Isolation and characterization of cucumber mosaic virus mutants defective in aphid vector transmission.

摘要

Cucumber mosaic virus (CMV) is transmitted by aphid vectors and the coat protein (CP) is a primary determinant of transmission. The stability of the aphid transmission phenotype of transmissible field isolates of CMV was evaluated using Aphis gossypii and Myzus persicae. All isolates were transmitted, with A. gossypii generally showing a higher efficiency of transmission than M. persicae. A spontaneous mutant, 2A1-MT-60x was obtained by repeated mechanical passaging of the parent, 2A1-AT. This mutant was poorly transmissible by A. gossypii and not transmissible by M. persicae. The mutant phenotype was conferred by an encoded amino acid change of alanine to threonine at position 162 of the CP. The physical basis for the defect in transmission was hypothesized to be virion instability. A urea disruption assay was developed to test for a correlation between changes in virion stability and defects in aphid transmission. In this assay, virions of transmissible strains 2A1-AT and CMV-Fny were more stable than transmission-defective mutants 2A1-MT-60x and CMV-M, consistent with the hypothesis that the basis for the defective transmission in some CMV strains was a reduction in virion stability. Unlike 2A1-MT-60x, full restoration of aphid transmissibility in CMV-M was shown to require the modification of CP amino acids at positions 129, 162 and 168 (Perry et al., 1998). The study on CMV-M was extended to determine if amino acid changes at these positions also increased virion stability as determined by urea disruption and infectivity assays. Virions with one (position 162) and two (positions 129 and 162) CP amino acid modifications exhibited intermediate levels of aphid (A. gossypii) transmissibility, stability and infectivity. Virions with modifications at all three amino acid positions showed wild-type levels of virion stability and aphid ( A. gossypii) transmission. Transmission-defective CMVs, with engineered modifications in a surface loop of the CMV-Fny CP, exhibited near wild-type levels of stability in the urea disruption assays. These studies suggest that, in these mutants, the basis for the defect in transmission is a change in virion surface properties rather than a reduction in virion stability.