J. Verver

Primary structure and gene organization of the middle-component RNA of cowpea mosaic virus.

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 cleaved at glutaminyl‐methionine and glutaminyl‐glycine, respectively, to produce the two viral capsid proteins.

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

The involvement of cowpea mosaic virus M RNA-encoded proteins in tubule formation.

On the surface of cowpea protoplasts inoculated with cowpea mosaic virus (CPMV), tubular structures containing virus particles have been found. Such tubular structures are thought to be involved in cell-to-cell movement of CPMV in cowpea plants. To study the involvement of the 58K/48K and capsid proteins of CPMV in the formation of the tubular structures, mutations were introduced into M cDNA clones from which infectious transcripts could be derived. No tubules were found on protoplasts inoculated with a mutant that fails to produce the 48K protein nor with a mutant that has a deletion in the 48K coding region, suggesting that the 48K protein is essential for this process. However, a possible role of the 58K protein in tubule formation could not be excluded. A mutant that fails to produce the capsid proteins did produce tubules and therefore the capsid proteins are not involved in the formation of the tubular structures. Electron microscopic analysis revealed that the tubules produced by this mutant are, apart from the absence of virus particles, morphologically identical to the tubules formed by the wild-type virus.

The sequence between nucleotides 161 and 512 of cowpea mosaic virus M RNA is able to support internal initiation of translation in vitro

Cowpea mosaic virus M RNA is translated in vitro as well as in vivo into two C-coterminal polyproteins of Mr 105K and 95K. Initiation of translation of the 95K protein gene occurs at an AUG codon at position 512 of M RNA, 351 nucleotides downstream of the initiation codon of the 105K protein gene at position 161. By employing an in vitro transcription and translation system it was determined that this 351 nucleotide sequence has the capacity to direct ribosomes to initiate translation at a downstream start codon. This effect is independent of the position of this sequence in an mRNA. Furthermore, evidence has been obtained that scanning ribosomes can bypass the AUG at position 161. Thus, both leaky scanning and internal entry are mechanisms for the initiation of translation of the 95K protein gene.

Study of the genetic organisation of a plant viral RNA genome by in vitro expression of a full-length DNA copy.

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 of viral RNA genomes.

Transgenic Plants Expressing HC-Pro Show Enhanced Virus Sensitivity While Silencing of the Transgene Results in Resistance

Nicotiana benthamiana plants were engineered to express sequences of the helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic potyvirus (CABMV). The sensitivity of the transgenic plants to infection with parental and heterologous viruses was studied. The lines expressing HC-Pro showed enhanced symptoms after infection with the parental CABMV isolate and also after infection with a heterologous potyvirus, Potato virus Y (PVY) and a comovirus, Cowpea mosaic virus (CPMV). On the other hand, transgenic lines expressing nontranslatable HC-Pro or translatable HC-Pro with a deletion of the central domain showed wild type symptoms after infection with the parental CABMV isolate and heterologous viruses. These results showed that CABMV HC-Pro is a pathogenicity determinant that conditions enhanced sensitivity to virus infection in plants, and that the central domain of the protein is essential for this. The severe symptoms in CABMV-infected HC-Pro expressing lines were remarkably followed by brief recovery and subsequent re-establishment of infection, possibly indicating counteracting effects of HC-Pro expression and a host defense response. One of the HC-Pro expressing lines (h48) was found to contain low levels of transgenic HC-Pro RNA and to be resistant to CABMV and to recombinant CPMV expressing HC-Pro. This indicated that h48 was (partially) posttranscriptionally silenced for the HC-Pro transgene inspite of the established role of HC-Pro as a suppressor of posttranscriptional gene silencing. Line h48 was not resistant to PVY, but instead showed enhanced symptoms compared to nontransgenic plants. This may be due to relief of silencing of the HC-Pro transgene by HC-Pro expressed by PVY.

Subcellular Location of the Helper Component-Proteinase of Cowpea Aphid-Borne Mosaic Virus

The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of HC-Pro expressed with other viral proteins in cowpea protoplasts in a natural CABMV infection, or in protoplasts transfected with a transient expression construct expressing HC-Pro separately from other viral proteins under the control of the 35S promoter. In both cases the protein showed a diffuse cytoplasmic location. Similar localisation patterns were shown in live protoplasts when the transient expression system was used to express HC-Pro as a fusion with the green fluorescent protein as a reporter. In an alternative expression system, the HC-Pro coding region was subcloned in-frame between the movement protein and large coat protein genes of RNA2 of Cowpea mosaic virus (CPMV). Upon transfection of protoplasts with this construct, HC-Pro was expressed as part of the RNA2 encoded polyprotein from which it was fully processed. In this case, the protein localised in broad cytoplasmic patches reminiscent of the typical CPMV induced cytopathic structures in which CPMV replication occurs, suggesting an interaction of HC-Pro with CPMV proteins or host factors in these structures. Finally, recombinant CPMV expressing HC-Pro showed a strongly enhanced virulence on cowpea and Nicotiana benthamiana consistent with the role of HC-Pro as a pathogenicity determinant, a phenomenon now known to be linked to its role as a suppressor of host defense responses based on post-transcriptional gene silencing.

The genomic sequence of cowpea aphid-borne mosaic virus and its similarities with other potyviruses

Summary The genomic sequence of a Zimbabwe isolate of Cowpea aphid-borne mosaic virus (CABMV-Z) was determined by sequencing overlapping viral cDNA clones generated by RT-PCR using degenerate and/or specific primers. The sequence is 9465 nucleotides in length excluding the 3′ terminal poly (A) tail and contains a single open reading frame (ORF) of 9159 nucleotides encoding a large polyprotein of 3 053 amino acids and predicted Mr of 348. The size of the genome and the encoded polyprotein is in agreement with other potyviruses and contains nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. The P1 and P3 were the most variable proteins while CI, NIb and CP were the most conserved.

The NTP-binding motif in cowpea mosaic virus B polyprotein is essential for viral replication.

We have assessed the functional importance of the NTP-binding motif (NTBM) in the cowpea mosaic virus (CPMV) B-RNA-encoded 58K domain by changing two conserved amino acids within the consensus A and B sites (GKSRTGK500S and MDD545, respectively). Both Lys-500 to Thr and Asp-545 to Pro substitutions are lethal as mutant B-RNAs were no longer replicated in cowpea protoplasts. Transiently produced mutant proteins were not able to support trans-replication of CPMV M-RNA in cowpea protoplasts in contrast to transiently produced wild-type B proteins. Therefore loss of viral RNA synthesis was a result of a protein defect rather than an RNA template defect. Mutant B polyproteins were correctly processed in vitro and in vivo and the regulatory function of the 32K protein on processing of B proteins was not affected by these mutations. Since regulation of processing by the 32K protein depends on interaction with the 58K domain, the mutations in the NTBM apparently do not interfere with this interaction. The Asp-545 to Pro substitution left intact the binding properties of the 84K precursor of the 58K protein, with respect to ATP-agarose, whereas the Lys-500 to Thr substitution decreased the binding capacity of the 84K protein, suggesting that the Lys-500 residue is directly involved in ATP binding. The Lys-500 to Thr substitution in the 58K domain resulted in an altered distribution of viral proteins, which failed to aggregate into large cytopathic structures as observed in protoplasts infected with wild-type B-RNA. However viral proteins containing the Asp-545 to Pro substitution showed a normal distribution in protoplasts.

Mutational analysis of AUG codons of cowpea mosaic virus M RNA

The involvement of the AUG codons at positions 115, 161, 512 and 524 in translation and infectivity of cowpea mosaic virus M RNA was studied. Mutations were introduced in each of these codons in a full length cDNA clone of M RNA and the effect of the mutations was examined by translation from in vitro transcripts of these mutant cDNAs in rabbit reticulocyte lysates and by checking the replication of these transcripts in the presence of B RNA in cowpea protoplasts and plants. It was found that AUG115, at the beginning of an open reading frame (ORF) for a putative 2-kDa protein, can be used in vitro to initiate translation, but mutation of this AUG codon in the M RNA does not affect the ability of the virus to infect cowpea plants. AUG161, located at the beginning of the large ORF, was shown to be essential for expression of the large 105-kDa polyprotein and for replication of M RNA. Translation of the second 95-kDa polyprotein was found to start at AUG512. Upon mutation of this AUG codon efficient initiation of translation occurred at AUG524. Results with M RNAs that lack AUG512 and/or 524 indicate that an intact 95-kDa polyprotein is not required for replication of M RNA but that this protein has an essential function in cell-to-cell movement of the virus.