Functional analysis of brome mosaic virus coat protein RNA-interacting domains |
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Authors: | S L Calhoun A L N Rao |
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Institution: | (1) Department of Plant Pathology, University of California, Riverside, CA, U.S.A. |
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Abstract: | Summary The coat proteins (CP) of cowpea chlorotic mottle (CCMV) and brome mosaic virus (BMV), two members of the genus Bromovirus, share 70% identity at the amino acid (aa) level and contain four highly conserved regions, identified as putative RNA-interacting
domains (RIDs). To assess the contribution of the conserved aa sequence within each RID and the structural features contained
therein toward virion assembly and RNA packaging, we engineered a set of fourteen independent mutations (deletions and substitutions)
encompassing all four RIDs. The effect of each mutation on viral biology, pathogenesis, and RNA packaging was analyzed in
whole-plant infection assays. Among the four RIDs, two mutations engineered into the N-proximal domain (RID I) and two of
the four mutations engineered into the C-proximal domain (RID IV) proved to be more debilitating (compared to wild-type) while
only selected regions in the central domains (RID II or III) showed a detectable effect. Neutral effects were observed when
aa residues that are predicted to affect calcium binding were mutated. To further analyze the importance of N and C terminal
interactions leading to virus assembly and RNA packaging, four CP hybrids were constructed by precisely exchanging either
the N-terminal 77 or the C-terminal 113/112aa between BMV and CCMV. Despite the fact that the CP composition of the hybrid
viruses is distinct from either of the parents, the symptom phenotype in Chenopodium quinoa, migration pattern of CP in Western blots and virion mobility in agarose gels was indistinguishable from the respective parent
providing the genetic background. Collectively, the data provide insight for assessing the relative importance of each RID
during genome packaging and in molecular processes regulating the overall architecture of the assembled virions.
Correspondence: A. L. N. Rao, Department of Plant Pathology, University of California, Riverside, CA 92521-0122, U.S.A. |
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