Virions of beet western yellows luteovirus contain a major capsid protein (P22.5) and a minor readthrough protein (P74), produced by translational readthrough of the major capsid protein sequence into the neighboring open reading frame, which encodes the readthrough domain (RTD). The RTD contains determinants required for efficient virus accumulation in agroinfected plants and for aphid transmission. The C-terminal halves of the RTD are not well conserved among luteoviruses but the N-terminal halves contain many conserved sequence motifs, including a proline-rich sequence separating the rest of the RTD from the sequence corresponding to the major coat protein. To map different biological functions to these regions, short in-frame deletions were introduced at different sites in the RTD and the mutant genomes were transmitted to protoplasts as transcripts and to Nicotiana clevelandii by agroinfection. Deletions in the nonconserved portion of the RTD did not block aphid transmission but had a moderate inhibitory effect on virus accumulation in plants and abolished symptoms. Deletion of the proline tract and the junction between the conserved and nonconserved regions inhibited readthrough protein accumulation in protoplasts by at least 10-fold. The mutants accumulated small amounts of virus in plants, did not induce symptoms, and were nontransmissible by aphids using agroinfected plants, extracts of infected protoplasts, or purified virus as a source of inoculum. Other deletions in the conserved portion of the RTD did not markedly diminish readthrough protein accumulation but abolished its incorporation into virions. These mutants accumulated to low levels in agroinfected plants and elicited symptoms, but could not be aphid-transmitted. A preliminary map has been produced mapping these functions to different parts of the RTD.

Brome mosaic bromovirus (BMV), a tripartite plus-sense RNA virus, has been used as a model system to study homologous RNA recombination among molecules of the same RNA component. Pairs of BMV RNA3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny RNA3 in a large number of lesions was analyzed. The majority of doubly infected lesions accumulated the RNA3 recombinants. The distribution of the recombinant types was relatively even, indicating that both RNA3 counterparts could serve as donor or as acceptor molecules. The frequency of crossovers between one pair of RNA3 variants, which possessed closely located markers, was similar to that of another pair of RNA3 variants with more distant markers, suggesting the existence of an internal recombination hot spot. The majority of crossovers were precise, but some recombinants had minor sequence modifications, possibly marking the sites of imprecise homologous crossovers. Our results suggest discontinuous RNA replication, with the replicase changing among the homologous RNA templates and generating RNA diversity. This approach can be easily extended to other RNA viruses for identification of homologous recombination hot spots.It is generally accepted that RNA recombination contributes significantly to the diversity of viruses with RNA genomes (37). However, little experimental evidence supports the occurrence of high-frequency recombination in the virus life cycle. The processes of RNA replication and RNA recombination have been studied extensively in brome mosaic bromovirus (BMV), a tripartite positive-strand RNA virus (12). BMV RNA1 and RNA2 code, respectively, for the 1a and 2a proteins (the viral components of the replicase complex), while RNA3 encodes the 3a (movement) and coat proteins (2). Both homologous and nonhomologous recombination events have been observed among different BMV RNAs (24). Homology-supported crossovers can occur between two nearly identical RNAs (or within nearly identical regions), while nonhomologous crosses can occur between nonrelated RNAs or dissimilar regions (8,16,29). The frequency of homologous intersegmental crosses in BMV is approximately 10-fold higher than that of the nonhomologous crosses (24). In addition to BMV, homologous RNA recombination has been demonstrated for picornaviruses (18-20, 35), coronaviruses (21, 23, 42), for cowpea chlorotic mottle bromovirus (3), tombusviruses (41), and bacteriophages (31). Homology-driven recombination of non-replicative RNA precursors has been reported for Sindbis virus within the overlapping sequences (34).Homologous crossovers among different BMV RNA segments appear to require common 15-to 60-nucleotide (nt) sequences (26) which are composed of GC-rich regions followed by AU-rich regions (27,28). A proposed templateswitching mechanism (27, 28) predicts that the replicase enzyme pauses (stalls) at the AU-rich sequence on a donor BMV RNA molecule and switches to the acceptor template while the upstream GC-rich region facilitate...

Luteoviruses and the luteovirus-like pea enation mosaic virus (PEMV; genus Enamovirus) are transmitted by aphids in a circulative, nonreplicative manner. Acquired virus particles persist for several weeks in the aphid hemolymph, in which a GroEL homolog, produced by the primary endosymbiont of the aphid, is abundantly present. Six subgroup II luteoviruses and PEMV displayed a specific but differential affinity for Escherichia coli GroEL and GroEL homologs isolated from the endosymbiotic bacteria of both vector and nonvector aphid species. These observations suggest that the basic virus-binding capacity resides in a conserved region of the GroEL molecule, although other GroEL domains may influence the efficiency of binding. Purified luteovirus and enamovirus particles contain a major 22-kDa coat protein (CP) and lesser amounts of an ϳ54-kDa readthrough protein, expressed by translational readthrough of the CP into the adjacent open reading frame. Beet western yellows luteovirus (BWYV) mutants devoid of the readthrough domain (RTD) did not bind to Buchnera GroEL, demonstrating that the RTD (and not the highly conserved CP) contains the determinants for GroEL binding. In vivo studies showed that virions of these BWYV mutants were significantly less persistent in the aphid hemolymph than were virions containing the readthrough protein. These data suggest that the Buchnera GroEL-RTD interaction protects the virus from rapid degradation in the aphid. Sequence comparison analysis of the RTDs of different luteoviruses and PEMV identified conserved residues potentially important in the interaction with Buchnera GroEL.

A model system of a single-stranded trisegment Brome mosaic bromovirus (BMV) was used to analyze the mechanism of homologous RNA recombination. Elements capable of forming strand-specific stem-loop structures were inserted at the modified 3 noncoding regions of BMV RNA3 and RNA2 in either positive or negative orientations, and various combinations of parental RNAs were tested for patterns of the accumulating recombinant RNA3 components. The structured negative-strand stem-loops that were inserted in both RNA3 and RNA2 reduced the accumulation of RNA3-RNA2 recombinants to a much higher extent than those in positive strands or the unstructured stem-loop inserts in either positive or negative strands. The use of only one parental RNA carrying the stem-loop insert reduced the accumulation of RNA3-RNA2 recombinants even further, but only when the stem-loops were in negative strands of RNA2. We assume that the presence of a stable stem-loop downstream of the landing site on the acceptor strand (negative RNA2) hampers the reattachment and reinitiation processes. Besides RNA3-RNA2 recombinants, the accumulation of nontargeted RNA3-RNA1 and RNA3-RNA3 recombinants were observed. Our results provide experimental evidence that homologous recombination between BMV RNAs more likely occurs during positive-rather than negativestrand synthesis.RNA recombination is a general phenomenon in animal, plant, and bacterial RNA viruses, and it plays an important role in the fitness and evolution of virus genomes (24,28,36). Model systems to study RNA recombination have been developed for the Brome mosaic bromovirus (BMV), coronaviruses, poliovirus, Turnip crinkle carmovirus (TCV), tombusviruses, and for RNA phage Q␤ (5, 6). Most of the RNA recombination events are thought to occur via a copy-choice mechanism. The evidence for participation of the replicase (RdRp) proteins in recombination has been provided for BMV (15)(16)(17)35). Other proposed mechanisms are cleavage/religation (25,28,45) and transesterification (10).According to a copy-choice model, prior to the actual switch the replicase stalls or dislodges from its template (21,28,36), which may occur at the 5Ј end of the template (47, 49), at a template break (39), or at the nascent strand stem-loop structure. The latter is analogous to rho-independent transcription termination (46, 50), although it may be distinct from a dislodging RdRp in a copy-choice model. Polymerase stalling may be induced by RNA secondary and/or tertiary structures, by homopolymer runs, or by the misincorporation of nucleotides (12,20,21,48,50).The success of the switch to the acceptor template can be influenced by a number of factors (5,6,23,26,28), such as (i) intermolecular RNA-RNA interactions (18, 31), (ii) intramolecular RNA-RNA interactions, or (iii) RNA-protein interactions (9, 36, 38). Altogether, RNA recombination appears to be a multistep process that involves primary and secondary structures, and the progeny recombinants are further selected on the basis of their fitness (43).The intersegmental...