Molecular characterization of the saguaro cactus virus RNA-dependent RNA polymerase and capsid protein

Abstract

Saguaro cactus virus (SCV) is a single-stranded RNA virus which belongs to the carmovirus genus within the family Tombusviridae. A full-length infectious clone of SCV has been generated and in this study was used to: (1) elucidate the role of the capsid protein (CP) in cell-to-cell and long distance movement, and (2) to better understand the various function(s) of the p26 and p86 proteins in viral replication. Analysis of a series of frameshift mutants and a deletion mutant has demonstrated that the CP is required for cell-to-cell movement in both Chenopodium amaranticolor and C. capitatum. This analysis also revealed a requirement of the CP coding region for viral replication in protoplasts. This is the first report of a cis-element, required for tombusvirus replication, which extends beyond the 3'-untranslated region into the CP coding region. The p26 and p86 constitute the putative SCV RNA-dependent RNA polymerase (RdRp). To better understand the structure and function of the RdRp, 16 clustered charged-to-alanine mutants were generated in the p26 and p86. The infectivity as well as the ability of each of these mutants to replicate in protoplasts was analyzed and compared to the infectivity and replication level of the wild type (pSCV15). Of the 16 mutants, five of them were nearly as infectious as wild type and were also able to replicate at near wild type levels. Four of the mutants consistently displayed a lower replication rate as determined by Northern analysis with two of these four demonstrating a lower level of infectivity on indicator plants. Two other mutants demonstrated a level of replication which was only able to be detected by RT-PCR. These mutants were not able to elicit the formation of local lesions on C. amaranticolor or induce symptoms on either inoculated or systemic leaves of C. capitatum. The ability of these mutants to synthesize negative-strand RNA, was examined. It was determined that all of the mutants which were able to produce positive-strand RNA were also able to synthesize negative strand RNA as determined by RT-PCR. Five of the mutants were not able to replicate in protoplasts and were not infectious on either host. These remaining five uninfectious mutants were also unable to replicate either negative or positive-strand RNA.