Mapping viral functional domains for genetic diversity in plants

Abstract

Cucumber mosaic virus (CMV) comprises numerous isolates with various levels of in-host diversity. Subgroup-distinctive features of the Fny and LS strains provided us with a platform to genetically map the viral control elements for genetic variation in planta. We found that both RNAs 1 and 2 controlled levels of genetic diversity, and further fine mapping revealed that the control elements of mutation frequency reside within the first 596 amino acids (aa) of RNA 1. The 2a/2b overlapping region of the 2a protein also contributed to control of viral genetic variation. Furthermore, the 3' nontranslated region (NTR) of RNA 3 constituted a hot spot of polymorphism, where the majority of fixed mutations found in the population were clustered. The 2b gene of CMV, a viral suppressor of gene silencing, controls the abundance of the fixed mutants in the viral population via a host-dependent mechanism.

Fig 1

Genomic organization of the three RNAs of Fny- and LS-CMV and schematic diagram of intermolecular recombinants and RNA 3 reporters. The lines represent noncoding regions, while the rectangles represent open reading frames. (A) L_{1 M}F_1H was constructed by exchanging sequences between RNAs 1 of Fny- and LS-CMV. The stippled and white rectangles indicate that the sequences are from LS- and Fny-CMV, respectively. The black rectangle represents a stretch of 22 nucleotides that are conserved between Fny-, LS-, and L_{1 M}F_1H. (B) F_2aLS_2b was constructed by replacing the 2b gene of Fny-CMV with that of LS-CMV. To create F_2aΔ2b with a deleted 2b ORF, all three AUGs that are found in the N terminus of the 2b protein were mutated. The dashed rectangles indicate that the 2b ORF is no longer encoding. (C) Portions of RNA 3 encoding the CP and flanking regions that were amplified and cloned for sequence analysis. The solid arrows indicate primer positions, and the numbers correspond to nucleotide positions.

Fig 2

Genetic variation in populations of parental and reassortant CMVs. The error bars correspond to the standard errors. HS, redundant mutations calculated as a mutational hot spot; F, redundant mutations calculated as fixed mutants.

Fig 3

Host factors determine the levels of genetic variation in CMV populations. The standard errors of the mean (error bars) of mutation frequencies, calculated irrespective of the viruses tested, emphasized the role of host factors in generating and maintaining viral genetic variations. HS, redundant mutations calculated as a mutational hot spot; F, redundant mutations calculated as fixed mutants; Nb, N. benthamiana; Tob, tobacco; Tom, tomato; Pep, pepper; Squ, squash.