A conserved capsid protein surface domain of Cucumber mosaic virus is essential for efficient aphid vector transmission

Abstract

A prominent feature on the surfaces of virions of Cucumber mosaic virus (CMV) is a negatively charged loop structure (the beta H-beta I loop). Six of 8 amino acids in this capsid protein loop are highly conserved among strains of CMV and other cucumoviruses. Five of these amino acids were individually changed to alanine or lysine (an amino acid of opposite charge) to create nine mutants (the D191A, D191K, D192A, D192K, L194A, E195A, E195K, D197A, and D197K mutants). Transcripts of cDNA clones were infectious when they were mechanically inoculated onto tobacco, giving rise to symptoms of a mottle-mosaic typical of the wild-type virus (the D191A, D191K, D192A, E195A, E195K, and D197A mutants), a systemic necrosis (the D192K mutant), or an atypical chlorosis with necrotic flecking (the L194A mutant). The mutants formed virions and accumulated to wild-type levels, but eight of the nine mutants were defective in aphid vector transmission. The aspartate-to-lysine mutation at position 197 interfered with infection; the only recovered progeny (the D197K(*) mutant) harbored a second-site mutation (denoted by the asterisk) of alanine to glutamate at position 193, a proximal site in the beta H-beta I loop. Since the disruption of charged amino acid residues in the beta H-beta I loop reduces or eliminates vector transmissibility without grossly affecting infectivity or virion formation, we hypothesize that this sequence or structure has been conserved to facilitate aphid vector transmission.

FIG. 1.

Ribbon model of the CMV capsid protein and sites of mutagenesis. (A) The CMV capsid protein is presented as a ribbon highlighting the location of the βH-βI loop structure in the center and the bounding βB-βC and βD-βE loops. The carboxy and amino termini of the protein are indicated. The orientation is such that the surface of the virus would be at the top of the figure and the N-terminal alpha helix is directed downward toward the center of the virion. The positions of amino acids in the βH-βI loop that were mutagenized as part of this study are depicted as black spheres. (B) The amino acid sequence of the βH-βI loop, corresponding to positions 191 to 198 of the CMV-Fny capsid protein, is shown. The 5 amino acids that were mutagenized are underlined, and the amino acids to which they were changed are shown below the sequence.

FIG. 2.

Agarose gel electrophoresis of CMV mutants. (A) Results for a 1% agarose-Tris-acetate-EDTA gel at 4°C in which virions were subjected to electrophoresis followed by staining with ethidium bromide; (B) same gel after being dried and stained with Coomassie blue. The virus identifications at the bottom of the figure apply to corresponding lanes of both panels.