The carboxy-terminal two-thirds of the cowpea chlorotic mottle bromovirus capsid protein is incapable of virion formation yet supports systemic movement.

Previous investigations into recombination in cowpea chlorotic mottle bromovirus (CCMV) resulted in the recovery of an unusual recombinant virus, 3-57, which caused a symptomless infection of cowpeas but formed no detectable virions. Sequence analysis of cDNA clones derived from 3-57 determined that mutations near the 5' terminus of the capsid protein gene introduced an early translational termination codon. Further mutations introduced a new in-frame start codon that allowed translation of the 3' two-thirds of the capsid protein gene. Based on the mutations observed in 3-57, wild-type CCMV clones were modified to determine if the carboxyl two-thirds of the capsid protein functions independently of the complete protein in long-distance movement. Analysis of these mutants determined that while virion formation is not required for systemic infection, the carboxy-terminal two-thirds of the capsid protein is both required and sufficient for systemic movement of viral RNA. This indicates that the CCMV capsid protein is multifunctional, with a distinct long-distance movement function in addition to its role in virion formation.

De novo generation of defective interfering-like RNAs in broad bean mottle bromovirus.

Broad been mottle virus (BBMV) is the only member of the bromoviruses that is known to accumulate defective-interfering (DI) RNAs (Romero et al., Virology 194, 576-584, 1993). De novo generation of DI-like RNAs was demonstrated during serial passages of BBMV in broad bean using either DI RNA-free virion RNA preparations or transcribed genomic RNA inocula. As for previously described DI RNAs, all but one of the characterized de novo generated DI-like RNAs were derived by a single in-frame deletion from the RNA2 component. The sole exception was derived by two shorter in-frame deletions from RNA2. The maintenance of an open reading frame by all DI-like RNAs suggests the importance of coding capacity and/or the shortened 2a protein in the accumulation of these RNAs during infection. The deletion junction sites were between nucleotides 1152 and 2366, suggesting that the retained regions are essential for the efficient accumulation of BBMV DI-like RNAs in planta. Short regions of sequence similarity and/or complementarity were revealed at the 5' and 3' junction borders. We speculate that these regions can facilitate DI (DI-like) RNA formation. In addition to DI-like RNAs, the full-length nucleotide sequences of RNA2 components of the Type and Morocco strains of BBMV are presented.

Host-specific alterations in viral RNA accumulation and infection spread in a brome mosaic virus isolate with an expanded host range.

To facilitate studies of virus-host interaction and the determinants of viral host range, we constructed full-length cDNA clones to all three genomic RNAs of an unusual brome mosaic virus (BMV) isolate with an expanded host range. While other BMV strains, including the previously cloned M1 strain, systemically infect barley and other grasses but not legumes, the expanded-host-range isolate and the set of transcripts from its cDNA clones, designated the M2 strain of BMV, systemically infect both barley and cowpea line TVu-612, a legume. All reassorted combinations of M1 and M2 genomic RNAs were equally competent for replication in barley protoplasts and systemic infection of barley plants but showed widely varying levels of viral RNA accumulation in cowpea protoplasts and systemic infection in TVu-612 cowpea plants. Systemic infection levels were influenced by all three genomic RNAs. M2 RNA2 and M2 RNA3 made independent and additive contributions to the frequency with which reassortants infected TVu-612 systemically. The greater individual effect segregated with M2 RNA3, which encodes functions required for infection spread (the 3a movement protein and coat protein). M2 RNA3 also directed accelerated expansion of BMV lesions in inoculated TVu-612 leaves. If the inoculum contained M2 RNA3, the frequency with which reassortants infected TVu-612 systemically could be further enhanced by the presence of M2 RNA1 rather than M1 RNA1. RNA1 encodes the 1a RNA replication protein, and despite similar accumulation in barley protoplasts, in cowpea protoplasts all reassortants bearing M2 RNA1 accumulated positive- and negative-strand RNAs to levels at least six- to eightfold higher than reassortants bearing M1 RNA1. Overall, the results indicate that changes in several distinct virus functions contribute to adapting BMV-M2 to systemically infect TVu-612 cowpea.