- Volume 75, Issue 11, 1994

The genomic RNA of potyvirases has a characteristic 5′ non-translated region (5′NTR) to which a viral protein, VPg, is covalently attached. This suggests that the viral RNA lacks a conventional cap structure and thus its translation may not proceed in the same way as most cellular mRNAs. To investigate the role of the 5′NTR during translation, various derivatives of the turnip mosaic potyvirus (TuMV) leader were fused to the reporter gene β-glucuronidase (GUS). These constructs were used to monitor the efficiency of translation in vitro in a rabbit reticulocyte lysate and in planta following microprojectile DNA delivery into tobacco cell suspensions. GUS transcripts fused with the TuMV 5′NTR, whether they were capped or not, were efficiently translated, whereas GUS transcripts without the viral leader needed to be capped for expression. When transcripts of the viral leader were supplied in excess over functional transcripts, translation was inhibited in a dose-dependent manner. Similarly, transcripts synthesized from the reverse complement of the 5′NTR inhibited translation to the same extent as the wild-type sequence, indicating that cap independence was not conferred by a specific sequence within the viral leader. A stable hairpin loop was placed in front or after the viral sequence. This hairpin loop normally prevented translation of control GUS transcripts but when the viral leader was positioned after it a significant level of GUS activity was measured, whether the transcripts were capped or not. On the other hand, when the hairpin loop was positioned after the viral leader, no GUS activity was measured. These results suggested that ribosomes bound to an internal site within the TuMV 5′NTR and then presumably scanned the sequence for the initiator AUG.

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.

Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV la and 2a proteins, as immunogens. Five out of eight monoclonal antibodies, which bound to the la and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro. Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the la protein containing putative helicase and methyltransferase domains respectively. Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps. Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase. Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the la and 2a proteins. The greatest inhibition was shown by antibodies to a peptide containing the GDD motif. These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates.

Cucumber mosaic cucumovirus (CMV) can be divided into two subgroups, I and II. LS-CMV and most other subgroup II strains cause a mild, systemic mottle on tobacco and can induce a necrotic etching (necrotic rings) symptom on inoculated tobacco leaves. In contrast, Fny-CMY and most other subgroup I strains cause severe, systemic mosaic symptoms on tobacco, but do not induce the necrotic etching symptom. Full- length cDNA clones of all three genomic RNAs of LS- CMV were constructed and infectious RNAs were generated from these clones. Using pseudorecombinants constructed from the infectious transcripts of LS-CMV and Fny-CMV, we found that both RNAs 1 and 2 of Fny-CMV are involved in determining the severity of systemic symptom on tobacco, and that LS-CMV RNA 3 contains the determinant for the necrotic etching symptom. Chimeras formed between Fny- and LS-CMV RNA 3 were used to demonstrate that the inducer of the necrotic etching symptoms mapped to the 5′ 618 nucleotides of LS-CMV RNA 3, and required sequences in both the 5′ non-translated region, as well as the 3a gene of CMV.

The 3a protein, encoded by RNA 3 of cucumber mosaic virus (CMV), is the putative movement protein of viral progeny in infected plants. An analysis of transgenic tobacco plants constitutively expressing the CMV 3a protein showed that the protein is accumulated in leaves at every stage of development. In fully expanded leaves the protein is immunodetectable mostly in a cell-wall- enriched fraction. Dye-coupling experiments using fluor- escent-dextran probes were performed on fully expanded leaves to study the modifying effect of CMV 3a protein on the gating capacity of plasmodesmata. Movement of fluorescein-isothiocyanate-labelled dex- tran with a mean molecular mass of 10000 Da, and an approximate Stokes’ radius of 2·3 nm, was detected between cells of the 3a protein transgenic plants, but not in the control plants. These results are consistent with the idea that the CMV 3 a protein is involved in the modification of plasmodesmata and, therefore, in the cell-to-cell spread of the virus.

In-frame contiguous deletions were created in the movement protein gene of alfalfa mosaic virus by site- directed mutagenesis. The mutated movement proteins were expressed in Escherichia coli, extracted and then purified by denaturing gel electrophoresis and then renatured. Their binding ability with RNA was assayed by electrophoretic retardation and u.v.-crosslinking. Results indicated that a domain included within amino acids 36 to 81 was necessary for RNA binding.

The nonanucleotide motif TAATATTAC occurs in the intergenic region of all geminiviruses that have been examined to date. The motif is invariably located within the loop of a potential stem-loop structure that has been implicated in viral DNA replication. To investigate the contribution of these sequences to virus proliferation, African cassava mosaic virus (ACMV) DNA B mutants have been screened for their ability to infect Nicotiana benthamiana when co-inoculated with DNA A. Mutants in which the putative stem structure was altered by the introduction of single nucleotide mismatches remained as infectious as the wild-type virus and the mutations were retained in the progeny. Mutants containing nucleotide substitutions within the loop sequences were similarly infectious but analysis of progeny showed that in most cases wild-type sequences were restored by recombination with DNA A. Stem-loop deletion mutants of both genomic components were not infectious when co-inoculated, although they were once again efficiently rescued by recombination when inoculated with the wild-type components. Co-inoculation of genomic components containing the motif TAGTAT- TAC did not result in a systemic infection while mutants containing the motif TAATACTAC were infectious and the mutation was stable. The results demonstrate that ACMV will tolerate some modification to this highly conserved region of the genome that might allow more precise mapping of the position at which the viral DNA is nicked during replication.