Mutation and Recombination Frequencies Reveal a Biological Contrast within Strains of Cucumber Mosaic Virus

Abstract

Recent in planta studies have shown that strains Fny and LS of Cucumber mosaic virus (CMV) display differential genetic diversities, Fny and LS having higher and lower mutation frequencies, respectively (J. S. Pita and M. J. Roossinck, J Virol 87:790 -797, 2012 http://dx.doi.org/10.1128/JVI.01891-12). In this article, we show that these virus strains have differential recombination frequencies as well. However, the high-diversity Fny strain is a low-recombination virus, whereas the very-low-diversity LS strain is instead a high-recombination virus. Unlike the mutation frequency that was determined by both RNAs 1 and 2, the control elements of recombination frequency reside predominantly within RNA 2, specifically within the 2a gene. IMPORTANCERecombination is an important mechanism in virus evolution that can lead to increased or decreased variation and is a major player in virus speciation events that can lead to emerging viruses. Although viral genomes show very frequent evidence of recombination, details of the mechanism involved in these events are still poorly understood. We show here that the reciprocal effects of high mutation frequency and low recombination frequency (and vice versa) involve the RNA-dependent RNA polymerase of the virus, and we speculate that these evolutionary events are related to differences in processivity for two strains of the same virus. G enetic recombination is the formation of chimeric molecules from segments previously separated on the same molecule or present on different parental molecules (1). RNA recombination is one of the major forces increasing diversity of RNA viruses. It has been well studied for many plant RNA viruses under natural or experimental conditions (for a recent review, see reference 2). In spite of extensive use of Cucumber mosaic virus (CMV) as a model system for virus evolution studies, not much about the role of recombination in the life cycle of CMV is known, other than a propensity for exchanges in the 3= end in reassorted viruses (3,4). CMV (genus Cucumovirus; family Bromoviridae) is an extremely successful virus that infects plants all around the world. The variety of available strains of CMV and the divided genome of the virus provide a useful and convenient system to study many general principles of virus evolution and ecology. CMV RNA 1 encodes the methyltransferase and helicase domains (1a), and RNA 2 encodes the polymerase (2a), together forming the viral components of the replicase complex (5, 6). RNA 2 also encodes the 2b protein (7) involved in RNA silencing suppression, systemic spread, and symptom production, and RNA 3 encodes the 3a (movement) and coat proteins (8, 9). Previously, we used the CMV system to elucidate the forces behind RNA virus population diversity. We identified the genes that are related to the different population diversity levels in different strains of CMV, and we mapped the regions that are associated with high and low levels of diversity (10). We used the same system to understand the mechanisms underlying t...

Impact of Cultivated Hosts on the Recombination of Cucumber Mosaic Virus

“…2). In tobacco, the calculated recombination frequency (22%) ( Table 1) ysis is in the same range as the recombination frequency previously calculated in N. benthamiana (19) and as that of Brome mosaic virus (BMV; Bromovirus) recombination frequency in quinoa (23). In pepper, we found an increase of LS-CMV recombination events, with 40.1% of the recovered population being recombinants.…”

“…In pepper, the two-site recombinants were positively selected over no-site recombinants, which were equivalent to the wild type. We cannot test selection directly in this system, but an unbalanced population structure was described previously in a different study in N. benthamiana, where the mutant LS-SpeI was selected over the mutant LS-BglII, although in that case the recombinant types were composed only of the regenerated wild type (19). The host role in controlling recombination events of (ϩ)ssRNA viruses has been studied using single-cell models.…”

“…Site-directed mutagenesis PCR with the primers XmnI-forward (GTTACCAGaACGGTTTCTACG) and XmnI-reverse (GGCACACAGCACTTGAGG) was used to introduce the XmnI restriction site by replacing guanine with adenine at position 387 (lowercase nucleotide on XmnI-forward primer) on the wild-type cDNA clone of LS-CMV, following a method described previously (31). The mutant LS-BglII was created and described in a previous study (19). The descriptions of the three mutants and their restriction sites are detailed in Fig.…”

“…The RT-PCR products were purified by electrophoresis on 5% polyacrylamide gels following the method previously described and cloned into the pGEM-T Easy vector (Promega). The transformed Escherichia coli DH5␣-competent cells were plated on LB agar, and positive clones were identified by colony PCR (19).…”

“…In a recent study, we calculated the recombination frequency of two CMV strains in wild tobacco (Nicotiana benthamiana) and showed that LS-CMV (subgroup II) has a high recombination frequency (28.2%), while recombination in strain Fny-CMV (subgroup I) was rare (1.85%). The same study identified RNA 2, and specifically the 2a gene, as controlling recombination in CMV (19).…”