Brome Mosaic Virus

All polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. Viral RNA-dependent RNA polymerases (RdRp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. We examined RNA synthesis by the brome mosaic virus RdRp on DNA, RNA, and hybrid templates and found that precise initiation of RNA synthesis can take place from all of these templates. Furthermore, initiation can take place from either internal or penultimate initiation sites. Using a template competition assay, we found that the BMV RdRp interacts with DNA only three- to fourfold less well than it interacts with RNA. Moreover, a DNA molecule with a ribonucleotide at position -11 relative to the initiation nucleotide was able to interact with RdRp at levels comparable to that observed with RNA. These results suggest that relatively few co...

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.

Positive-strand RNA viruses are known to rearrange the endomembrane network to make it more conducive for replication, maturation or egress. Our previous transmission electron microscopic (TEM) analysis showed that ectopic expression of wild type (wt) capsid protein (CP) of Brome mosaic virus (BMV) has an intrinsic property of modifying endoplasmic reticulum (ER) to induce vesicles similar to those present in wt BMV infection. In this study, we evaluated the functional significance of CP-mediated vesicle induction to the BMV infection cycle in planta. Consequently, cytopathological changes induced by wt CP or its mutants defective in virion assembly due to engineered mutations in either N- or C-proximal domains were comparatively analyzed by TEM in two susceptible (Nicotiana benthamiana and Chenopodium quinoa) and one non-host (N. clevelandii) plant species. Results showed that in susceptible hosts, CP-mediated ER-derived vesicle induction is contingent on the expression of encapsidation-competent CP. By contrast, unlike in N. benthamiana and C. quinoa, transient expression of wt CP in non-host N. clevelandii plants eliminated vesicle induction. Additionally, comparative source-to-sink analysis of virus spread in leaves of N. benthamiana and N. clevelandii co-expressing wt BMV and Cucumber mosaic virus (CMV) showed that despite trans-encapsidation, CMV failed to complement the defective cell-to-cell movement of BMV. The significance and relation of CP-mediated vesicle induction to virus cell-to-cell movement is discussed.

Brome mosaic virus (BMV) is a representative member of positive-strand RNA viruses. The 1a replicase from BMV is a membrane protein of unknown structure with a methyltransferase N-terminal domain and a putative helicase activity in the C-terminal domain. In order to make a functional prediction of the helicase activity of the BMV 1a C-terminal domain, we have built a model of its structure. The use of fold recognition servers hinted at two different superfamilies of helicases [superfamily 1 (SF1) and superfamily 2 (SF2)] as putative templates for the C-terminal fragment of BMV 1a. A structural model of BMV 1a in SF2 was obtained by means of a fold recognition server (3D-PSSM). On the other hand, we used the helicase motifs described in the literature to construct a model of the structure of the BMV 1a C-terminal domain as a member of the SF1. The biological functionality and statistic potentials were used to discriminate between the two models. The results illustrate that the use of sequence profiles and patterns helps modeling. Accordingly, the C-terminal domain of BMV 1a is a potential member of the SF1 of helicases, and it can be modeled with the structure of a member of the UvrD family of helicases. The helicase mechanism was corroborated by the model and this supports the hypothesis that BMV 1a should have helicase activity.

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can...

Non-homologous RNA recombination is a process enabling the exchange of genetic material between various (related or unrelated) RNA-based viruses. Despite extensive investigations its molecular mechanism remains unclear. Studies on genetic recombination in brome mosaic virus (BMV) have shown that local hybridization between genomic RNAs induces frequent non-homologous crossovers. A detailed analysis of recombinant structures suggested that local complementary regions might be involved in two types of non-homologous recombination in BMV: site-specific and heteroduplex-mediated. To verify the above hypothesis and better recognize the mechanism of the phenomenon studied we have tested how the putative types of recombination are affected by a specific mutation in the BMV polymerase gene or by changes in RNA structure. The experiments undertaken revealed substantial differences between site-specific and heteroduplex-mediated recombination, indicating that they occur according to different...

The biochemical and functional properties of the movement protein (MP) of brome mosaic virus (BMV) were investigated. Expression and purification of the BMV MP from Escherichia coli resulted in a pure and soluble protein preparation. Sucrose gradient centrifugation revealed that BMV MP forms oligomers consisting of two or more copies but no higher order multimers even when different ionic strengths and pHs were applied. Nitro-cellulose filter binding and gel retardation studies showed that in vitro the BMV MP preferentially bound to ss nucleic acids (RNA and DNA); the affinity to ssRNA was lower compared to BMV coat protein. The binding to ss nucleic acid was cooperative and not sequence specific and the hypothetical binding site was calculated to be around three to six nucleotides per MP monomer. The nucleic acid binding properties of the BMV MP are discussed in relation to the recent finding that this protein is also able to form tubular structures in infected protoplasts.

The nucleotide sequences of the RNA 3 of fifteen isolates of Prunus necrotic ringspot virus (PNRSV) varying in the symptomatology they cause in six different Prunus spp. were determined. Analysis of the molecular variability has allowed, in addition to study the phylogenetic relationships among them, to evaluate the minimal requirements for the synthesis of the subgenomic RNA in Ilarvirus genus and their comparison to other members of the Bromoviridae family. Computer assisted comparisons led recently to Jaspars (Virus Genes 17, 233-242, 1998) to propose that a hairpin structure in viral minus strand RNA is required for subgenomic promoter activity of viruses from at least two, and possibly all five, genera in the family of Bromoviridae. For PNRSV and Apple mosaic virus two stable hairpins were proposed whereas for the rest of Ilarviruses and the other four genera of the Bromoviridae family only one stable hairpin was predicted. Comparative analysis of this region among the fifteen ...