The cucumovirus 2b gene drives selection of inter-viral recombinants affecting the crossover site, the acceptor RNA and the rate of selection.

RNA-RNA recombination is an important pathway in virus evolution and has been described for many viruses. However, the factors driving recombination or promoting the selection of recombinants are still unclear. Here, we show that the small movement protein (2b) was able to promote selection of RNA 1/2-RNA 3 recombinants within a chimeric virus having RNAs 1 and 2 from cucumber mosaic virus, and RNA 3 from the related tomato aspermy virus, along with heterologous 2b genes. The source of the 2b also determined the selection of the acceptor RNA and the crossover site, as well as affecting the rate of selection of the recombinant RNAs. The nature of the RNA 3 also influenced the selection of the recombinant RNAs. A 163-nt tandem repeat in RNA 3 significantly affected the rate of selection of the recombinant RNA, while a single nucleotide within the repeat affected the crossover site. The recombination occurred in a non-random manner, involved no intermediates and probably was generated via a copy-choice mechanism during (+) strand RNA synthesis.

Stability and competitiveness of interviral recombinant RNAs derived from a chimeric cucumovirus.

We previously described interviral recombinant RNAs derived from a chimeric virus having RNAs 1 and 2 of cucumber mosaic virus (CMV) with RNA 3 from the related tomato aspermy virus (TAV) and the 2b gene from either TAV or another strain of CMV. Here, we show that these interviral recombinant RNAs 3 were stable in the infected plants and could co-exist with their wild-type parental viral RNAs in the same plants, but their de novo generations were inhibited in the presence of the wild-type parental viral RNAs. The recombinant viral genomes did not prevent the replication of other viral RNAs or vice versa, but one of the interviral recombinant viruses induced different symptoms in Physalis floridana from those induced by the parental chimeric virus without the interviral RNA 3 recombinant. Factors such as the nature of the 2b gene and/or the presence or absence of competing wild-type parental RNAs influenced the generation of the recombinant RNAs described. Our data provide additional mechanistic insight into generation, stabilization and competition of recombinant viral RNA in infected host plants.

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid.

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P(L) and P(R), of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P(L) is the pathogenicity-modulating domain. The role of the P(R) domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.

Differential virulence by strains of Cucumber mosaic virus is mediated by the 2b gene.

The approximately 12-kDa 2b protein, encoded by all cucumoviruses, had been shown to play an important role in viral long-distance movement, hypervirulence, and suppression of post-transcriptional gene silencing. The role of the 2b gene in the hypervirulence of Cucumber mosaic virus (CMV) and whether hypervirulence was linked to movement were analyzed using a hybrid virus (CMV-qw), generated by replacing the 2b gene in a subgroup II strain, Q-CMV, with the 2b gene from a subgroup IA strain, WAII-CMV. CMV-qw was more virulent than Q-CMV or WAII-CMV on most of the host plant species tested. Northern blot and nucleotide sequence analyses demonstrated that CMV-qw was stably maintained during the course of infection and upon passage. Kinetic studies revealed that the hypervirulence induced by the hybrid virus was associated with neither increased viral RNA accumulation nor more rapid viral movement per se, suggesting that other functions of the 2b protein are important in determining the hypervirulence.

The 2b protein of cucumoviruses has a role in promoting the cell-to-cell movement of pseudorecombinant viruses.

Pseudorecombinant viruses (i.e., those containing a reassorted genome of closely related multipartite viruses) are often not as competitive as the parental viruses. The role of the 2b gene in hypervirulence and maintenance of a progressive infection was assessed in a pseudorecombinant virus formed between RNAs 1 plus 2 of Cucumber mosaic virus (CMV) and RNA 3 of Tomato aspermy virus (TAV). The presence of RNA 3 of TAV was found to affect the level of RNA accumulation but not the level of virulence. By contrast, the 2b genes of both TAV and a hypervirulent strain of CMV (WAII-CMV) were found to affect the virulence of the pseudorecombinant viruses but not the levels of viral RNA accumulation. The 2b gene rather than the overlapping open reading frame encoding the C-terminal 41 amino acids of 2a protein of the corresponding virus was found to be essential for promoting infection of the pseudorecombinant viruses in planta. However, the 2b gene was not essential for replication of pseudorecombinant viruses containing CMV RNAs 1 plus 2 and TAV RNA 3. These results indicate that the 2b protein is involved in promoting the cell-to-cell movement of the pseudorecombinant viruses. These data also suggest the existence of specific interaction between the TAV 2b protein and either RNA 3 or its encoded proteins, which may be critical for promoting or maintaining infection or both.

The conserved, 5' termini of RNAs 1 and 2 of Tomato aspermy virus are dispensable for infection but affect virulence.

The 5' terminus of each of the three genomic RNAs (RNAs 1, 2 and 3) of Tomato aspermy virus (TAV) begins with the sequence 5'-GUUU, which is also shared by a number of other viruses. Mutagenic analyses showed that the 5'-GUUU sequence of RNAs 1 and 2 of TAV was dispensable for viral infection and did not prevent symptom induction. On the other hand, substitution of U at position 5 for G in RNA 1, but not RNA 2, induced veinal necrosis symptoms in Nicotiana glutinosa. The mutants constructed included insertion of UUU into the 5'-GUUU sequence of TAV RNAs 1 and/or 2. All RNA 2 mutants induced more severe symptoms than viral RNAs containing either mutated RNA 1 or most combinations of mutated RNAs 1 and 2. Some combinations of mutated RNAs 1 and 2 also induced veinal necrosis in N. glutinosa. Virulence was unrelated to the levels of viral RNA accumulation. Sequence analysis of progeny viral RNAs showed that only the mutant viral RNAs with a G to U substitution in RNA 1 and the deletion of the 5'-GUUU in both RNAs 1 and 2 were able to maintain the same sequence as the inoculum. The other mutants either reverted to the wildtype sequence or underwent further deletion or insertion. None of the constructed mutants were able to compete for accumulation with the wildtype virus after co-inoculation to the plant species tested.

Stable and unstable mutations in the 5' non-translated regions of tomato aspermy virus RNAs 1 and 2 generated de novo from infectious cDNA clones containing a cauliflower mosaic virus 35S promoter.

Tomato aspermy virus RNAs derived from infectious cDNA clones exhibited a number of sequence alterations in the 5' non-translated region (NTR). These included a deletion of the first four residues in both RNAs 1 and 2, transversion of residue 5 from a G to a U in RNA 1, and transversion of A to C at position of 50 of RNA 1. These alterations were not stable in the infected plants while the insertion of a U residue between nucleotides 1 and 5 of RNA 1 was stable in the infected plants. Generation of these sequence alternations was not dependent upon either the host species or the concentration of the inoculum. The sequence alterations also did not occur on passage of wildtype virus. Rather, the sequence alterations related to transcription from the cauliflower mosaic virus 35S RNA promoter-driving infectious cDNAs. The alternations observed had no impact on symptoms or infectivity, but did affect the accumulation of specific viral RNAs. The data also demonstrated the existence of some plasticity in the sequence of the 5' NTR.

Extensive variation of sequence within isolates of Grapevine virus B+.

Four regions covering 1247 nucleotides of the RNA genome of 20 isolates of a Vitivirus, Grapevine virus B (GVB), from three countries were analyzed. All the regions in these isolates varied in sequence as compared to the published GVB sequence. Of these, the intergenic region varied the most, with 73.2% nucleotide sequence homology, while ORF4 encoding coat protein varied the least when compared both at nucleotide sequence (80.3% homology) and at amino acid sequence levels (90.6% homology). The variations were scattered along each region length and were higher at the nucleotide level than at the amino acid level, but none resulted in a frame shift or stop codon. These results indicate that GVB may exist as a heterogeneous population, possibly resulting from mixing different strains by grafting practices or by RNA-RNA recombination in the grapevine, the only known natural host of this virus. Although it has been reported that GVB is associated with corky bark disease, no corky bark symptoms were observed in any of the GVB positive grapevine sample collected from Australia.