The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RN... more The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.
A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic viru... more A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B RNA was constructed with the aim to study the role of viral proteins in the proteolytic processing of the primary translation products. For the same purpose cDNA clones were constructed having sequences derived from both M and B RNA of CPMV. In vitro transcripts prepared from these clones with T7 RNA polymerase, were efficiently translated in rabbit reticulocyte lysates. The translation products obtained were processed in the lysate by specific proteolytic cleavages into smaller products, which made it possible to study subsequently the effect of the various mutations on this process. The results obtained indicate that the B RNA-encoded 24K polypeptide represents a protease responsible for all cleavages in the polyproteins produced by both CPMV B and M RNA. For efficient cleavage of the glutamine-methionine site in the M RNA encoded polyprotein the presence of a second B RNA encoded protein, the 32K polypeptide, is essential, although the 32K polypeptide itself does not have proteolytic activity. A number of cleavage-site mutants were constructed in which the coding sequence for the glutamine-glycine cleavage site between the two capsid proteins was changed. Subsequent in vitro transcription and translation of these cleavage site mutants show that a correct dipeptide sequence is a prerequisite for efficient cleavage but that the folding of the polypeptide chain also plays an important role in the formation of a cleavage site.
Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are ... more Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.
Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles... more Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein ('60K') plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-beta, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.
Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue hav... more Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.
Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-s... more Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically c...
The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by ... more The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by the lack of methods to select desired mutants following random mutagenesis. An alternative might be to copy RNA genomes into DNA and use methods for site-directed mutagenesis to modify specific regions of the DNA copy. Transcription of the DNA copy will subsequently produce viral RNA with desired mutations. We have constructed a full-length DNA copy of the smaller of the two cowpea mosaic virus (CPMV) RNAs, referred to as M RNA. The DNA copy was positioned downstream from the promoter of bacteriophage SP6 and using SP6 RNA polymerase, this copy and two derivatives of it containing a specific deletion and insertion, respectively, have been transcribed into RNA molecules which are efficiently translated in rabbit reticulocyte lysates. The results obtained show that the subsequent in vitro transcription and translation of DNA copies may be a powerful tool to unravel the genetic properties o...
The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RN... more The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.
The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection... more The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.
The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RN... more The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.
A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic viru... more A series of specific deletion mutants derived from a full-length cDNA clone of cowpea mosaic virus (CPMV) B RNA was constructed with the aim to study the role of viral proteins in the proteolytic processing of the primary translation products. For the same purpose cDNA clones were constructed having sequences derived from both M and B RNA of CPMV. In vitro transcripts prepared from these clones with T7 RNA polymerase, were efficiently translated in rabbit reticulocyte lysates. The translation products obtained were processed in the lysate by specific proteolytic cleavages into smaller products, which made it possible to study subsequently the effect of the various mutations on this process. The results obtained indicate that the B RNA-encoded 24K polypeptide represents a protease responsible for all cleavages in the polyproteins produced by both CPMV B and M RNA. For efficient cleavage of the glutamine-methionine site in the M RNA encoded polyprotein the presence of a second B RNA encoded protein, the 32K polypeptide, is essential, although the 32K polypeptide itself does not have proteolytic activity. A number of cleavage-site mutants were constructed in which the coding sequence for the glutamine-glycine cleavage site between the two capsid proteins was changed. Subsequent in vitro transcription and translation of these cleavage site mutants show that a correct dipeptide sequence is a prerequisite for efficient cleavage but that the folding of the polypeptide chain also plays an important role in the formation of a cleavage site.
Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are ... more Cowpea mosaic virus moves from cell-to-cell in a virion form through tubular structures that are assembled in modified plasmodesmata. Similar tubular structures are formed on the surface of protoplasts inoculated with cowpea mosaic virus. The RNA 2-encoded movement protein (MP) is responsible for the induction and formation of these structures. To define functional domains of the MP, an alanine-substitution mutagenesis was performed on eight positions in the MP, including two conserved sequence motifs, the LPL and D motifs. Results show that these two conserved motifs as well as the central region of the MP are essential for cell-to-cell movement. Several viruses carrying mutations in the N- or C-terminal parts of their MP retained infectivity on cowpea plants. Coexpression studies revealed that mutant MPs did not interfere with the activity of wild-type MP and could not mutually complement their defects.
Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles... more Cowpea mosaic virus (CPMV) replication occurs in close association with small membranous vesicles in the host cell. The CPMV RNA1-encoded 60 kDa nucleotide-binding protein ('60K') plays a role in the formation of these vesicles. In this study, five cellular proteins were identified that interacted with different domains of 60K using a yeast two-hybrid search of an Arabidopsis cDNA library. Two of these host proteins (termed VAP27-1 and VAP27-2), with high homology to the VAP33 family of SNARE-like proteins from animals, interacted specifically with the C-terminal domain of 60K and upon transient expression colocalized with 60K in CPMV-infected cowpea protoplasts. eEF1-beta, picked up using the central domain of 60K, was also found to colocalize with 60K. The possible role of these host proteins in the viral replicative cycle is discussed.
Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue hav... more Tubular structures extending from plasmodesmata in cowpea mosaic virus (CPMV)-infected tissue have been implicated to play an important role in cell-to-cell movement of this virus. Using a cauliflower mosaic virus 35S promoter-based transient expression vector, we show that expression of only the CPMV M RNA-encoded 48-kDa protein (48K protein) in cowpea protoplasts is sufficient to induce these structures. Strikingly, expression of the 48K protein in protoplasts from a number of nonhost plant species, such as barley, Arabidopsis thaliana, and carrot, also resulted in tubular structure formation. Thus, it is not likely that the viral 48K protein, though playing a key role in cell-to-cell movement of CPMV, has a role in determining the host range of CPMV.
Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-s... more Middle component RNA (M RNA) of cowpea mosaic virus (CPMV) was transcribed into cDNA and double-stranded cDNA was inserted into the EcoRI site of plasmid pBRH2. The nucleotide sequence of inserts was determined, after subcloning in bacteriophages M13mp7, M13mp8 or M13mp9, by the dideoxy chain termination method. The complete sequence of CPMV M RNA, up to the poly(A) tail, is 3481 nucleotides long. The sequence contains a long open reading frame starting at nucleotide 161 from the 5' terminus and continuing to 180 nucleotides from the 3' terminus. The sequence does not contain a polyadenylation signal for the poly(A) tail at the 3' end of CPMV RNA. The initiation site at position 161 together with AUG codons in the same reading frame at positions 512 and/or 524 account for the two large colinear precursor polypeptides translated in vitro from M RNA. The amino acid sequence deduced from the nucleotide sequence suggests that both precursor polypeptides are proteolytically c...
The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by ... more The genetic approach for elucidating functions encoded by RNA plant viruses has been hampered by the lack of methods to select desired mutants following random mutagenesis. An alternative might be to copy RNA genomes into DNA and use methods for site-directed mutagenesis to modify specific regions of the DNA copy. Transcription of the DNA copy will subsequently produce viral RNA with desired mutations. We have constructed a full-length DNA copy of the smaller of the two cowpea mosaic virus (CPMV) RNAs, referred to as M RNA. The DNA copy was positioned downstream from the promoter of bacteriophage SP6 and using SP6 RNA polymerase, this copy and two derivatives of it containing a specific deletion and insertion, respectively, have been transcribed into RNA molecules which are efficiently translated in rabbit reticulocyte lysates. The results obtained show that the subsequent in vitro transcription and translation of DNA copies may be a powerful tool to unravel the genetic properties o...
The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RN... more The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.
The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection... more The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.
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