferred to nitrocellulose filteres which are washed twice in distilled water and dried and the amount of 2-5A
probe bound to the 2-5A-~l l.o~.A~ RNase on the f ilters is measured by scintillation counting, Silverman, R.H. and Krause, D., In, Clemens, M.J., Morris, A.G., and Gearing.
A.J.}~., (eds. ), Lvm~hokines and Interferons - A Practical Al~roach. I.R.L. Press, ûxford, pp. 149-193 (1987). Data is presented as counts per min of labeled 2-5A bound to 2-5A ~-L~ ~A~nt RNase e~Le~ç-ed in the trslnc~onic plants.
Ba~kuLul~.d rA~is~-t-vity from extracts of control plants, 705 counts per min, consisting of n~ncpocif j ~ binding of 2-5A, is subtracted from these data.

WO9512224~ 2 1 8346 ~ P~

To further confirm that the trAn-~on; r.
plants cnntA;n;n~ 2-~A ~ L RNase cDNA express fllnr~ ns~l 2-Sa~ L RNase protein or an amino acid ~ c~ e, an affinity lAhol inj method i5 pcLL- ' (data not shown). In thi6 method, 2-5A-binding activity is ~Dto~; nPd on a Western blot with a bromin~ Lituted, 32P-labeled 2-5A analog (the "probe"), as described in Nolan-Sorden, N.L. et al.: ~nAl. Biochem.. 184:298-304 (19go). More particularly, leaves are col l ort~o~ f rom LL A ~ ~ - J~ ; r plants r~n~A;n;n~ 2-5~ A~ _" l~"L RNase cDNA and they are h- J ; "od in NP40 lysis buffer, see Silverman, R.H. and Rrause, D. (1987) In, Clemens, ~5.J., Morris, A.G., and Gearing, A.J.E~., (eds. ), Lv ~nkino~:
Tnterferons - A Practical AP~roach. I.R.I., Press, Oxford, pp. 14g-193, supplemented with about 5m~
ascorbic acid, about 1 mM cysteine, about 2 llg per ml leupeptin, about 100 11 per ml phenylmethyleulfonyl fluoride, and about 2 llg per ml pepstatin. Extracts are clarified by centrifugation at about 10,000 x g for about 10 min. Supernatants of the extracts, about 100 llg of protein per assay, are separated by SDS/10~6 polyacrylamide gel electrophoresis, followed by transfer of the proteins to Immobilon-P membrane filters (M;ll;r~re Corp., Bedford, MA). The filter is then incubated with about 4 X 105 c.p.m. per ml of 32P-labeled 2-SA probe for about 24 h at about 4-C, Wo 9512224~ 2 1 ~ 3 4 6 ~ nCA
- ~s -According to Zhou, A. et al .: Cell 73: 753-765 (1993). The autoradiogram8 of the washed ~nd dried filters show the ~tJL._S~ of run-t; nn~l human 2-5~ d~ RNase visible to ~bout 80 kDa bands, in plants Z3, Z5, and Z6 (data not shown).
Antiviral activity of the plants are det~rmin~-3 by rubbing celite powder coated with Tobacco mosaic virus (A'ACC) and Tobacco Etch virus tfrom Dr. Amit Mitra, Nebra8ka). The plants are monitored for sy ; ~ of viral infection on leaves from control and ~L~ J~ ;c plants and are ~c ted in photographs.
The plAF~;tlC described and the ~L~r~r~ ' Argobacterium strains can be used to transform any other plants into virus-resistant plants. r 1A~
of plants that may be tran~rur r~ in accordance with the present invention include vegetable plants like corn, potato, c~rrot, lettuce, cabbage, broccoli, cau1if1ower, bean, squash, pumpkin, pepper, onion, tomato, pea, beet, celery, c~ ` , turnip and radish plants, fruit plant6 like banana, apple, pear, plum, apricot, peach, nectarine, cherry, key lime, orange, lemon, lime, grapefruit, grape, bQrry-, and melon plants, grain plants like wheat, barley, rice, oat and rye plants, grass, flowers, trees, shrubs and weeds such as laboratory weeds like Arabidopsis. It should therefore be ~ L~-LDO~ that the present wos~2n4s 21 834 61 r~
invention i nrl ~AD~ any plant into which any nucleotide 5~ e -nro~ an amino acid having antiviral activity has been i~ u luCe~ to form LL''I'-'J .; r plants having immunity or resistance against viral infection.

C~ LL~ -ion of pAM943 rr. -- l u-;L A~ and r`~943 MI-PgR (~;ul~LLu~ L B~
The pl n~ c pgS(+)PRR and pgS(+)muPgR, enro~; n~ wild type PgR and a lysine to arginine at codon 296 mutant form of PgR, respectively, present in 3;. çoli cells (obtained from Dr. B.R.G. Williams, Cleveland Clinic, Cleveland, Ohio) are ~re~ared by standard methods. See, for example, gatze, N.G. et al.: Nol. Cell Biol., 11:5497-5505 (1991) for generation of muPgR, lysine - 296 ~ arginine mutant (K296R), by site sp~ri f;c mutagenesis as described.
The PKR nucleotide se~ e utilized to cu~ LU~;L
rli~ pgs(+)pgR and pKS(+)muPgR is depicted in FIG. 18 . To dQt~rmi ne the ability of a plant translation apparatus to synthesize P~R protein, capped PgR mRNA is pLu-luced from linearized pgS(+)PgR
by ;n vitro LLelns~;Liption. The RNA is then translated in wheat germ extract (obtained from Promega Corp., Madison, W. I . ) in the ~L. 3~1~Ce of 35S_methinnin~-. Synthesis of the 35s-labeled PKR is cle~ec ~ed in an autoradiogram of the dried, SDS/polyacrylamide gel.

The cDNAs -n~o~l i n~ PKR and muPKR are excised from plr-~;r4c pKS(+)PKR and pKS(+)muPKR by digesting with KpnI and XbaI. The resulting DNA
containing the entire coding se l ~nc~ for PKR and muP~ are purified from a low melting point ~garose gel . To ~- n~e.te cDNAs containing at the 5 ' end XbaI and at the 3 ' end ClaI sites, the PRR cDNA
and muPKR cDNA are then digested with ClaI and purif ied . The resulting digested PKR cDNA and muPKR
cDNA are then f orce cloned into XbaI and ClaI
digested pAN943 by DNA ligation. The resulting plAP~ C, FIG. 13, constructs A and B, are used to transform ~raobacterium tumefaciens strain LBA4404 (Clonetech, Plao Alto, CA). Recombinant plasmids are prepared from transformed Ar~obacterium tumefaciens bacteria by standard methods and the ~ S~I~Ct: of PRR
and muPRR cDNA is conf; ' by PCR analysis and restriction enzyme digests of the isolated p Cu.l,Llu~ion of DAM943:5vl-u~_ l~se ~CO~Dl- U1~ C1 The plasmid ptac-15 containing the human cDNA illustrated in FIG. 20 for a small form of 2-5A-synthetase (producing a 1.8 kb mRNA) (obtained from Dr. B.R.G. Williams, Cleveland Clinic, Cleveland, Ohio) is prepared by stand~rd methods and is digested with BamHI and EcoRI. The synthetase cDNA is purified from a low melting point agarose gel by standard methods and is then subcloned into wo95/2224s 21 8346 I r~

plasmid pRS (+) (Strategene, La Jolla, CA) in BamHI
and EcoRI sites. The resulting recombinant plasmid DNA (pRS (+) synth~Ace) is r~ Ct~tl with XbaI and ClaI
and the 2-5A synt~Pt~ce cDNA is purified from a low melting point agarose gel and is then subcloned into XbaI and ClaI digested pAM943 to produce construct C
(FIG. 13). r ;nAnt p~ c are prepared from ~Lal~ari ~ Ar~obacterillm tumefaciens bacteria by -L~ laLd methods and the ~L.~ e of 2-5A sy~LI~eL~se cDNA is conf irmed by PCR analysis and by restriction enzyme digests of the isolated r~ c.

CJ...,LLuL:~ion of pA~1943:2--5Adep.RNase sense (._.~..DLL..~L D) and pA~943:2-SAdep.RNase nn1-1 C~n~ r~,~",~LLu~.~ D/a~
The plasmid pXS(+)ZC5 ~nro~;nq a complete coding s~ for human 2-5A-~C~r~n~ nt RNase is digested with HindIII. The 2.5kbp cDNA for 2-5A-C1~ I L RNase is purif ied in a low melting point agarose gel and is then subcloned in HindIII
digested pAM943 in both sense (forward) and antisense (reverse) orientations to produce pAM943:2-5Adep.RNase sense (~ IDLLU~;L D) and pAM943:2-5Adep.RNase antisense (c~alaLL~ L D/a~, as ict~cl in FIG- 13D and D/a, respectively.
I-~.",ro -~ Ar~obacterium are ~t~rminc-d to contain the 2-5A-A~r~-n~ nt RNase cDNA by restriction enzyme digests and by PCR analysis.
t WO 95/2224~; 2 1 8 3 4 6 1 _7g_ r ~ Lion of pA11822:2-5Adep.RN~e J~T'~il:~n~:D r~'....,.~ ",_L ~) Polymerase chain rDArl inn~: (PCR) are pe rf~ ~` on pla~mid pRS(+)ZC5 enr~oAinq human 2-5A d~ D~ I RNaqe to generate HindIII and BamHI
~ites on the two ends Or the cDNA and to reduce 5 ' and 3 ~ untrAn~l A~ S~ "e ~`efj . The PCR primers used are:

ID SEQ NO: 7:

2DR-5 5 ' -TcATGcTrr~r~AAGcTTGGATccAccATGr~Ar~Gr7 3 '; and ID SEQ NO: 8:
H2DR-4 5 ' -GATACTCr-~r~Ar-C"rTGCATCCTCATr~r-r~rCCAr-GGCTGG
--3' .
The PCR product (about 2.25 kbp) is purified on a low melting point agarose gel and is then digested with HindIII and is then sl~h~lonDd into HindIII digested plasmid pKS(+). The resulting plasmid, pRS:pZC5 is digested with BamHI and the 2-5A A-l~e~ RNase cDNA
LL, is puri~ied and cloned into BglII digested pAM822. Recombinants isolated in the reverse (antisense) orientation give pAM822:2-5Adep.RNAse antisense (cor, ~lu~ E). See FIG. 13E.

wogs/2224s 21 8346 1 As to the nucleotide Lqerr~nr~C disclosed herein, A means adenine; C means cytosine; G means guanine; T means thymine; and lJ means uracil. With respect to the 1; crl o5c-~1 amino acid s~Tl~nr~, A
means ala or alanine; R means arg or arginine; N
means asn or asparagine; D means asp or aspartic acid; C means cys or cysteine; E means glu or glutamic acid; Q means gln or glutamine; G means gly or glycine; H means his or histidine; I means ile or isoleucine; L means leu or leucine; K means lys or Lysine; N means met or ~ n i n~; F means phe or phenylAlAn;nP; P means pro or proline; S means ser or serine; T means thr or threonine; W means trp or tryptophan; Y means tyr or tyrosine; and v means val OF valine.

The following listed materials are on deposit under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, USA, and have been assigned the following ArCc~.qsion Numbers.

Plasmid DNA ATCC No. Deposit Date Viabilitv Date pAM943:PK68 ~Plasmid pA~ 75996 21 Dec. 1994 13 Jan. 1995 pAM943:muPK68 (Plasmid pB) 75997 21 Dec 1994 13 Jan. 1995 ~.r'^1^~ , (Plasmid pCI 75998 21 D~c. 1994 13 Jan. 1995 pAM943:2-5Adep.RNase (Plasmid pDI 75999 21 Dec. 1994 13 Jan. 1995 Z9-, exptessing, human 97047 01 Feb. 1995 07 Feb. 1995 2 5A d."_.,J..,l RNase cDNA
l~i/2-- expressing human 97041 01 Feb. 1995 07 Feb. 1995 2 5A ~, ' cDNA
this seed contains construct D, shown in Fig. 13~ which is pAM943:2-5Adep.RNase-this seed contains construct C, shown in Fig. 13, which is pr.r~,s^~

wo gsn224s 2 1 ~ 3 4 6 l . ~ 5~

= :L
~a~n 2-5A-dtved_.lL RNase SEQ ID N0 : 1 ~, SEQ ID N0 : 2 :, SEQ ID N0 : 3 : and SEQ ID N0 : 4 :
-103 aatccc~l~cttA~actcAA~ ct t~LLLyatLdagtgct~ gelLaa~ y~ zca A~ yyL~9~ u~ accgtc Iet Glu Ser Arq Asp His Asn Asn Pro Gln 10 Glu Gly Pro Thr Ser Ser Ser Gly Arg Arg 20 Ala Ala Val Glu Asp Asn His Leu Leu Ile 30 Lys Ala Val Gln Asn Glu A&p Val Asp Leu 40 Val Gln Gln Leu Leu Glu Gly Gly Ala Asn 50 Val Asn Phe Gln Glu Glu Glu Gly Gly Trp 60 Thr Pro Leu His Asn Ala Val Gln Met Ser 70 Arg Glu Asp Ile Val Glu Leu Leu Leu Arg 80 ~is Gly Ala Asp Pro Val Leu Arg Lys Lys 90 (CCT) ~
AAT GGG GCC ACG CTT TTT ATC CTC GCA GCG 3 o 0 Afin Gly Ala Thr Leu Phe Ile Leu Ala Ala 100 (Pro) ~

Ile Ala Gly Ser Val Lys Leu Leu Lys Leu 110 TTC CTT TCT AaA GGA GCA GAT GTC AAT GAG 360 Phe Leu Ser Lys Gly Ala Asp Val Asn Glu 120 Cys Asp Phe Tyr Gly Phe Thr Ala Phe Met 130 Glu Ala Ala Val Tyr Gly Lys Val Lys Ala 140 Wo 95t2223s ' 2 1 8 3 4 6 1 p~"" ~ ~

I~u Lys Phe Leu Tyr Lys Arg Gly Ala Asn 150 Val Asn Leu Arg Arg Lys Thr Lys Glu Asp 160 Gln Glu Arg Leu Arg Lys Gly Gly Ala Thr 170 Ala Leu llet Asp Ala Ala Glu Lys Gly His 180 Val Glu Val Leu Lys Ile Leu Leu Asp Glu 190 Net Gly Ala Asp Val Asn Ala Cys Asp Asn 200 Net Gly Arg Asn Ala Leu Ile His Ala Leu 210 CTG AGC TCT G~C GAT AGT GAT GTG GAG GCT 660 Leu Ser Ser Asp Asp Ser Asp Val Glu Ala 220 Ile Thr His Leu Leu Leu Asp His Gly Ala 230 Asp ~Tal Asn Val Arg Gly Glu Arg Gly Lys 240 Thr Pro Leu Ile Leu Ala Val Glu Lys Lys 250 His Leu Gly Leu Val Gln Arg Leu Leu Glu 260 Gln Glu His Ile Glu Ile Asn Asp Thr Asp 270 Ser Asp Gly Lys Thr Ala Leu Leu Leu Ala 280 Val Glu Leu Lys Leu Lys Lys Ile Ala Glu 290 Leu Leu Cys Lys Arg Gly Ala Ser Thr Asp 300 Cys Gly Asp Leu Val ~Set Thr Ala Arg Arg 310 Asn Tyr Asp His Ser Leu Val Lys Val Leu 320 -~WO 95/22245 ~ 2 1 8 3 4 6 1 r~

C T CAT GGA GCC AaA GAA GAT TTT CAC 990 Leu Ser His Gly Ala Lys Glu Asp Phe His 330 Pro Pro Ala Glu Asp Trp Ly6 Pro Gln Ser 340 Ser His Trp Gly Ala Ala Leu Lys Asp Leu 350 His Arg Ile Tyr Arg Pro Net Ile Gly Lys 360 C AAG TTC m ATT GAT GAA AAA TAC AaA 1110 Leu Lys Phe Phe Ile Asp Glu Lys Tyr Lys 370 Ile Ala Asp Thr Ser Glu Gly Gly Ile Tyr 380 Leu Gly Phe Tyr Glu Lys Gln Glu Val Ala 390 Val Lys Thr Phe Cys Glu Gly Ser Pro Arg 400 Ala Gln Arg Glu Val ser Cy5 Leu Gln Ser 410 Ser--Arg Glu Asn Ser His Leu Val Thr Phe 420 Tyr Gly Ser Glu Ser His Arg Gly His Leu 430 Phe Val Cys Val Thr Leu Cys Glu Gln Thr 440 Leu Glu Ala Cys Leu Asp Val His Arg Gly 450 GAA GAT GTG GAA AAT GAG GAA GAT GAA m 1380 Glu Asp Val Glu Asn Glu GlU Asp Glu Phe 460 Ala Arg Asn Val Leu Ser Ser Ile Phe Lys 470 Ala Val Gln Glu Leu His Leu Ser Cys Gly 480 Tyr Thr His Gln Asp Leu Gln Pro Gln Asn 490 ATC TTA ATA GAT T AAG AaA GCT GCT CAC 1500 Ile Leu Ile Asp Ser Lys Lys Ala Ala His 500 Wo95/22245 . 2 1 8346 1 P~

Leu Ala Asp Phe Asp Lys Ser Ile Lys Trp 510 Ala Gly Asp Pro Gln Glu Val Lys Arg Asp 520 Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr 530 GTG GTA AAG AAG GGA AGC A~C TCA TTT GAG 1620 Val Val Lys Lys Gly Ser Ile Ser Phe Glu 540 Asp Leu Lys Ala Gln Ser Asn Glu Glu Val 550 Val Gln Leu Ser Pro Asp Glu Glu Thr Lys 560 Asp Leu Ile His Arg Leu Phe His Pro Gly 570 Glu His Val Arg Asp Cys Leu Ser Asp Leu 580 Leu Gly His Pro Phe Phe Trp Thr Trp Glu 590 Ser--Arg Tyr Arg Thr Leu Arg Asn Val Gly 600 Asn Glu Ser Asp Ile Lys Thr Arg Lys Ser 610 Glu Ser Glu Ile Leu Arg Leu Leu Gln Pro 620 Gly Pro Ser Glu His Ser Lys Ser Phe Asp 630 Lys Trp Thr Thr Lys Ile Asn Glu Cy5 Val 640 ATG A~A AAA ATG AAT AAG TTT TAT GAA AAA 1950 ~Set Lys Lys Iqet Asn Lys Phe Tyr Glu Lys 650 AGA GGC A~T TTC TAC CAG AAC ACT GTG GGT 1980 Arg Gly Asn Phe Tyr Gln Asn Thr Val Gly 660 Asp Leu Leu Lys Phe Ile Arg Asn Leu Gly 670 Glu His Ile Asp Glu Glu Lys His Lys Lys 680 .

WO 95/22245 Y~~ 'O.C~u lSet Lys Leu Lys Ile Gly Asp Pro Ser Leu 690 TAT m QG AAG ACA m CQ GAT CTG GTG 2100 Tyr Phe Gln LyEi mr Phe Pro Asp Leu Val 700 Ile Tyr Val Tyr Thr Ly6 Leu Gln A6n Thr 710 Glu Tyr Arg Lys His Phe Pro Gln mr His 720 Ser Pro Asn Lys Pro Gln Cys Asp Gly Ala 7 3 0 Gly Gly Ala Ser Gly Leu Ala Ser Pro Gly 740 TGC 2223 tgat~ L~ L~J-~J~c,r~J~Artact 2258 Cys 741 t d L L z ~ ~ Le~ ~ L I ~J J ~ A ll~t r A r A A r ~ t 2 2 9 2 t ~ ~ J~JJ ~ ~LLI AArtrA~I~.J~3LLc3~_LLyLga~3~3yaL 2330 ~3ayLLy~aL~3~.~yaLdLy~ 3L~ J~J~ t~3~g 2367 tatt~:- aLaLy Lc;~dt-Ar~ gcAAtatatacccag 2405 acL~ A~rA~tA~tCCat~CtttarrartAArt~g~a 2442 gga~aL~ J~ JaLh;~;LLLLyL~,aat~cA~I~cAA 2480 aagaatga-3 LL3.;. L ~ ~J-rccrt ~Aty~ L~3. atat~3 LL 2517 açaattct~ L~ _Attt~ rC~ tgatC~ttqrAAA 2555 a~l~3~3~3aLLat~aL~ aLLl ~A~AArt~ AACC 2592 t~ 3rtr~ J- -JL~3l ~J-.-J~ J~J~ ~7~ttat 2630 tcaatttatacctA~r~rttt~taaaLLLatgL-3~3~ 2667 LLaLL~3~3Ldcct~ aL~l~3~J~3~rctfAAaArttaac 2705 tatctt ,F~g~"t_tt , _L~3 ~JJ ~-~--AA~r~t 2~42 atataggggttrraq~At- Ll_aLk.aLL~atLcagta 2780 LLLaLL~3_~3catctagtataagtc~q~gcactq~atg 2817 catgaatt 2825 *It is believed that the original codon number 95, i . e . CTT F~nro~; n~ the amino acid number 95, i . e.
leucine, i5 cQrrect, however the alternative codon in parenthesis shown above codon number 95, i.e. CCT
o~ i n~ the alternative amino acid in parPnth~
shown below amino acid number 95, i.e. proline may also exist at this position (see page 81).
SEQ ID No:l: _~L___ Ls~ the DNA encoding sequence for the hun~n 2-'~ dor~n~1~nf RNase protein. SEQ ID No:2: L~ L~___ Ll, the anino ~cid 3equence encoded by the DNA Bequence ~ r~ d SEQ ID No:l:.
SEQ I3 NO:3: 1~ ' the DNA sequence, L~L~__..L.I by SEQ ID
No:l:, h~ving the Illt~rn~tive codon number 95, CCT. SEQ ID N0:4:
- L~ __ LB the aT~lino ~cid Bequence encoded by SEQ ID No:3:, h~ving the ~ltern~tive amino ~.cid nu ~ber 95, proline .
.
.. ..... . _ _ _ _ _ _ _ _ _ WO 95122245 2 1 8 3 4 6 1 ~", Ml-rin~ 2--5A .1~ RNage (~
SEQ ID N0: 5: and SEQ ID N0: 6:

n l ~ ~ J J~ ~ ,r~J~,-J J - A J~J Lyuue~ -J~ L L~L L La LL-,y Ly La uLyaty- ~aty Lr - J.~J ~ .Ji.A. ~~ ~lat~ ~.-J~ ~tc~c~c~ ~rtrr~
~J L~ L~ -LLyLyL~-Jl~ r- -r--r~JLyLy~ LLl~c~ _A7~AA-IA
yy~ L~J---J~---`c ATG GAG ACC CCG GAT TAT 18 15et Glu Thr Pro Asp Tyr 6 Asn Thr Pro Gln Gly Gly Thr Pro Ser Ala 16 Gly Ser Gln Arg Thr Val Val Glu Asp Asp 2 6 Ser Ser Leu Ile Lys Ala Val Gln Lys Gly 36 Asp Val Val Arg Val Gln Gln Leu Leu Gl,u 4 6 Lys Gly Ala Asp Ala Asn Ala Cys Glu Asp 56 Thr Trp Gly Trp Thr Pro Leu His Asn Ala 66 Val Gln Ala Gly Arg Val Asp Ile Val Asn 76 Leu Leu Leu Ser His Gly Ala Asp Pro His 86 Arg Arg Lys Lys Asn Gly Ala Thr Pro Phe 96 Ile Ile Ala Gly Ile Gln Gly Asp Val Lys 106 Leu Leu Glu Ile Leu Leu Ser Cys Gly Ala 116 Asp Val Asn Glu Cys Asp Glu Asn Gly Phe 126 WO 95n224s ~ 2 1 8 3 4 6 1 r l",~ IQ2~R

Thr Ala Phe Met Glu Ala Ala Glu Arg Gly 136 AAC GCT GAA GCC TTA AGA TTC CTT m GCT 438 Asn Ala Glu Ala Leu Arg Phe Leu Phe Ala 146 Lys Gly Ala Asn Val A~n Leu Arg Arg Gln 156 Thr Thr Lys Asp LYB Arg Arg Leu Lys Gln 166 Gly Gly Ala Thr Ala Leu Net Ser Ala Ala 176 Glu Lys Gly His Leu Glu Val Leu Arg Ile 186 Leu Leu Asn Asp Met Lys Ala Glu Val Asp 196 Ala Arg Asp Asn Met Gly Arg Asn Ala Leu 206 Ile Arg Thr Leu Leu Asn Trp Asp Cys Glu 216 Asn Val Glu Glu Ile Thr Ser Ile Leu Ile 226 Gln His Gly Ala Asp Val Asn Val Arg Gly 236 Glu Arg Gly Lys Thr Pro Leu Ile Ala Ala 246 Val Glu Arg Lys His Thr Gly Leu Val Gln 256 Met Leu Leu ser Arg Glu Gly Ile Asn Ile 266 Asp Ala Arg Asp Asn Glu Gly Lys Thr Ala 276 Leu Leu Ile Ala Val Asp Lys Gln Leu Lys 286 Glu Ile Val Gln Leu Leu Leu Glu Lys Gly 296 GCT GAT AAG TGT GAC GAT CTT GTT TGG Aq~A 918 Ala Asp Lys Cys Asp Asp Leu V~l Trp Ile 306 ~ = = =

Wo ss/2224~ 2 1 8 3 4 6 1 ~ o~x Ala Arg Arg Asn His Asp Tyr His Leu Val 316 Lys Leu Leu Leu Pro Tyr Yal Ala Asn Pro 326 Asp Thr Asp Pro Pro Ala Gly Asp Trp Ser 336 Pro His Ser Ser Arg Trp Gly Thr Ala Leu 346 Lys Ser Leu His Ser Net Thr Arg Pro Met 356 Ile Gly Lys Leu Lys Ile Phe Ile ~is Asp 366 Asp Tyr Lys Ile Ala Gly Thr Ser Glu Gly 376 Ala Val Tyr Leu Gly Ile Tyr Asp Asn Arg 386 Glu Val Ala Val Lys Val Phe Arg Glu Asn 396 Ser~ro Arg Gly Cys Lys Glu Val Ser Cys 406 Leu Arg Asp Cys Gly Asp His Ser Asn Leu 416 Val Ala Phe Tyr Gly Arg Glu Asp Asp Lys 426 Gly Cys Leu Tyr Val Cys Val Ser Leu Cys 436 GAG TGG ACA CTG GA~ GAG TTC CTG AGG TTG 1338 Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu 446 Pro Arg Glu Glu Pro Val Glu Asn Gly Glu 456 Asp Lys Phe Ala His Ser Ile Leu Leu Ser 466 Ile Phe Glu Gly Val Gln Lys Leu His Leu 476 Nis Gly Tyr Ser His Gln Asp Leu Gln Pro 486 W095/22245 2 1 8346 1 I~",~ c~

Gln Asn Ile Leu Ile Asp Ser Lys Lys Ala 496 GTC CGG G GCA GAT m GAT CAG AGC ATC 1518 Val Arg Leu Ala Asp Phe Asp Gln Ser Ile 506 Arg Trp Net Gly Glu Ser Gln llet Val Arg 516 Arg Asp Leu Glu Afip Leu Gly Arg Leu Val 5Z6 CTC TAC GTG GTA ATG AaA GGT GAG ATC CCC 1608 Leu Tyr Val Val Net Lys Gly Glu Ile Pro 536 m GAG ACA CTA AAG ACT CAG AAT GAT GAA 1638 Phe Glu Thr Leu Lys Thr Gln Asn Asp Glu 546 Val Leu Leu Thr Net Ser Pro Asp Glu Glu 556 Thr Lys Asp Leu Ile His Cyc Leu Phe ser 566 Pro Gly Glu Asn Val Lys Asn Cys Leu Val 576 Asp~Leu Leu Gly His Pro Phe Phe Trp Thr 586 Trp Glu Asn Arg Tyr Arg Thr Leu Arg Asn 596 Val Gly As~ Glu Ser Asp Ile Lys Val Arg 606 Lys Cys Lys Ser Asp Leu Leu Arg Leu Leu 616 Gln His Gln Thr Leu Glu Pro Pro Arg Ser 626 Phe Asp Gln Trp Thr Ser Lys Ile Asp Lys 636 Asn Val Met Asp Glu Net Asn His Phe Tyr 64 6 Glu Lys Arg Lys Lys Asn Pro Tyr Gln Asp 656 ACT GTA GGT GAT CTG CTG AAG m ATT CGG 1998 Thr Val Gly Asp Leu Leu Lys Phe Ile Arg 666 Wog5/~2245 21 83461 r~ O
--9 o--Asn Ile Gly Glu His Ile Asn Glu Glu Ly~ 676 Ly~ Arg Gly 679 SEQ ID NQ : 5 : represents the DNA sequence encodin Murine 2-5A-dependent RNase (partial) . SEQ ID N0: 6:
represents the amino acid sequence encoded by SEQ ID N0:5:, WO 95/22245 _ 2 1 8 3 4 6 1 PCTI~IS95102058 SEQI~ENOE LISTING
( 1 ) GENE11AL _ --Ttll~
~i) APPLIC~'NT: 8ilverman, Ro~ert ~.
S~nGupt~, Di~yer,du N.
Iii) TITLE OF INYEI~TION: Antivir~l' r Plu~t6, Vector~
Cells and Metho~
( iii ) NUMBER OF SEQIIENOES: ll tiv) Cur~;~l ADDRESS:
~A~ ADD~ESSEE: Ruden, Barnett, McCloL~ky, Smith, Schuster &
~u~
,~' S-R-ET: 200 E. Bro~Tard Boulevard C C'~: Fort Lauderd~le E: Florid~
COL~TRY: USA
~_~: 33301 (v~ COM~HT~ REA IAB E FORM:
A M~ ~IrJM `YP: Floppy di~k B l ~ O~PDl~ ~: BM PC - ~ hl ~
C OP~ATING ' YSTEM: PC NS/I~S-NS
D So TWA~: ~ate3tIn ReleA~e #1.0, VerL~ion #1.25 Ivi) C~RRENT APPLICaTION DATA:
(A) APPLICATION NllMBER: US 08~198, 973 (B) FIL~NG DATE: 18-FEB-1994 (C) CL~:.~r~ un: 1808 (viii) ATTORNEY/AGE'NT lNrl ~lun:
(A) NAME: M~n~, Pet6r J.
( B ) ~ NOMBER: 3 2 2 6 4 (C) REFERENOE~DOCRET NHM3ER; CL11363-16 (lx) l~T. _.JNlCATION lL.. _ lUN:
(A) ~ELEPHONE: 30s/527J2qg8 (B) TELEFAX: 305/764/4996 (2) lNr~ UN FOR SEQ ID NO:1:
(i) SEQ~ OE t~ rFRTq~I~C
A ~ GTH: 292 R h~L8e pairs B' ---'E: nucle: c ~cid C ~ )T~ ingl~
I D' --OPOLOGY: l near (ii) MOLEC~LE TYPE: DN-A (genomic) (ix) FEAT~RE:
(A~ NAME/I~EY: CDS
(B) LOCATION: 104..2326 (xi) SEQI~ENOE irr,UK~e~lUn: SEQ ID NO:1:

W0 95/22245 ~ 2 1 8 3 4 6 1 P~ n~sg AATCCChaCT T~r~rTr~'a~ A TTAAGTGCTA r~ T~ .T ~ 60 SCAAGGAAAA rrr T~ -T r~ ~nr~rrT ~ GTC ~G GAG AGC AGG
Met Glu Ser Arg 115 GAT CAT AAC AAC CCC CAG GAG GGA CCC ACG TCC TCC AGC GGT AGA AGG
A p Eis A~m Asn Pro Gln Glu Gly Pro Thr S~r Ser Ser Gly Arg Arg 163 GCT GCa GTG GAA GaC AAT CAC TTG CTG ATT AaA GCT GTT CAA AAC GAA 211 Ala Ala Val Glu A p Asn Hi~l Leu Leu Ile Lys Ala V~l Gln Asn Glu GAT GTT GAC CTG GTC CAG CAA TTG CTG GAA GGT
Asp Val Asp Leu V~l G13 Gln Leu L~u Glu Gly Gly Ala Asn Val Asn 259 TTC CAG GAA GAG G~A GrG GGC TGG ACA CCT CTG CAT AAC GCA GTA CAA
Phe Gln Glu GlU Glu Gly Gly Trp Tkr Pro Leu Eis A~n Ala Val Gln ATG AGC AGG GAG GAC ATT GTG GAA CTT CTG CTT CGT CAT GGT GC~ GAC 355 Met Ser Arg Glu Asp Ile Val Glu Leu Leu L u Arg Eis Gly Ala Asp CCT GTT CTG Ak G AAG AAG AAT GGG GCC aCG CTT m ATC CTC GCA GCG 403 Pro Val Leu Arg Ly~ Lys Asn Gly Ala Thr Leu Phe Ile Leu Ala Al~
85 _ ~ 90 95 100 ATT GCG GGG AGC GTG AAG CTG CTG AAA CTT TTC CTT TCT AAA GGA GCA
Ile Ala Gly Ser Val Lys Leu Leu Lys Leu Phe Leu Ser Lys Gly Ala 451 GAT GTC AAT GAG TGT GAT TTT T~T GGC TTC ACA GCC TTC ATG GAA GCC
Asp V~l Asn Glu Cy- ~p Ph~ Tyr Gly Phe Thr Al~ Phe M~t Glu Ala G''T GTG TAT GGT AAG rTC AAA GCC CTA AAA TTC CTT TAT AAG AGA GGA 547 Ala Val Tyr Gly Lys Val Lys Al Leu Lys Ph~ Leu Tyr Lys Arg Gly GCA AAT GTG AAT l~G AGG CGA AAG ACA AAG GAG GAT CAA GAG CGG CTG 595 Ala Asn Val Asn Leu Arg Arr Lys Thr Lys Glu Asp Gln Glu Arg Leu AGG A~A GGA GGG GCC ACA GCT CTC ATG GAC GCT GCT GAA A~A
Arg Lys Gly Gly A1A Thr Ala Leu Met Asp Ala Ala Glu Lys Gly His 643 165 ~ 170 175 180 GTA GAG GTC TTG AAG ATT CTC CTT GAT GAG ATG GrG GCA GAT
Val Glu Val Leu Lys Ile Leu Leu Asp Glu Met Gly Ala Asp Val Asn 691 185 190 l9S
GCC TGT GAC AaT ATG GGC AGA AAT GCC TTG ATC CAT GCT CTC CTG AG
Ala Cys Asp Asn Met Gly Arg Asn Ala Leu Ile Eis Ala Leu Leu Ser .

TCT GAC GAT AGT GAT GTG GAG GCT ATT ACG CAT CrG CTG CTG GAC CAT 787 ~ W0 95/222~5 2 1 8 3 4 6 1 r~
-g3-Ser Asp A~p Ser Asp Val Glu Aln ~1~ Thr ~ Leu Leu Leu Asp }iis OEly Al~l Asp V~l Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu Ile Leu Ala Val Glu Lys Ly~ Pis Leu Gly Leu Yal G13 Arg Leu Leu Glu 245 . 250 ' 255 260 CAA GAG CAC ATA GAG ATT AAT GAC ACA GAC AGT GAT GGC A~A ACA GCA 931 Gla Glu !iis Ile Glu Ile A~3 Asp Thr A~p S~r Asp Gly Lys Thr Al~

G CTG CTT GCT GTT GAA CTC AaA CTG AaG AAA ATC GCC GAG TTG CTG 979 Leu Leu Leu Ala Val Glu Leu Lys Leu Lys Lys Ile Ala Glu Leu Leu GC AaA CGT GGA GCC AGT ACA GAT TGT GGG GAT CTT GTT ATG ACA GCG 1027 cy6 Lys Arg Gly Ala Ser Thr Asp Cy~ Gly Asp Leu Val Met Thr Al~

Arg Arg Asn Tyr Asp ~ Ser Leu Val Lys Yal Leu Leu S~r ilis Gly GCC AAA GAA GAT m CAC CCT CCT GCT GAA GAC TGG AAG CCT CAG AGC 1123 Ala Lys Glu Asp Phe }li8 Pro Pro Ala Glu ASp Trp Lys Pro Gl3 Ser 325 _ 330 335 340 TCA CAC TGG GGG GCA GCC CTG AaG GAT CTC CAC AGA ATA TAC CGC CCT 1171 ser ~3is Trp Gly Ala Ala L~u Lys A~p Leu Eli~ Arg Ile Tyr Arg Pro ATG ATT GGC A~A CTC AAG TTC m ATT GAT GAA A~A TAC A~A ATT GCT 1219 Met Ile Gly Lys Leu-Lys Phe Ph~ Ile A8p Glu Lys Tyr Lys Ile Ala Asp T}lr Ser Glu Gly Gly Ile Tyr Leu Gly Phe Tyr Glu Lys Gl3 Glu Val Ala Val Lys Thr Phe Cy8 Glu Gly Ser Pro Arg Ala Gl3 Arg Glu Val S~r Cys Leu G13 S~r Ser Arg Glu Asn Ser }lis L~u Val Thr Phe TAT GGG AGT GAG AGC CAC AGG GGC CAC TTG m GTG TGT GTC ACC CTC 1411 Tyr Gly Ser Glu S~r ~is Arg Gly E~ia Leu Phe Val Cys Val Thr L~u TGT GAG CAG ACT CTG GAA GCG TGT TTG GAT GTG CAC AGA GGG GAA GAT
Cys Glu G13 Thr Leu Glu Ala Cys Leu Asp Vlll llis Arg Gly Glu Asp 1459 GTG GAA 7~S GAG GAA GAT GAA TTT GCC CGA AAT ~C CTG TCA TCT ATA 1507 wo g5n22qs 2 1 ~ 3 4 6 1 . ~

V~l Glu A~3 Glu Glu A p Glu Phe Al~ Arg A~3 Val L~u Ser Ser Il~

m AAG GCT GTT CAA GAA CTA CAC TTG TCC TGT GGA TAC ACC CAC CaG 5 Phe Lys Ala Val Gln Glu Leu Eiis Leu Ser Cy~ Gly Tyr Thr ilis Gl3 l 55 GAT CTG ChA CCA CAA AAC ATC TTA ATA GAT TCT AaG AaA GCT GCT CAC 0 A~p Leu Gl3 Pro Gln Ae3 Ile Leu Ile Asp Ser Ly~ Ly~ Al~ Ala i~i~ 16 3 485 ~90 ~g5 500 CTG GCA GAT m GAT AAG AGC ATC AAG TGG GCT GGA GAT CCA CAG GAA 1651 Leu Al~ Asp Phe Asp Lys S~r }le Ly- Trp Al~ Gly Asp Pro Gl3 Glu Val Lys Arg Asp Leu Glu A p Leu Gly Arg Leu Val ~eu Tyr Val V~1 AAG AAG GGA AGC ATC TCA TTT GAG GAT CTG AaA GCT CAA AGT AAT
Lys Lys Gly S~r Il~ Ser Ph~ Glu A p Leu Ly~ Ala Gl3 Ser Asn Glu 1747 GAG GTG GTT CaA CTT TCT CCA GAT GAG GAA ACT AAG GAC CTC ATT CAT 7 Glu Val V~l Gl3 Leu Ser Pro Asp Glu Glu Thr Lys Asp Leu Ile ~li8 1 95 Arg Leu Phe ~li8 Pro Gly Glu ~i6 V~l Arg A557p5 Cys Leu 8~r AGP 5Le80u 585 Trp Tyr Arg u CGG AAT GTG GGA AAT GaA TCC GAC ATC AAA ACA CGA AaA TCT GAA
Arg Asn Val Gly bs3-Glu S~r Asp Ile Lys Thr Arg Ly6 Ser Glu Ser 1939 GAG ATC CTC AGA CTA CTG CaA CCT GGG CCT TCT GAA CAT TCC AAA AG
Glu Ile Leu Arg Leu Leu Gl3 Pro Gly Pro Ser Glu }~is Ser LYB Ser 1987 . TT GAC AAG TGG ACG ACT AAG ATT AAT GAA TGT GTT ATG AAA AaA
Phe Asp LYL Trp Thr Thr Lys Ile As3 Glu Cys Val Met Lys Lys Met 2035 AAT AAG TTT TAT GAA AAA AGA GGC AAT TTC TAC CAG AAC ACT
Asn Lys Phe Tyr Glu Lys Arg Gly A 3 Phe Tyr Gl3 As3 Thr Val Gly 2083 A6p Leu Leu Lys Phe Ile Arg AL3 Leu Gly Glu i~is Ile Asp Glu Glu 2131 AAG CAT AAA AAG ATG AaA TTA AAA ATT GGA
GAC CCT TCC CTG TAT m 2179 CAG Al~G ACA m CCA GAT CTG GTG ATC TAT GTC TAC ACA AaA CTA CAG 2227 W0 95/22245 - 2 1 8 3 4 6 1 ~ S

Gln Ly~ Thr Phe Pro Aep Leu V~l Ile Tyr Val Tyr Thr Ly8 Leu Gln AAC AC~ GAA TAT A6A AAG CAT TTC CCC QA ACC QC AGT CQ AAC AAA 2275 Asn Thr Glu Tyr Arg Lys Hi8 Phe Pro Gln T~r Hi~ Ser Pro Asn Ly~

Pro Gln Cy~ A~p Gly Al~ Gly Gly Ala Ser Gly Leu Al~ Ser Pro Gly TGC TGATr~CTG A~-U~lul~A ~ CTACTTATTA 6CTGTAGAGT 2376 Cys CCTTGGCA~LA TCAChACATT ~U~ L TAACTCACQ ~L~LLU~ 6AGGGATGAG 2436 TTGCATAGCT GATATGTQG ~ u~ AI ~_~ LUL~L~ A~ L~' ~L~7`7`rr`~L 2496 ~T~T~T~rrr ~- rT~rDrT AGTCQTAAG CTTTACCCAC T~ rTr-~r~ GAQTTCTGC 2556 TAAGATTCCT mGTCAATT rr~rr~ r ~Tr `QTrrr~ TTGACCCCTA i;lU~l` ~LA 2616 TGTTACAATT CTCTCACrTA ATTTTCCQA ~ CA ~ r~ `T LJ~L~l ~-~- 2676 ATTTA~GAAC TGAGGAACCT GAGACTCAGA GAGTGTGAGC T~rTr.r:rrr~ AGATTATTCA 2736 ATTTATAC AGCACmAT A~AmATGT ~L~L~ LLU GTACCTCTCA TTTGGGQCC 2796 TTAAAACTTA ACTATCTTCC AG6GCTCTTC CAGATGAGGC rr~ TD T3T~rrrr~TT 2B56 CCAGGAATCT CATTCATTCA TTCAGTATTT ATTGAGCATC T~r.TDT~ T rTrr~rDrTr. 2916 .
~2) INFORMATION FOR-SEQ ID NO:2:
(i) SEQUENCE r~ ~TcTIrc ~A) LENGTH: 7Ll ~mino ~cid~
(B) TYPE: a~ino acid ~D) TOPOLOGY: line~r ~ii) MOLECULE TYPE: protein (xi) SEOIIENCE D~ LlL~ SEQ ID NO:2:
et Glu Ser Arg Asp His Asn Asn Pro Gln Glu Gly Pro Thr Ser Ser er Gly Arg Arg Ala Al~ Val Glu Asp Asn His Leu Leu Ile Ly~ Al al Gln Asn Glu Asp Val Asp Leu Val Gln Gln ~eu Leu Glu G
35 40 45 ly G y Ala Asn Val Asn Ph~ Gln Glu Glu Glu Gly Gly Trp Thr Pro Leu Hi~

Asn Ala V~l Gln Met S-r Arg Glu A~p Il~ V~l Glu Leu L u Leu Arg _ _ . . . . . _ _ . ... ... . _ . _ _ _ _ _ W0 95122245 2 1 8 3 4 6 1 P~ l~u~

~!i- Gly Ala A~lp Pro Val Leu Ar5 Ly~ Ly A9n Gly Ala Thr Leu Phe 65 go gs le Leu ~la A1A Ile Ala Gly 8-r Val Lys Leu Leu Ly~ Leu Phe Leu loo 105 llo 8 Ly~ Gly Al~ A~p V~l Asn Glu CSn Asp Phe Tyr Gly Phe Thr Ala llS 120 125 Phe Met Glu Ala Ala Val Tyr Gly Lya Val Ly Ala Leu Lys Phe Leu Tyr Lys Ars qly Ala Asn Val Asn Leu Arg Arg Lys Thr Lys Glu Asp 145 lSo 155 160 Gln Glu Arg Leu Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala lu Lys Gly ~lis V~l Glu Val Leu Lyn Ile Leu Leu Asp Glu Met Gly 180 185 lgo Ala Asp Val Asn Ala Cys Asp Asn Met Gly Arg Asn Ala Leu Ile Elis 19S` 200 205 Ala Leu Leu Ser Ser Asp Asp Ser A~p Val Glu Ala Ile Thr ilis Leu Leu Leu Asp i~is Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu Ile Leu Ala Val Glu Lys Lys i~i~ Leu Gly Leu Val Gln 2~5 250 255 rg Leu Leu Glu Gln Glu }!is Ile Glu Ile Asn Asp Thr Asp Ser As Gly Ly~ Thr Ala Leu Lcu Leu Ala Val Glu LeU Lys Leu Lys Lys Il~
27s 280 z8s Ala Glu Leu-LeU Cys Lys Arg Gly Al~ Ser Thr Asp Cys Gly Asp Leu Val Met Thr Ala Arg Arg Asn Tyr Asp i~is Ser Leu Val Lys Val Leu Leu Ser }li5 Gly Ala Lys Glu Asp Phe ~i8 Pro Pro Ala Glu Asp Trp 32s 330 33s ys Pro Gln Ser Ser Pi6 Trp Gly Ala Ala Leu Lys Asp Leu i~is Ar 340 34s 350 Ile Tyr Arg Pro Met Ile Gly Lys Leu Lys Phe Phe Ile Asp Glu Lys 355 360 36s Tyr Lys ~le Ala Asp Thr Ser GlU Gly Gly Ile Tyr Leu Gly Phe Tyr 370 . 375 380 Glu Lys Gln GlU Val Ala Va1 Lys Thr Phe Cy~ GlU Gly Ser Pro Ars W095/22245 2 1 8 3 4 6 ~ r~

385 - 390 3gS 400 la Gln Arg Glu Val Ser Cy8 Leu Gln Ser Ser Arg Glu Asn Ser His Leu Val Thr Phe Tyr Gly Ser G1u Ser His Arg Gly Hi6 Leu Phe Val Cys Val Thr Leu Cy3 Glu Gl:l T~r Leu Glu Ala Cys Leu Asp Val His Arg aly Glu Asp Val Glu Asn Glu Glu A p Glu Phe Ala Arg Asn Val Leu Scr Ser Ile Ph~ Lys Ala Val Gln Glu Leu His Leu Ser Cys Gly yr Thr His Gln Asp Leu Gln Pro Gln Asn Ile Leu }le Asp Ser Lys Lys Ala Ala His Leu Ala Asp Phe Asp Lys Ser Ile T Ala Gl 500 505 Ly8 SrlPO Y
Asp Pro Gln Glu Val Lys Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr Val Val Lys Lys Gly Ser Ile Ser Phe Glu Asp Leu Lys Ala Gln Ser Asn Glu Glu Val Val Gln Leu Ser Pro Asp Glu Glu Thr Lys Asp Leu Ile His Arg Leu Phe }~is Pro Gly Glu His Val Arg Asp Cys Leu Ser Asp Leu Leu Gly His Pro Phe Phe Trp Thr Trp Glu Ser Arg ~yr Arg Thr Leu Arg Asn Val Gly Asn Glu Ser Asp Ile Lys Thr Arg Lys Ser Glu Scr Glu Ile Leu Arg Leu Leu Gln Pro Gly Pro Ser Glu His Ser Lys Ser Phe Asp Lys Trp Thr Thr Lys Ile Asn Glu Cys Val Met Lys Lys Met Asn Lys Phe Tyr Glu Lys Arg Gly Asn Phe Tyr Gln Asn Thr Val Gly Asp Leu Leu Lys Phe Ile Arg Asn Leu Gly Glu His Ile Asp Glu Glu Lys His Lys Lys Met Lys Leu Lys le Gly Asp Pro Ser Leu Tyr Phe Gln Ly~ Thr Phe Pro Asp Leu Val Il~ Tyr Val Tyr Thr Lys Leu Gln Asn Tbr Glu Tyr Arg Lys l~i~ Phe Pro Gln Thr His W0 95l222~5 2 1 ~ 3 4 6 1 r~

~05 710 715 720 8er Pro Asn Ly~ Prc~ Gln Cys Asp Gly Al~ Gly Gly Al~ Ser Gly Leu la Ser Pro Gly Cys 7i0 ~2) lC.r~ FOR SEO ID NO:3:
(i) SE ~ ~a~-~GIX: 292 b~ne p~ir~
. l ~E: nucle .~ ~cid C : ~ingle r) rl ~OLOGY: 1 ~ear ~ii) MOLEC;lLE TYPE: DNA (ger:o~ic) (ix) FEATUP~E:
(A) l~aME/l~EY: CDS
(B) LOCATION: 104..2326 ~xi) SEQ~NOE Dr.~ ON: 8EQ ID NO:3:
AATCCCAACT TACACTCAaA ~ .~ rrDaC'TGrTA r''`"~T7~ T L~ 60 TCAAGGAAAA GnrTPa~'`''T r,rTan~arr~T GGCATTTACC GTC ATG GAG AGC AGG 115 ~5et Glu Ser Arg GAT CAT AAC AP,C CCC CAG GAG GGA CCC AW TCC TCC AGC GGT AGA AGG 163 Asp ~is Asn Asn Pro Gln Glu Gly Pro Tbr Ser Ser Ser Gly Arg Arg s 10 15 20 GCT GCA GTG GAA GAC-AAT CAC TTG CTG ATT AaA GCT GTT CAA AAC GAA 211 Ala Ala Val Glu Asp Asn ~is Leu Leu Ile Lys Ala Val Gln Asn Glu Asp Val Asp Leu Val Gln Gln Leu Leu Glu Gly Gly Ala Asn Val Asn TTC CAG GAA GAG GAA GGG GGC TGG ACA CCT CTG CAT AAC GCA GTA CAa. 307 Ph~ Gln Glu Glu Glu Gly Gly Trp Thr Pro Leu }iis Asn Ala Val Gln s5 60 65 Met Ser Arg Glu Asp Ile Val Glu Leu Leu Leu Arg }li~ Gly Al~ Asp CCT GTT CTG AGG AAG AAG AAT GGG GCC ACG CCT TTT ATC CTC GCA
Pro Val Leu Arg Lys Lys Asn Gly Al~ Tbr Pro Ph~ Ile Leu Ala Al~
85 90 g5 100 ~
ATT GCG GGG AGC GTG AaG CTG CTG AaA CTT TTC CTT TCT AaA GGA GCA 451 Ile Ala Gly S-r Val Lys Leu Leu Ly~ Leu Phe Leu Ser Lys Gly Ala . _ . _ _ . _ . _ _ . , . , _, _ .

WO95/22245 2 1 8 3 4 6 1 r~"l ~h _99 _ GAT GTC AAT GAG TGT GAT m TAT GGC TTC ACA GCC TTC ATG GAA GCC 499 Asp Val Asn Glu Cy~ Asp Phe Tyr Gly Phe Thr Ala Phe Met Glu Ala ~20 125 130 GCT GTG TAT GaT AaG GTC AaA GCC CTA Aaa TTC CTT TAT
Ala Val Tyr Gly Lys Val Lys Al~ Leu Ly8 Phe Leu Tyr LYL Arg Gly 135 140 ~ 145 -GCA AAT GTG AAT TTG AGG CGA AaG ACA AaG GaG GAT CAA GAG CGG CTG SgS
Ala Asn Val Asn Leu Arg Arg Lya Thr Lys Glu Asp Gln Glu Arg Leu AGG AAA GGA GGG GCC ACA GCT CTC ATG GAC GCT GCT GAA AaA GGA CAC 643 Arg Lys Gly Gly Ala Thr Al~ Leu Met Asp Ala Ala Glu Lys Gly ~lis GTA GAG GTC TTG AaG ATT CTC CTT GAT GAG ATG GGG GCA GAT GTA AAC 691 Val Glu Val Leu Lys Ile Leu Leu Asp Glu Met Gly Ala Asp Val A
185 ~90 l9S
GCC TGT GAC AaT ATG GGC AGA AAT GCC TTG ATC CAT GCT CTC CTG AGC 739 Ala Cys A~p Aan M~t Gly Arg A8D Ala Leu Ile E~is Ala Le L
200 20S u eu 9er ser Asp Asp Ser Asp Val Glu Ala Ile Thr Xia Leu Leu Leu As ~i 215_ 220 225 P ~8 GGG GCT GAT 5TC AAT GTG AGG GGA GAA AGA GGG A~G ACT CCC CTG ATC 835 Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu Il~

Cl'G GCA GTG GAG AaG A~G CAC TTG GGT TTG

245 ~ 250 255 260 CAA GAG CAC ATA GAG-ATT AAT GAC ACA GAC AGT GAT GGC AAA ACA
Gln Glu ~lis Ile Glu Ile A8D Aap Thr Asp Ser Asp Gly Lys Thr Ala 931 -TG CTG CTT GCT GTT GAA CTC Aaa CTG AaG AAT~
L~u Leu L~u Ala V~-l Glu Leu Lys Leu Lys Lys ~le Ala Glu Leu Leu --lG~ AAA CGT GGA GCC AGT ACA GAT TGT GGG GAT CTT GTT ATG ACA
Cys Lys Arg Gly Ala Ser Thr Asp Cys Gly Asp Leu Val Met Thr Ala lOZ7 Prg Arg A8D Tyr Asp ~is Ser Leu Val Lys Val Leu Leu Ser ~is Gly GCC AAA GAA GAT m CAC CCT CCT GCT GAA GAC TGG Al~G CCT CAG AGC 1 3 Ala Lys Glu Asp Phe ~is Pro Pro Ala Glu Asp Trp Lys Pro Gln Ser 12 TCA CAC TGG aGG GCA GCC CTG AaG GAT CTC CAC AGA ATA TAC CGC CCT 111 ser ~lis Trp Gly Al Ala Leu Lys Aap Leu ~is Arg Ile Tyr Arg Pro , . , ., ,, , . ~

W095l22245 ' ' 2 1 8 34 6 1 ATG ATT GGC AAA CTC AaG TTC TTT PTT GAT GAA AAA TAC AAA ATT GCT ~1 M~t Ile Gly Ly- Lcu Ly Phe Phe Ile A~p alu Ly~ Tyr Lyn Ile Al~

GAT ACT TCA GAA GGA GGC ATC TAC CTG GGG TTC ~T GAG AAG CAa GAA
Asp Thr Ser Glu Gly Gly Ile Tyr Leu Gly Pbe Tyr Glu Lys Gln Glu 267 375 3ao 385 GTA GCT GTG AAG ACG TTC TGT GAG GGC AGC CCA CGT GCA CAG CGG GAa Val Ala Val Ly-l Thr Phe Cys Glu Gly Ser Pro Arg Ala Gln Arg Glu 315 GTC TCT TGS CTG CAA AGC AGC CGA GAG AAC AGT CAC TTG GTG ACA TTC
Val Ser cy8 Leu Gln Ser Ser Arg Glu ADn Ser His Leu Val Thr Phe 1363 TAT GGG AGT GAG AGC CAC AGG GGC CAC TTG m GTG TGT GTC ACC CTC
Tyr Gly Scr Glu Ser His Arg Gly Hi~ Leu Ph- Val Cys Val Thr Leu 1411 TGT GAG CAG ACT CTG GAA GCG TGT TTG GAT GTG CAC AGA GGG GAA GAT
Cy8 Glu Gln Thr Leu Glu Ala Cys Leu ADP Val l~iD Arg Gly Glu Asp GTG GAA AaT GAG GAA GAT GAA TTT GCC CGA AAT GTC CTG TCA T ATA
Val Glu ADn Glu Glu Asp Glu Phe Ala Arg Asn V~l Leu Ser Ser Ile 455 460 ~,65 TTT AAG ~G~ GTT CAA GAA CTA CAC TTG TCC TGT GGA TAC ACC CAC CAG
Phe Lys Al~ Val Gln Glu Leu Hi~ Leu Ser Cy5 Gly Tyr Thr Hi3 Gln S55 GAT CTG CAA CCA CaA AAC ATC TTA ATA GAT TCT AAG A~A GCT GCT CAC
Asp Leu Gln Pro Gln ADn Ile Leu Ile Asp Ser Lys LYB Ala Ala Hi~ 1603 CTG GCA GAT T~T GAT-AAG AGC ATC AAG TGG GCT GGA GAT CCA CAG GAA
Leu Ala ADp Phe Asp Lys Ser Ile Lys Trp Ala Gly ADP Pro Gln Glu 1651 505 S10 SlS
GTC AAG AGA GAT CTA GAG GAC CTT GGA CGG CTG GTC CTC TAT GTG GTA
Val Lys Arg Asp Leu Glu A8p Leu Gly Arg Leu Val Leu Tyr Val Val s20 525 530 AaG AAG GGA AGC ATC TCA TTT GAG GAT CTG AAA GCT CAA AGT AAT GAA 17 Lys Lys Gly S Ile Ser Phe Glu Asp Leu Lys Ala Gln Ser ADn Glu GAG GTG GTT CAA CTT TCT CCA GAT GAG GAA ACT AAG GAC CTC ATT CaT 17 Glu Val Val Gln Leu Ser Pro Asp Glu Glu Thr Lys ADp Leu Ile Xis Arg Leu Phe Hi8 Pro Gly Glu Hi8T vTalG AarGG AAC TGT CTG AGT GAC CTG 1~43 s65 570 575 580 Leu Gly HiD Pro Phe Phe Trp Thr ~rp Glu Ser Arg Tyr Arg Thr Leu 1891 W0 95l2224~ 2 l -a~3 4 6 1 r ~

CGG AAT GTG GQA AAT GAA TCC GAC ATC MA ACA CGA AaA TCT GAA AGT 1939 Arg Asn V~l Gly Asn Glu Ser A p Ile Ly~ Thr Arg Ly~ Ser Glu Ser Glu Ile Leu Asg Leu Leu Gln Pro Gly Pro Ser Glu Ni6 Ser Lys ser 615 620 , 625 m GAC AAG TGG ACG ACT AaG ATT AAT GAA TGT GTT ATG AaA AaA ATG 2035 Phe Asp Lys Trp Thr Thr Ly~ Ile Asn Glu Cy~ Val Met Lys Lys M~t 630 635 6~.0 A~T AAG m TAT GAA AaA AGA GGC AAT TTC TAC CAG AAC ACT GTG GGT 2083 Asn Lys Phe Tyr Glu Lys Arg Gly Asn Pke Tyr Gl~ Asn Thr Val Gly G~T CTG CTA A~G TTC ATC CGG AAT TTG GGA GAA CAC ATT GAT GAA GAA 2131 Asp Leu Leu Lys Phe Ile Arg Asr Leu Gly Glu Nis Ile Asp Glu Glu AAG CAT AAA AAG ATG AAA TTA MA ATT GGA GAC CCT SCC CTG TAT m 2179 Lys Nis Lys Ly6 Met Ly6 Leu Lys Ile Gly Asp Pro Ser Leu Tyr Phe ~AG AAG ACA m CCA GAT CTG GTG ATC TAT GTC TAC ACA A~A CTA CAG 2Z27 Gln Lys Thr Phe Pro Asp Leu Val Ile Tyr Val Tyr Thr Lys Leu Gln 695 ~00 705 AAC ACA GA~ TAT AGA AAG CAT TTC CCC CaA ACC CAC AGT CCA AAC AAA 2275 Asn Thr Glu Tyr Arg Lys Nis Phe Pro Gln Ths Nis Ser Pro Asn Ly6 CCT CAG TG~ GAT GGA GCT GGT GGG GCC AGT GGG TTG GCC AGC CCT GGG 2323 Pro Gln Cys Asp Gly Ala Gly Gly Ala Ser Gly Leu Ala Ser Pro Gly TGC TGA~GGACTG A~ ~A GTTCAGGGAA CTACTTATTA ÇrT~r.TA~ '~-T 2376Cys CC~TGGCAAA TCACAACATT L~ TAACTCACCA ~,~ ,, r.Arrr.DTr~-. 2436 TTGCATAGCT GATATGTCAG ~ AT ~ e~ AT.~ D'`"D7```'"'D 2496 AT~T~TArrr Ar~arArT AGTCCATAAG CTTTACCCAC TDl~rT--'`'`- GACATTCTGC 2556 TAAGATTCCT mGTCAATT çrDrr~ `Tr`-Tr-rC TTGACCCCTA ATGCTGCATA 2616 TGTTACAATT CTCTCACTTA AmmCCCAA ~w~T.~ ''7.rDr.Gt:AT TATCATCCCC 2676 AmATACCT AGCACT~TAT MAmATGT ~,~i,,~.,~i GTACCTCTCA TTTGGGCACC 2796 TTA~AACTTA ACTATCTTCC AGGGCTCTTC CAGATGAGGC rr~ "" DTA TATAGGGGTT 2856 CCAGGaAT ~ A TTCAGTAm ATTGAGCATC TArTATD~-T CTGGGCA~TG 2916 WO 95/2224~ ~, t ~ 2 t ~ 3 4 6 1 1~ , 'n7n~8 ~2) lrlr~ FOR SEQ ID NO~
(i) SEOtlENOE r"~
(A) LEN: 741 ~mino acid (~3) mE~ ino ~cid ~D) TOPOLOGY: linear (ii) MOLECIILE mE: protein (xi) SEOUENOE l~ lr~lUDI: SEQ ID NO:4:
et Glu Ser Arg A~lp His A6n Asn Pro Gln Glu Gly Pro Thr Ser Ser er Gly Arg Arg Al~ Ala Vzll Glu Al~p Asn Hie Leu Leu Ile Ly~ Ala al Gln Asn Glu Anp Val A~p Leu Val Gln Gln Leu Leu Glu Gly Gly Ala Asn Val Asn Phe Gln Glu Glu Glu Gly Gly Trp Thr Pro Leu His Asn Al~ Val Gln Met Ser Arg Glu Asp Ile Val Glu Leu Leu Leu Arg is Gly-Ala Asp Pro Val Leu Arg Lys Lys Asn Gly Ala Thr Pro Phe le Leu Ala Ala Ile Ala Gly Ser Val Lys Leu Leu Lys Leu Phe Leu Ser Lys Gly Ala Asp Val Asn Glu Cys Agp Phe Tyr Gly Phe Thr Ala Phe Me~ Glu Ala Ala-Val Tyr Gly Lys V~l Ly~ Ala Leu Lys Phe Leu Tyr Lys Arg Gly Ala Asn Val Asn Leu Arg Arg Lys Thr Lys Glu A~

ln Glu Arg Leu Arg Lys Gly Gly Ala Thr Ala Leu Met Asp Ala Ala lu Lys Gly ~lis Val Glu Val Leu Lys Ile Leu Leu As Glu Met Gl lao las P 190 Y
Ala Asp Val Asn Ala Cys Asp Asn Met Gly Arg Asn Ala Leu Ile His Ala Leu Leu Ser Ser Asp Asp Ser Asp Val Glu Ala Ile Thr His Leu Leu Leu Asp His Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys 225 . 230 235 240 Thr Pro Leu Ile Leu Ala Val Glu Ly~ Lys Hi- Leu Gly Leu Val Gln WO95/22245 21 8346 1 P ~

Ars Leu Leu Glu Gln Glu Hi~ Glu Ile A~r ~sp Thr Asp Ser ~sp Gly Ly~ Thr Ala LeY Leu Leu Ala Val Glu Leu LYR Leu Ly~ Ly6 ~le 275 2~0 285 Ala Glu Leu Ieu Cys Lys Arg Gly Ala Ser Thr Asp Cys Gly ASp Leu 290 295 ' 300 Val Met Thr Ala Arg Arg A~n Tyr Asp HiD Ser Leu V~l Lys Val Leu Leu Ser His Gly Al~ Lys Glu Asp Phe His Pro Pro Ala Glu ASp Trp Lys Pro GlD Ser Ser His Trp Gly Al~ A1A Leu Lys Asp Leu His Arg Ile Tyr Arg Pro Met Ile Gly Lys Leu Lys Phe Phe Ile ADP Glu Lys Tyr Lys Ile Ala Asp Thr Ser Glu Gly Gly Ile Tyr Leu Gly Phe Tyr 3~0 375 380 Glu Ly~ Gln Glu V~l Ala Val Lys Thr Phe Cy8 Glu Gly ser Pro Arg Ala Gln Arg Glu Val Ser CYD Leu Gln Ser Ser Arg Glu Asn Ser His Leu Val Thr Phe Tyr Gly Ser Glu S~r His Ar~ Gly His Leu Phe Val Cys Val Thr Leu Cy8 Glu Gln Thr Leu Glu Ala Cy~ Leu Asp Val Hi6 Ar~ Gly Glu Asp Val~Glu A~n Glu Glu ADp Glu Phe Ala Arg Asn Val Leu Ser Ser Ile Pbe Ly~ Ala Val Gln Glu Leu His Leu ser CyD Gl Tyr Thr His Gln Asp Leu 61n Pro Gln Asn Ile Leu Il~ Asp Ser Lys 4~5 490 495 Lys Ala Al~ His Leu Ala Asp Phe ADp Ly~ Ser Ile Lys Trp Al~ Gl Asp Pro 5615n Glu Val Lys Arçl A p Leu Glu Al~p Leu Gly Arg Leu Val Leu Tyr Val Val Lys Lys Gly Ser Ile Ser Phe Glu Asp Leu Lys Ala Gln Ser Asn Glu Glu Val Val Gln Leu Ser Pro Glu Glu Thr s 5 50 A8p 5Ly6 o Asp Leu Ile His Arg Leu Phe His Pro Gly Glu Hi~ Val Arg Asp Cys .

W0 9S/22245 `-- -Leu 8er A p Leu Leu Gly ~i~ Pro Phe Phe Thr Glu Ser S80 SBS Trp Trp Arg Tyr Arp, ~ Leu Arg Asn V~l Gly AJr, Glu Ser A~p Ile Lya Thr Arg Ly~ Ser Glu Ser Glu Ile Leu Arg Leu Leu Gln Pro Gly Pro Ser Glu 610 615 ~ 620 Xi8 Ser Lys Ser Phe Asp Lyll Trp Thr Thr Ly~ Ile A D. Glu CYD Val . 625 630 635 640 Met Lys Ly~ Met As3 Ly~ Phe Tyr Glu Ly~l Arg Gly Asn Phe Tyr Gln Asn Thr Val Gly Asp Leu Leu Ly~ Phe Ile Arg Asn Leu Gly Glu His Ile Asp Glu Glu Lys ~i8 Lyl~ Ly~ Met Lys Leu Lys Ile Gly Asp Pro ser Leu Tyr Phe Gl~ Ly8 Thr Phe Pro AYP Leu Val Ile Tyr Val Tyr Thr Lys Leu Gln A~n Thr Glu Tyr Ar~ Ly~ ~is Phe Pro Gl~ Thr lli~
70s 710 715 720 Ser Pro Asn Lya Pro Gln Cys ALP Gly Al~ Gly Gly Al~ Ser Gly Leu Al~ Ser Pro Gly Cy ~2) lNronl_.llUN FOR SEQ ID Nù:S:
( i ) SEnDEYC 3 ~
. . ~; ~GT~r 220n oa~e pairu ~ "E: nucle.c ~cid C s ~ : single D --O'OLOGY: l_ e~r (ii) MOLEC~ILE TYPE: DNA (genomic) ( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 164..2200 ~xi) SEQUENCE Ll~-~Ll~l~llIJDI: SEQ ID N~O:S:
ATTCGGCACG AGGA~GGTGC CAATTACTAG L~L~ V-A CTGATGAGAT 60 GTCAGAAGAC ~ DT~7 T CAGCCCAATC CCTACTCCAA GACTCTCATT GTGTCCCI~AA 120 r~rllro. ""~"",,,~ rr~ GGCATTGAGG ACC ATG GAG ACC CCG 175 Met Glu Thr Pro WO 95/2224~ 2 1 8 3 4 6 1 P ., L~

Asp Tyr Asn Thr Pro Gln Gly Gly Thr Pro Ser Al~ Gly Ser Gln Arg ACC GTT GTC GAA GAT GaT TCT TCG TTG ATC AAA GCT GTT CAG AAG GGA 2~1 Thr Yal Val G1U Agp A5p SCr Ser Leu Ile Ly8 Ala Val Gln Lys Gly 25 30 , 35 Asp Val Val Arg Val Gln Gln Leu Leu Glu Lys Gly Ala Asp Ala A~n Ala Cys Glu Asp Thr Trp Gly Trp Thr Pro Leu }~is A5n Ala Val Gln Ala Gly Arg Val A5p Ile Val Asn Leu Leu Leu Ser ~lis Gly Ala Asp 70 ~S eo CCT CAT CGG AGG AAG AaG AAT GGG GCC ACC CCC TTC ATC ATT GCT GGG 463 Pro ~is Arg Arg Lys Ly~ A5n Gly Ala Thr Pro Phe Ile Ile Ala Gly es so ss 100 ATC CAG GGA GAT GTG AaA CTG CTC GAG ATT CTC CTC TCT TGT GGT GCA Sll Ile Gln Gly Asp Val Lys Leu Leu Glu Ile Leu Leu Ser Cy5 Gly Ala lOS llO 115 Asp Val Asn Glu Cys Asp Glu A~n Gly Phe Thr Ala Phe Met Glu Ala G''T GAG CGT GGT AAC GCT G'A'A GCC TTA AGA TTC CTT TTT GCT AAG GGA 607 Ala Glu Arg Gly A5n Al~ Glu Ala Leu Arg Phe Leu Phe Ala Lys Gly GCC AAT GTG AAT TTG CGA CGA CAG ACA ACG AAG GAC A~AA AGG CGA TTG 655 Ala Asn Val Asn Leu-Arg Arg Gln Thr Thr Ly~ Asp Lys Arg Arg Leu Lys Gln Gly Gly Ala Thr Ala Leu Met Ser Ala Ala Glu Lys Gly ~i8 ~eu Glu Val Leu Arg Ile Leu Leu Asn A~p Met Lys Ala Glu Val Asp le5 190 195 Ala Arg Asp Asn Met Gly Arg Asn Ala Leu Ile Arg Thr Leu Leu Asn Trp Asp Cys Glu Asn Val Glu Glu Ile Thr Ser Ile Leu Ile Gln }lis GGG GCT GAT GTT AAC GTG AGA GGA GAA AGA GGG AAA ACA CCC CTC ATC
Gly Ala Asp Val Asn Val Arsl Gly Glu Arg Gly Lys Thr Pro Leu Ile egs wo gs/~s 6 1 P.

Al Al~ Val Glu Arg Ly Hi~ Thr Gly Leu Val Gln Met Leu leu ~3er CGG GAA GGC ATA AAC ATA GAT GCC AGG GAT AaC GAG GGC AAG ACA GCT
Arg Glu Gly Ile Asn Ile A8p Ala Arg Alrp Aun Glu Gly Lys Thr Ala CTG CTA ATT GCT GTT GAT AaA CAA CTG AAG GAA ATT GTC CAG TTG CTT
Leu Leu Ile Al~l V~l Asp Ly~ Gln L u Lys Glu Il- Val Gln Leu Leu CTT GAA aAG GGA GCT GAT AAG TGT GAC GAT CTT GTT TGG ATA GCC AGG
Leu Glu Lys Gly Al~ A~p Lys Cy~ Asp A p Leu V~l Trp Ile Al~ Ar~ 1087 AGG AaT CAT GAC TAT CAC CTT GTA AAG CTT CTC CTC CCT TAT GTA GCT
Arg Asn Hi~ Asp Tyr His L u V~l Lys Leu Leu Leu Pro Tyr Val Ala 135 AAT CCT GAC ACC GAC CCT CCT GCT GGA GAC TGG TCG
Asn Pro Asp Thr Asp Pro Pro Ala Gly Asp Trp 8er Pro Hls Ser Ser 1183 325 330 335 3',0 CGT TGG GGG ACA GCC TTG AaA AGC CTC caC AGT ATG ACT CGA CCC ATG
Arg Trp Gly Thr Ala Leu Ly~ Ser Leu His S~r Met Thr Arg Pro M~t lZ31 ATT GGC AaA CTC LAaG IAlTC pThe Ile Hi A5p A5p Tyr Lys Ile Ala Gly lZ79 ~CT TCC GaA GGG GCT GTC TAC CTA GGG ATC TAT GAC AAT CGA GAA GTG
Thr S~r Glu Gly Ala Val Tyr Leu Gly Ile Tyr Asp Asn Arg Glu Val 375 . 380 385 Ala al Ly5 Val Phé-Arg Glu A~Tn 5AGerC prA AGT GlA TGT AAG GAA GTC 1375 TCT TGT CTG CGG GAC TGC GGT GAC CAC AGT AAC TTA GTG GCT TTC TAT
~er Cys Leu Arg Asp Cys Gly Asp ~i~ Ser Asn Leu Val Ala Phe Tyr 1423 405 = 4l0 415 420 GGA AGA GAG GAC GAT AAG GGC TGT TTA TAT GTG
Gly Ars Glu Asp A~p Lys Gly Cys Leu Tyr Val Cys Val Ser Leu Cys 1471 GAG TGG ACA CTG GaA GAG TTC CTG AGG TTG CCC
Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu Pro Arg Glu Glu Pro Val 1519 GAG AAC GGG GaA GAT AAG m GCC CAC AGC ATC
Glu Asn Gly Glu Asp Lys Phe Ala His Ser Ile Leu Leu S~r Ile Phe 1567 4s5 460 465 GAG GGT GTT CAA AhA CTA CAC TTG CAT GGA TAT TCC CAT CAG GAC CTG 1615 CAA CCA CaA AAC ATC TTA ATA GAT TCC AaG A~A GCT GTC CGG CTG GCA 1663 wo ssm24s 2 18 3 4 6 1 r~-,u. ~ x Gln Pro Gln A6n Ile Leu Ile A~p 8er Ly~ Lys Ala V~l Arg Leu Ala 485 ~.90 4g5 500 GAT m GAT CAG AGC ATC CGA TGG ATG GGA GAG TCA CAG ATG GTC AGG 1~11 A~p Pb~ Al~p Gln Ser Ile Arg Trp Met Gly Glu Ser Gln Met Val Arg AGA GAC TTG GAG GAT CTT GGA CGG CTG GTT CTC TAC GTG GTA ATG A~A 1~59 Arg Aap Leu Glu Aap Leu Gly Arg Leu Val Leu Tyr Val Val Met Lys GGT GAG ATC CCC m GAG ACA CTA AAG ACT CAG AAT GAT GAA GTG CTG 180 Gly Glu Ile Pro Phe Glu Tbr Leu Lys Thr Gln Asn Asp Glu Val Leu CTT A~A ATG TCT CCA GAT GAG GAG ACT A~G GAC CTC ATT CAT TGC CTG 1855 Leu Thr Met Ser Pro A5p Glu Glu Tbr Lys Asp Leu Ile His Cya Leu s50 555 560 Plle Ser Pro Gly Glu A5n Val Lys A6n Cy~ Leu Val Asp Leu Leu Gly 565 5~0 5~5 580 ~is Pro Phe Phe Trp Tbr Trp Glu Asn Arg Tyr Arg Thr Leu Arg A~n GTG GGA AAT GAA TCT GAC ATC i~AA GTA CGG AAA TGT A~A AGT GAT CTT 1999 Val Gly A-;rr Glu Ser Asp Ile Lys Val Arg Ly6 Cy8 Lys Ser Asp Leu t-~C AGA CTA CTG CAG CAT CAG ACA CTT GAG CCT CCC AGA AGC TTT GAC 2047 Leu Arg Leu Leu Gln His Gln Tbr Leu Glu Pro Pro Arg Ser Phe Asp 615 620 62s GAG l'GG ACA TCT AAG ATC GAC AaA AAT GTT ATG GAT GAA AT
Gln Trp Thr S~r Lys-Ile Asp Lys Asn Val Met ABP Glu Met Asn Hls 2095 . C --,AC GAA AAG AGA AAA AAA AAC CCT TAT CAG GAT ACT GTA GGT GAT 43 Pne Tyr Glu Lys Arg Lys Lys Asn Pro Tyr Gln A~p Thr Val Gly Asp 21 CT.G CTG AAG TTT ATT CGG AAT ATA GGC GAA CAC ATC AAT GAG GAA A~A 2191 Leu Leu Lys Phe Ile Arg Asn Ile Gly Glu His Ile Asn Glu Glu LYB

AAG CGG GGG
Lys Arg Gly 2200 ~2) ~NFORMATION FOR SEQ ID NO:6:
(i) SEQUENOE ~ , ,u ~
~A) LENGTI~: 679 amino ~cids ~B) TYPE: amino acid ~D) TOPOLOGY: lin~ar (ii) MOLEC~LE TYPE: protein wo g ! . . 2 1 8 3 4 6 ~
s~2t24s r~~ 7n~Q

`
~xi) 8EOI~EIIOE vl~a~KLr~ SEQ Iv ~0:6 Met Glu Tbr Pro ABP Tyr Asn Thr Pro Gln &ly Gly Tkr Pro Ser ~
ly Ser Gln Ars Tbr Val V~l Glu Asp A p Ser Ser Leu Ile Ly~ Al-al Gln Ly- Gly Asp Yal Val Arg V~l Gln Gln Leu Leu Glu Lys Gly Al~ Asp Ala Asn Al~ Cy- Glu ABP Thr Trp Gly Trp Thr Pro Leu Hi~
sO SS 60 Asn Ala Val Gln Ala Gly Arg Val Asp Ile Val Asn Leu Leu Leu Ser 65 70 ~S 80 His Gly Al~ Asp Pro His Arg Arg Lys Lys Asn Gly Al a Thr Pro Phe le Ile Ala Gly Ile Gln Gly Asp Val ~ys Leu Leu Glu Ile Leu Leu Scr Cy~ Gly Ala A p V~l Asn Glu Cys Asp Glu Asn Gly Phe Thr Ala l1S 120 125 Phe Met Glu Ala Ala Glu Arg Gly Asn Ala Glu Ala Leu Arg Phe Leu Phe Ala Lys Gly Ala Asn Val Asn Leu Arg Arg Gln Thr Thr Lys As 14~ lS0 lSS 160 y~ Arg Arg Leu Lys Gln Gly Gly Ala Thr Ala Leu Met Ser Ala Ala lu Lys Gly His Leu-Glu Val Leu Arg Ile Leu Leu Asn Asp Met Lys 150 lBS 190 Ala Glu Val Asp Ala Arg Asp Asn Met Gly Arg Asn Ala Leu Ile Ar l9S 200 2~5 Thr Leu Leu Asn Trp Asp Cys Glu Asn Val Glu Glu Ile Thr Ser Ile Leu Ile Gln Nis Gly Ala Asp Val Asn Val Arg Gly Glu Arg Gly Lys Thr Pro Leu Ile Ala Ala Val Glu Arg Lys His Thr Gly Leu Val Gln Gly Lys Thr Ala Leu Leu Ile Ala V~l Asp Lys Gln Leu Lys Glu Ile Val 2GlOn Leu Leu Leu Glu 2Lgy5 Gly Al~ Asp Ly- Cys Asp Asp Leu V~l WO 9Sl22245 ; 2 1 8 3 4 6 ~
~ P~ u~.

. Trp Ile Al~ Arg Arg Asr, lii- A~p Tyr ~li8 Leu Val Ly Leu Leu Leu Pro Tyr Val Ala A8 Pro A8p Thr A8p Pro Pro Ala Gly Asp Trp Ser Pro ~is 8~r Ser Arç Trp Gly Thr Al- Leu Lys Ser Leu ~is Ser 340 345 350 Met Thr Ar~ Pro Met Il~ Gly Lys Leu Lys Ile Phe Il~ llis Asp Asp Tyr Lys Ile Ala Gly Thr Ser Glu Gly Ala Val Tyr Leu Gly Ile Tyr Asp 370 375 3~0 Asn Arg Glu Val Ala Val Lys Val Phe Arg Glu Asn Ser Pro Arg Gly Cys Lys Glu Val S~r Cy8 Leu Arg Asp Cys Gly Asp His Ser Asn Leu Val Ala Phe Tyr Gly Arg Glu Asp Asp Lys Gly Cys Leu Tyr Val Cy8 420 . 4Z5 430 Val Ser Leu Cys Glu Trp Thr Leu Glu Glu Phe Leu Arg Leu Pro Ar 435 4~0 445 Glu Glu ~ro Val Glu Asn Gly Glu Asp Lys Ph~ Ala His Ser Ile Leu Leu Ser Ile Phe Glu Gly Val Gl~ Lys Leu His Leu ilis Gly Tyr Scr 465 ~.70 475 4~0 His Gln Asp Leu Gln Pro Gl~ A~n Ile Leu Ile Asp Ser Lys Lys Ala Val Arg Leu Ala Asp~Ph~ A~p Gln Ser Ile Arg Trp Met Gly Glu Ser Gln Met Val Arg Arg Asp Leu Glu Asp Leu Gly Arg Leu Val Leu Tyr Val Val Met Ly6 Gly Glu Ile Pro Phe Glu Thr Leu Lys Thr Gln A3n Asp Glu Val Leu Leu Thr M~t Ser Pro Asp Glu Glu Thr Lys Asp Leu Ile H~s Cy8 Leu 5P6h5 Ser Pro Gly Glu Asn Val Lys Asn Cys Leu Val Asp Leu Leu Gly His Pro Phe Ph~ Trp Thr Trp Glu Asr. Arg Tyr Arg Thr Leu Arg Asn Val Gly Asn Glu S~r Asp Ile Lys Val Arg Lys Cys Lys Ser Asp LeU Leu Arg Leu Leu Gln His Gln Thr Leu Glu Pro Pro ~10 615 620 W095/22245 ~ 2 1 8346 1 I~

Arg 8er Ph~ A p Gln Trp Thr 8cr Ly8 ~le A~p l.ys AGn V~l Met AGP

Glu Met A~n ~ Phe Tyr Giu Ly~ Arg Ly3 Lyll Asn Pro Tyr Gln Asp 6~5 650 655 A n Glu Glu Lys Ly~ Arg Gly ( 2 ) _ FOR SEQ ID NO 7 ( i ) SE- ~ " r ~
~GT~ 190 amino ~cids ~E ~mino cid C single D ~O 'OLOGY linear (ii) MOLECULE TYPE protein (xi) SEOUENCE ~ LUI!I: SEQ ID NO ~
AGP Arq Arg Ly- Pro Arg Gln Asn Asn Arg Arg Asp Arg Asn Glu Arq Arg Asp Thr Arg Ser Glu Arg Thr Glu Gly Ser Asp Asn Arq Glu Glu Asn Arg Arg AGn Arg Arg Gln Ala Gln Gln Gln Thr Ala Glu Thr Ary Glu Ser Arg Gln Gln Ala Glu Val Thr Glu LYG Al~ Ars Thr Ala Asp Glu Gln Gln Al~ Pro Arg Arg Glu Arg Ser Arg Arg Arg AGn Asp Asp Ly~ Arg Gln Ala Gln Gln Glu Ala Lys Ala Leu Asn Val Glu Glu Gln Ser Vlll Gln Glu Thr Glu Gln Glu Glu Arg V~l Arg Pro Val Gln Pro Arg Arg LYG Gln Arg Gln Leu Asn Gln LYG Val Arg Tyr Glu Gln Ser V~l Ala Glu Glu Ala V l Val Ala Pro Val Val OElu Glu Thr V~l Ala Ala Glu Pro Ile Val Gln Glu Ala Pro Ala Pro Arg Tbr Lys Val Pro Leu Pro Val Val Ala Gln Tbr Ala Pro Glu Gln Gln Glu Glu Acn AGn Al~ AGP A n Arg A~p A n Gly Gly Met Pro Ser W09512Z245 21 83461 r~ 2~S8 ' 180 185 l90 2 ) . ~ FOR SEQ ID NO 8 ~i) SE~ OE rl~ p., ." ~
~Gl~: 256~ bal~e pairs ., l'E: nucle.c ~cid siQgl~
P "'O ?O~Ot~.': l_near (ii) MODECGLE TY~?E: CDNA
(xi) SEQWiNOE IJ~ 8 l~l~llUI~ SEQ ID NO 8 CAGTTTCTGG Pr~rr~TTrP ~ AAATTGTAAT GACCTCAaAA 60 CTTTAGCAGT L~ll-- ~L~l GACTCAGGTT l~ G--~ --. ~ GAATCAaCAT 120 CCPCACTTCC ~7~ Trrl~--P7~lrr TTCCAACCAG r~Tprrr~ 180 r~ Trr. ~,~., -~G~- -.--~I~ AGGAACTTAA TprpTprt~rT 240 r~r~ rrPr~r~ r~ r,Tpr,TprT TAaATATCAA r~rTGrrTp ATTCArGACC TCCACATGAT 300 AGGAGGTTTA CATTTCAAGT TPTr~Tpr~T r~r~r~T TTCCAGAAGG TGAPGGTAGA 360 TCAAAGAAGG ~r~ TGCCGCAGCC AaATTAGcTG TTGAGATACT Tp~Tp~rr~ 420 ~r~lrrr~G TTAGTccTTT PTTPTTr"rP ACAAC~r~aATT CTTCAGAAGG b----Ic~l~7 480 G,GAATTACA Tprrr,r~rT~T CAaTAGaATT Grrr~r~"r~ r-~rT~l~r TGTAaATTAT 540 GAACAGTGTG CATCGGGGGT ~ GA~GGAmc ATTATAaATG CAAAATGGGA 600 r~r~rAAT ATAGTATTGG TACAGGTTCT PrTP~rDrr A~-r~ rp A~ ~1 660 A~ACTTGCAT ATCTTCAGAT ATTATCAGAA GAAAccTcaG TGAAATCTGA CTACCTGTCC 720 l TTGCTACTAC GTGTruAGTCC r~ rrp ~rT cTrTAGTGAc CAGCACACTC 780 G TTCTGAAT ~'~ A ~ A AGGTGACTTC TCAGcaGATA CATCAGAGAT AAATTCTAAC 1340 AGTGACAG~T TAAACAGTTC ll-,7ll~ l ATGAATGGTC TCaGAaATAA TCaAa~GAAG 9oo ,rr~ r~ T CTTTGGCACC CPGATTTGAC CTTCGACA TGAaAGAaAC 7~ ~T~TPrT 960 GTGGAcaAGA ~l ~u AT GGATTTTAaA r~ Tp~~ T TAATTGGCTC AGGTGGATTr 1020 GGCCaAGTTT TCAaAGCAAA ACaCAGAATT r~Cr~'`'`'`~'` CTTACGTTAT TAaACGTGTT 1480 ~TATAAT~ prr~ GGAGCGTGAA rTP'`1`7'~~PT TGGCAAAA ~.sI~u--, 1140 AATATTGTTC PrTprpl~Tr~ u~1~T r7GATTTGATT A~ uA r~-rPrerr~T 1200 . PnPnrPGTr~ ~PrrP AaAaTAGTTC AaGGTCAaAG 1260 ACTAAGTGCC llL-~' A AATGGaATTC TrTr''T~l'~ GGACCTrGGA ACAATGGATT 1320 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .....

W095122145 ' 2 1 8 3 4 6 1 r~

.
.r~.~ r~ rr~ rTr-l~rr~ L~ TGGaACT_TT TG}iAr~ATA 1380 ~rr.~ r~~r ~ T Ar,ATTCAaaA AaATTAATTC ATAGA6ATCT T7~--rr.7~rT 1440 AATATATTCT T~rT~r~rT~r l~r7~ AAGATTGGA6 AClTrGGACT TrTr~Tr,T 1500 rTr~ T~r~ l,Trr.~ -rr~ rr.-.rr~-T AGGGGaACTT Tr~rr~T~rrT r~-rrr.-~ 1560 rAGAmCTT rrrr~ rTI~ Trr'`'`~ rTr-~-rTrT AC6CTTT6r6 6CrAATTC~T 1620 GcTGAacTTc ~L~;r~L-~L~ r~rTa~rT mrAaACAT C~AAGTTm rr~rr-~-rTa 1680 r-GG -aATGGr-A TCATCTCaGA ~r~L L ~W~T /~ r~ AaArTCTTCT ACAGaAATTA 1~4 0 r-Tr-Tr~AAGA ~ r rrTr~ TCGACCTAAC AcATcTGAaA Tr rTr ~ rr-~ r CTTGACT6TG 18 0 0 TGGAaGAaAA arrr~ Tr~ rr~r~ CACACATGTT AGA6CCrTTr TGAAaAAGTA 1860 1~L~ L nr-Tr-TGrr-r-T mCCTT~A TTATCTAAAA TCTGrTAGGG AATATChATA 1920 GATATTTACC ~-~LLLL~ J L /~ L-LL TAATmTTA rTAmTTAC TAATCTT5~CT 19~0 r~rr.~ rrr. AaAGGTTTTC L~LL~VC TTra~aACA TTCTTACATT TTACTTTTTC 2040 ~L~I~L~L~ l~LLLr,~LLL ~L~ ~L mTAAA6AC AGA6TCTCGC L~-L ~L~ 2100 AGGCTGGAGT nrr~Tr~-rr AGTCTT66r~T cArT6cAacT L~L~L~L~ 6r6TTCaA6T 2160 GATTrTCCTG~ ccrcAGccTc rTr~-Tr r~r G6ATTACAGG CATGT6CCAC rr~rrr~rT 2220 AATTTTTGTG TTTTTAATAA AGACA6G6TT TCACCATGTT 66CCAG6rTG GTrTC~AArT 2280 ccTGAccTr-A AGTAATCCAC L~ ~L CTCCCAaA6T ~ L--- r~LrGr.r~Tr~r 2340 rrrrrr,rr,rr CAGCCTCATC ~ L~L~L r~ r.rTrr~ rrrrr~rr CAAATTTTrT 2400 T~TTATACTA TTAATGAATC AATCaATTCA T~ LLL~ TTAaAmrT ACCGrTTTTA 2460 rr,rr~ ATGTAAGATC GLL~L~ L TCACATA6rT T/~rr~r~r~rrr~ CTGGAGAAAT 2520 ATGGTACTCA TTAaAAaAaA r ~ r TGATGTACAA CC 2562 2 ) INFORMATION FOR SEQ ID NO: 9:
(i) SEQ-~ 3 rT~ r.,, r~
A .E~GT~: 55~L ~Lmino a~ids 'E: ~mino acid c ~~ : fiingle D `O?OLOGY: lin~r (ii) MOLEC~E TYPE: protein (xil SEa~ENOE l~ -lL~h: SEQ ID NO:9:
Met Ala Gly Asp L~u Ser Al~ Gly Phe Phe M~t Glu Glu L~u Asn Thr Tyr Arg Gln Ly~l Gln Gly V~-l V~l Leu Ly~ Tyr Gln Glu L~u Pro Asn W09~122245 ~ u~,~r 8er Gly Pro Pro ~io Asp Arg Arg Phe Thr Phe Gln V~l Ile Ile Asp Gly Arg Glu Phe Pro Glu Gly Glu Gly Arg Scr Ly~ Ly- Glu Ala Lys As3 Ala Ala Ala Ly~ Leu 1~1~ Val Glu Ile Leu Asn Lys Glu Lys LYB

Ala Val Ser Pro Leu Leu Leu Thr Tkr Thr Asn Ser Ser Glu Gly Leu Ser M~t Gly A~n Tyr Ile Gly Leu Ile A~n Arg Ile Ala Gln Lys Lys Arg Leu Thr Val A~n Tyr Glu Gln Cys Ala Ser Gly Val ~lis Gly Pro Glu Gly Phe }ii8 Tyr Lys CYG Lys Met Gly Gln Lys Glu Tyr Ser Ile Gly Thr Gly Ser Thr Ly~ Gln Glu Al~ Lys Gln Leu Ala Ala Lys Leu Ala Tyr Leu Gln Ile Leu Ser Glu Glu Thr Ser Val Lya seF Asp Tyr Leu Ser Ser Gly 9er Phe Ala Thr Thr Cys Glu Ser Gln Ser Asn Ser Leu Val Thr Ser Thr Leu Ala Ser Glu Ser Ser Ser Glu Gly Asp Phe Ser Ala A~p Thr Ser Glu Ile Asn Ser Asn Ser Asp Ser Leu Asn Ser Ser Ser Leu Leu Met Asn Gly Leu Arg Asn Asn Gln Arg Ly~ Ala Lys Arg Ser Leu Ala Pro Arg Phe A~p Leu Pro Asp Met Lys Glu Thr Lys Tyr Thr Val Asp Lys Arg Phe Gly Met Asp Phe Lys Glu }le Glu Leu Ile Gly Ser Gly Gly Phe Gly Gln Val Phe Lys Ala Lys His Arg Ile A~p Gly Lys Thr Tyr Val Ile Lys Arg Val Lys Tyr Asn Asn Glu Lys Ala Glu Arg Glu Val Lys Al~l Leu Ala Lys Leu Asp }~is Val Asn Ile Val i~is Tyr Asn Gly Cys Trp Asp Gly Phe Asp Tyr Asp Pro Glu Thr Ser Asp Asp Ser Leu Glu Ser Ser A~p Tyr Asp Pro Glu Asn Ser Ly~

W0 95122245 I ~ n AJn Ser Scr Arg S~r Ly6 Thr Ly Cy~ Leu Phe Ile Gln Met Glu Phe Cy5 Asp Ly6 Gly Thr Leu Glu Gln Trp Ile Glu Lys Arg Arg Gly Glu Lys Leu Asp Lys V~l Leu Al~ Leu Glu Leu Phe Glu Gln Ile Thr Lys 3~5 390 395 400 Gly V~l Asp Tyr Ile Xi~ Ser Lys Ly6 Leu Ile Xis Arg Asp Leu Lys Pro Ser Asn Ile Ph~ Leu Val Asp Thr Lys Gln V~l Lys Ile Gly Asp Phe Gly Leu Val Thr Ser Leu Ly6 A6r, Asp Gly Lys Arg Thr Arg S~r Lys Gly Thr Leu Arg Tyr Met Ser Pro Glu Gln Ile Ser Ser Gln Asp Tyr Gly Lys Glu Val Asp Leu Tyr Ala Leu Gly Leu Ile Leu Ala Glu Leu Leu Xia Val Cys Asp Thr Ala Phe Glu Thr S~r Lys Phe Phe Thr q8s 490 495 Asp Leu Arg Asp Gly Il~ Ile Ser Asp Ile Phe Asp Lys Lys Glu Lys Thr Leu Leu Gln Lys Leu Leu Ser Lys Lys Pro Glu Asp Arg Pro Asn Thr Ser Glu Ile Leu Arg Thr Leu Thr Val Trp Lys Lys Ser Pro Glu Lys Asn Glu Arg Xis Thr Cys ~2~ INFORMATION FOR SEQ ID NO:10:
~i) SEr~ E ~-;r~lDD~ I ~1 I r''~
sNGT~l: 165S b~2~e pair~
, -- PE: nucle_c l~cid C ,~-D~ n~TrC~ gingle 3 ~ -oPOLOGY: l_ne~r (ii) MOLECtlLE TYPE: cDNA
(xi) SEWENCE l~ lUN: SE0 ID NO:10:
P7~rTr~rr AACAGCAGTC CAAGCTCAGT rDnrD~ r~TD'`~--D AACAGGTG 60 GGAGGCAGT~ CTGTTGCCAC I.-.-~ l GTCAATGATG C'`TrTrD-~'I DTDrrrr~nc 120 CAaATCTCTG GACAAGTTCA TTGA7~GACTA ~ ,~ GACACGTGTT ~ A 180 wo ss~s 2 1 8 3 4 6 1 ~ n?n~s AATCGACCAT GCCaTTGACP ,~ r;l~rrrTr~~ rr'`~-~GrT TCCGAGGTAG 240 CTCCTACCCT ~ Vl~ .VI crD --TrrT D ---Trnr TCCTCAGGCA ~~~~rDrrDr 300 CCTCaGaGGC CGATCTGACG rT-~-rTGr,T "",,,,~,, AGTCCTCTCA GCACTTTTCA 360 GGATCaGTTA AATCGCCGGG Gh5AGTTCPT rr~--`"'`TT P--~-'`-Prr TGGAAGCCTG 420 TCAAaGAGAG AGAGCACTTT CCGTGAAGTT, - ~ ,~ ~ -~ GCTCChCGCT rrrGrDr-r~r 480 AGCTTCGTAC ~ , rr~rrT~rr~r GAGGGGGTGG AGTTCGATGT 540 TGGGTCAGTT r'~-TrrrPnc TPTr~ -rTD DrrrrrP ~7,T 600 CTATGTCPAG CTCATCGAGG DnTrr1~rrrr CCTGCAGaAA r~ rnpr-T TCTCCACCTG 660 CTTCACAGAA rTPrD~ ArTTCCTGAA GrPnrnr~rrr ACCAAGCTCA AGAGCCTCP~T 720 CrGCCTAGTC ~r----PrTrrT ACCAAAATTG TP~`~rr~rr~ CTTGGGAAGC TGCCACCTCA 780 , GAGCTCCTGA CW1~ L TTGGGAGCGA GGGAGCaTGA AAACACAm 840 r~rDrDrCC CAU;G~mC GGACGGTCTT GGAATTAGTC DTr~ rTD~rr AGCAACTCTG 900 CPTCTACTGG DrD~arTDTT ATGACmAA AAACCCCATT ATTGAaAAGT prrTrr~ 960 GCAGCTCACG r~~,rr~r~~ ~ -W lC~l r,-~-rrrrr~lG r~-rrTDrD~ GAaACTTGGG 1020 TGGTGGAGAC CCAaaGGGTT r,~~--DnrT rrrPrD~-r- GCTGAGGCCT GGCTGAATTA 1080 CCCATGCTTT AaGAATTGGG AI~v..~c~ AGTGAGCTCC l~all.l~,~ TGGCTGAAAG 1140 rD~rDrTPrP rr~nDTrr~D CcGAcr~Tcc rDrr~ rTPT caGAAATATG GTTACATTGG 1200 AACACATGAG TACCCTCATT TCTCTCATAG DrCrDr-rDrG CTCCAGGCAG CATCCACCCC 1260 ArDrrrA"~r GAGGACTGGA rrTrrDrrDT CCTCTGAATG CCAGTGCATC TTGGGGGAAA 1320 vvGCTCCAGT i~ iVA CCAGTTCCTT CATTTTCAGG TGGGACTCTT GATCChGAGA 1380 AvACAaAGCT CCTCAGTGAG ~ lALI~ ATCCAAGACA r~rrrDrGT CTCCTGACTC 1440 1 A1~ 1A -- J, ~I,~ GATAACATTC TCCACAGCCT CACTTCATTC 1500 CACCTATTrT CTGAAAATAT TCCCTGAGAG Drr~rDnpnD GAmAGATA Dr~ Trr~ 1560 ATTCCAGCCT TGACTTTCTT CTGTGCAACCT nDTrr--r~-~r TAATGTCTAA T~ l~llAI A 1620 ATPDrD~TP~ l'r'7'TP~`~r--D P~ TDrrD~ 1650 ~2) INFORMAT}ON FOR SEQ ID NO ll ( i ) SliQl:~;r3 rPr--Dl , r:l~ I ~11~
A ~ ~GTH 400 amino ncids ' 'E amino cid si Iyle n ~OLOGY linenr (ii) MOLEC~E TYPE protein WO95l22245 _ r~.,. s (xi~ SEQUEr8:E Lc~lr~ SEQ ID liO:ll:
Met Met Asp Leu Arg Asn Thr Pro A12 Lys Ser Leu Asp Lys Phe Ile Glu Asp Tyr Leu Leu Pro Al~p T r Cy~ Phe Arg Met Gln Ile Aap Ni~

Ala Ile Asp Ile Ile Cy Gly Ph~ Leu Ly~ Glu Arg Cy Phe Arg Gly Ser Ser Tyr Pro Val Cy6 Val Ser Lys VA1 V~1 Lyl~ Gly Gly Ser Ser Gly Lys Gly Thr Thr Leu Arg Gly Arg S A~:p Ala Asp Leu Val V~l 65 70 75 . ~o Phe Leu Ser Pro Leu Thr Thr Phe Gln A-lp Gln Leu Asn Arg Arg Gly e5 90 95 Glu Phe Thr Gln Glu Ile Arg Arg Gln Leu Glu Ala cy8 Gln Arg Glu Arg Ala Leu S~r Val Lys Phe Glu V~l Gln Al- Pro Arg Trp Gly Asn Pro Arg Ala Leu Ser Phe val Leu Ser ser Leu Gln Leu Gly Glu Gly Val Glu Phe Asp Val Leu Pro Ala Phe Asp Ala Leu Gly Gln Leu Thr 145 ... 150 155 160 Gly Ser ~yr Lys Pro Asn Pro Gln Ile Tyr Val Lys Leu Ile Glu Glu - 165 170 17~
Cys Thr Asp Leu Gln Lys Glu Gly Glu Phe Ser Thr Cys Gly Thr Glu 180 la5 190 L~u Gln Arg Asp Phe Leu Lys Gln Arg Pro Thr Lya Leu Ly3 Ser Leu ~95 200 205 Ile Arg ` Leu Val Lys Xi- Trp Thr Gln Asn Cyl; Lys Lys Lys Leu Gly Lys Leu Pro Pro Gln Tyr Ala Leu Glu Leu Leu Thr Val Tyr Ala T

Glu Arg Gly Ser Met Lys Thr ~is Phe A~n Thr Ala Gln Gly Phe Arg Thr Val Leu Glu Leu Val Ile Asn Tyr Gln Gln Leu Cys Ile Tyr Trp 260 265 270 .
Ile Lys Tyr Tyr Asp Phe Ly~ As~n Pro Ile Ile GlU Lys Tyr Leu Arg Arg Gln Leu Thr Lya Pro Arg Pro Val Ile Leu Ly- Pro Ala A~p Pro _ . _ ~ wO95122 5 218 3 4 6 t 290 2g5 300 Thr Gly A~n Leu Gly Gly Gly A~p Pro Ly Gly Trp Arg Gln Leu Al Gln Glu Al~ Glu Al~ Trp Leu Asn Tyr Pro Cy~ Phe Lys A~n As 325 330 Trp p Gly Ser Pro V~l Ser Ser Trp Ile Leu Leu Ala Glu Ser Asn Ser Shr 340 3~15 350 Asp Asp Glu T} r Asp A~p Pro Arg Thr Tyr Gln Lys yr Gly Tyr ~le Gly Thr HiG Glu Tyr Pro Hi~ Phe Ser His Arg Pro Ser Thr Leu Gln Ala Ala Ser Thr Pro Gln Ala Glu Glu Asp Trp Thr Cy~ Thr Il~ I,eu -~ 21 83461 The present invention may, Or course, be cArri~d out in other ero~ifi~ ways than thos~ herein set forth without departing ~rom the spirit and D~ t~ r., I ~ istics of the invention. For exampl~, the nucleotide , - ~1; QC] oced herein ~ay be ; nD~i with other nucleotide r.~ DQ to ~te heterologous nucleotide r.~ R for i.-LL~,du. Lion into the genomes Or plants to form LL~n ~ ; C plants. The present ~ f are, th~ ULt, to be c~nc;~4D~ed in all L~=D~e~LS as illustrative and not restrictive and all changes coming within the meaning and equivalency range of the z~ cl claim6 are i ntDn~lD~ to be embraced herein .
~ aving described our invention, we clai~:

Claims (163)

1. A transgenic plant all of whose cells contain at least one nucleotide sequence introduced into said transgenic plant, or ancestor of said transgenic plant, said introduced nucleotide sequence encoding an amino acid sequence having antiviral activity for conferring to the transgenic plant immunity or resistance against viral infection.

2. A transgenic plant of claim 1, said nucleotide sequence includes the nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

3. A transgenic plant of claim 1, said nucleotide sequence being selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

4. A transgenic plant of claim 1, said nucleotide sequence includes the nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

5. A transgenic plant of claim 1, said nucleotide sequence includes the nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

6. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

7. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A synthetase.

8. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to PKR.

9. A trasgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A-dependent RNase, said nucleotide sequence further encoding a second amino acid sequence, said second amino acid sequence having activity similar or identical to 2-5A synthetase.

10. A transgenic plant of claim 9, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase and nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

11. A transgenic plant of claim 9, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase and nucleotides selected from the group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

12. A transgenic plant of claim 9, said nucleotide sequence further encoding a third amino acid sequence, said third amino acid sequence having activity similar or identical to PKR.

13. A transgenic tobacco plant of claim 12, said nucleotide sequence including nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase, nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase and nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA
binding domain of PKR.

.

14. A transgenic plant of claim 11, said nucleotide sequence including nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase, nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR and nucleotides selected from the group of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028, and 1-884 in Table 2.

15. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A synthetase, said nucleotide sequence further encoding a second amino acid sequence, said amino acid sequence having activity similar or identical to PKR.

16. A transgenic plant of claim 15, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase and nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

17. A transgenic plant of claim 1, said amino acid sequence having activity similar or identical to 2-5A-dependent RNase, said nucleotide sequence further encoding a second amino acid sequence, said amino acid sequence having activity similar or identical to PKR.

18. A transgenic plant of claim 17, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase and designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

19. A transgenic plant of claim 17, said nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA
binding domain of PKR and nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

20. A transgenic plant of claim 1, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

21. A transgenic plant of claim 2, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

22. A transgenic plant of claim 3, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

23. A transgenic plant of claim 4, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

24. A transgenic plant of claim 5, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

25. A transgenic plant of claim 6, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

26. A transgenic plant of claim 7, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

27. A transgenic plant of claim 8, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

28. A transgenic plant of claim 9, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

.

29. A transgenic plant of claim 12, said transgenic plant being selected from the group of transgenic plants congisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

30. A transgenic plant of claim 15, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

31. A transgenic plant of claim 17, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

32. A transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

33. A transgenic tobacco plant of claim 32, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

34. A transgenic tobacco plant of claim 32, said nucleotide sequence includes nucleotides selected from the group of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

35. A transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-synthetase.

36. A transgenic tobacco plant of claim 35, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

37. A transgenic tobacco plant all of whose cells contain a nucleotide sequence introduced into said transgenic tobacco plant, or an ancestor of said transgenic tobacco plant, said nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR.

38. A transgenic tobacco plant of claim 37, said nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PRR.

39. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least three nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-dependent RNase, said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A
synthetase, and said third introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR.

40. A transgenic plant of claim 39, said transgenic plant being a transgenic tobacco plant.

41. A transgenic plant of claim 39, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

42. A transgenic plant of claim 39, said first nucleotide sequence including nucleotides designated as 1-2223 in Table I or any part of this nucleotide which contains the complete or partial coding sequence for 2-5A-dependent RNase.

43. A transgenic plant of claim 42, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A synthetase and said third nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

44. A transgenic plant of claim 39, said first nucleotide sequence includes nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

45. A transgenic plant of claim 44, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase, and said third nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

46. A transgenic plant of claim 42, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

47. A transgenic plant of claim 43, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

48. A transgenic plant of claim 44, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

49. A transgenic plant of claim 45, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

50. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least two nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-dependent RNase, and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A
synthetase.

51. A transtenic plant of claim 50, said transgenic plant being a transgenic tobacco plant.

52. A transgenic plant of claim 50, said first nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

53. A transgenic plant of claim 52, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

54. A transgenic plant of claim 50, said first nucleotide sequence includes nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

55. A transgenic plant of claim 54, said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

56. A transgenic plant of claim 50, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

57. A transgenic plant of clsim 52, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

58. A transgenic plant of claim 53, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

59. A transgenic plant of claim 54, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

60. A transgenic plant of claim 55, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

61. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least two nucleotide sequences, each said nucleotide sequence being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR, and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A synthetase.

62. A transgenic plant of claim 61, said transgenic plant being a transgenic tobacco plant.

63. A transgenic plant of claim 61, said first nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of said nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA
binding domain of PKR and said second nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

64. A transgenic plant of claim 61, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

65. A transgenic plant of claim 63, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

66. A transgenic plant of claim 1, said transgenic plant all of whose cells contain at least two nucleotide sequences, each said nucleotide sequence, being introduced into said transgenic plant, or an ancestor of said transgenic plant, said first introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to 2-5A-dependent RNase and said second introduced nucleotide sequence encoding an amino acid sequence having activity similar or identical to PKR.

67. A transgenic plant of claim 66, said transgenic plant being a transgenic tobacco plant.

68. A transgenic plant of claim 66, said first nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

69. A transgenic plant of claim 68, said second nucleotide sequence including nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA
binding domain of PKR.

70. A transgenic plant of claim 66, said first nucleotide sequence includes nucleotides selected from a group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, 1-1095, 1-1028 and 1-884 in Table 2.

71. A transgenic plant of claim 70, said second nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA
binding domain of PKR.

72. A transgenic plant of claim 66, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

73. A transgenic plant of claim 68, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

74. A transgenic plant of claim 69, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

75. A transgenic plant of claim 70, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

76. A transgenic plant of claim 71, said transgenic plant being selected from the group of transgenic plants consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

77. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

78. A. plant transformation vector of claim 77, said nucleotide sequence includes nucleotides designated as 1-2223 in Table 1 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-dependent RNase.

79. A plant transformation vector of claim 77, said nucleotide sequence includes nucleotides selected from the group consisting of nucleotides designated as 1-2037, 1-1968, 1-1858, 1-1546, 1-1422, 1-1210, l-1095, q-1028 and 1-884 in Table 2.

80. A plant transformation vector of claim 77, said vector being plasmid pAM943:2-5A-dep. RNA sense.

81. A plant cell containing said plant transformation vector of claim 77.

82. A plant cell of claim 81, said plant transformation vector being plasmid pAM943:2-5A-dep.
RNase sense.

83. A plant cell of claim 81, said plant cell being a tobacco plant cell.

84. A differentiated tobacco plant comprising said tobacco plant cell of claim 83.

85. A differentiated tobacco plant of claim 84, said plant transformation vector being plasmid pAM943:2-5A-dep. RNase sense.

86. A plant cell of claim 81, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.

87. A bacterial cell containing said plant transformation vector of claim 77.

88. A bacterial cell of claim 87, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

89. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR.

90. A plant transformation vector of claim 89, snid nucleotide sequence includes nucleotides designated as 1-2562 in FIG. 18 or any part of this nucleotide sequence which contains the complete or partial coding sequence for PKR or the double stranded RNA binding domain of PKR.

91. A plant transformation vector of claim 89, said vector being plasmid pAM943: PK68.

92. A plant cell containing said plant transformation vector of claim 89.

93. A plant cell of claim 92, said plant cell being a tobacco plant cell.

94. A tobacco plant comprising said tobacco plant cell of claim 93.

95. A tobacco plant of claim 94, said plant transformation vector being plasmid pAM943:PK68.

96. A plant cell of claim 92, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.

97. A plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A synthetase.

98. A plant transformation vector of claim 97, said nucleotide sequence includes nucleotides designated as 1-1650 in FIG. 20 or any part of this nucleotide sequence which contains the complete or partial coding sequence for 2-5A-synthetase or the double stranded RNA binding domain of 2-5A-synthetase.

99. A plant transformation vector of claim 97, said vector being plasmid pAM943: 2-5A synthetase.

100. A plant cell containing said plant transformation vector of claim 97.

101. A plant cell of claim 100, said plant cell being a tobacco plant cell.

102. A plant cell of claim 100, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.

103. A tobacco plant comprising said tobacco plant cell of claim 101.

104. A tobacco plant of claim 94, said plant transformation vector being plasmid pAM943:synthetase.

105. A bacterial cell containing said plant transformation vector of claim 97.

106. A bacterial cell of claim 105, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

107. A plant cell of claim 81, said plant cell containing a second plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to 2-5A synthetase.

108. A plant cell of claim 107, said plant cell containing a third plant transformation vector which comprises a nucleotide sequence which encodes an amino acid sequence having activity similar or identical to PKR.

109. A plant cell of claim 107, said plant cell being a tobacco plant cell.

110. A plant cell of claim 108, said plant cell being a tobacco plant cell.

111. A plant cell of claim 107, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.

112. A plant cell of claim 108, said plant cell being selected from the group consisting of vegetable, fruit, grain tree, flower, grass, weed and shrub plant cells.

113. A bacterial cell containing said plant transformation vector and said second plant transformation vector of claim 107.

114. A bacterial cell of claim 113, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

115. A bacterial cell containing said plant transformation vector, said second plant transformation vector and said third plant transformation vector of claim 108.

116. A bacterial cell of claim 114, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

117. A transgenic plant comprising said tobacco plant cell of claim 109.

118. A transgenic plant comprising said tobacco plant cell of claim 110.

119. A transgenic plant comprising said plant cell of claim 31.

120. A transgenic plant comprising said plant cell of claim 109.

121. A transgenic plant comprising said plant cell of claim 110.

122. A transgenic plant comprising said plant cell of claim 111.

123. A transgenic plant comprising said plant cell of claim 112.

124. A method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of:
a.) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A-dependent RNase;
b. ) obtaining a transformed plant cell; and c. ) regenerating from the transformed plant cell a genectically transformed plant which expresses the amino acid sequence encoded by the construct.

125. A method of claim 124, said method including the further step of inserting into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A
synthetase.

126. A method of claim 125, said method including the further step of inserting into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A
synthetase.

127. A method of claim 124, said method including the further step of inserting into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to PKR.

128. A method for producing genetically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of:
a. ) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to PKR;
b. ) obtaining a transformed plant cell; and c. ) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct.

129. A method of claim 128, said method including the further step of inserting into said genome of said plant cell a second construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A
synthetase,

130. A method for producing genectically transformed plants which are resistant or immune to infection by a virus, said method comprises the steps of:
a. ) inserting into the genome of a plant cell of a plant susceptible to a virus a construct having a nucleotide sequence which encodes for an amino acid sequence having activity similar or identical to 2-5A synthetase;
b. ) obtaining a transformed plant cell; and C. ) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the construct.

.

131. A method of claim 124 in which the plant is a tobacco plant.

132. A method of claim 125 in which the plant is a tobacco plant.

133. A method of claim 126 in which the plant is a tobacco plant.

134. A method of claim 127 in which the plant is a tobacco plant.

135. A method of claim 128 in which the plant is a tobacco plant.

136. A method of claim 129 in which the plant is a tobacco plant.

137. A method of claim 130 in which the plant is a tobacco plant.

138. A method of claim 124 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

139.- A method of claim 125 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

140. A method of claim 126 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

141. A method of claim 127 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

142. A method of claim 128 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

143. A method of claim 129 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

144. A method of claim 130 in which the plant is selected from the group consisting of vegetable, fruit, grain, flower, tree, grass, weed and shrub plants.

145. A method for producing genectically transformed plants, which are resistant or immune to infection by a virus, said method comprises the steps of:
a. ) inserting into the genome of a plant cell of a plant susceptible to a virus a nucleotide sequence which encodes for an amino acid sequence having the ability to inhibit or interfere with viral replication;
b.) obtaining a transformed plant cell; and C.) regenerating from the transformed plant cell a genetically transformed plant which expresses the amino acid sequence encoded by the nucleotide sequence.

.

146. A method of claim 145, the amino acid sequence having activity similar or identical to 2-5A-dependent RNase.

147. A method of claim 145, the amino acid sequence having activity similar or identical to 2-5A-synthetase.

148. A method of claim 145, the amino acid sequence having activity similar or identical to PKR.

149. A transgenic plant all of whose cells contain a nucleotide sequence introduced into said transgenic plant, or an ancestor of said transgenic plant, said introduced nucleotide sequence encoding an antisense 2-5A-dependent RNase amino acid sequence.

150. A plant transformation vector which comprises said nucleotide sequence of claim 149.

151. A plant transformation vector of claim 150, said plant transformation vector being plasmid pAM943: 2-5A-dep. RNase antisense.

152. A plant transformation vector of claim 150, said plant transformation vector being plasmid pAM822: 2-5A-dep. RNase antisense.

153. A construct which comprises said nucleotide sequence of claim 149, said construct being the construct as described in FIG. 13 D/a.

154. A construct which comprises said nucleotide sequence of claim 149, said construct being the construct as described in FIG. 13E.

155. A plant cell containing said plant transformation vector of claim 150.

156. A plant cell of claim 155, said plant cell being a tobacco plant cell.

157. A plant cell of claim 155, said plant cell being selected from the group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub plant cells.

158. A bacterial cell containing said plant transformation vector of claim 150.

159. A bacterial cell of claim 158, said bacterial cell being an Argobacterium tumefaciens bacterial cell.

160. A transgenic plant of claim 149, said transgenic plant being a tobacco plant.

161. A transgenic plant of claim 149, said transgenic plant being selected from a group consisting of vegetable, fruit, grain, tree, flower, grass, weed and shrub transgenic plants.

162. An isolated nucleotide sequence encoding an amino acid sequence having human 2-5A-dependent RNAse activity, or an active fragment or analog thereof, said nucleotide sequence being identified as SEQ ID NO:3: and comprising:

163. An amino acid sequence having human 2-5A-dependent RNAse activity, or an active fragment or analog thereof, said amino acid sequence being identified as SEQ ID NO: 4: and comprising: