Exploring Different Mutations at a Single Amino Acid Position of Cucumber green mottle mosaic virus Replicase to Attain Stable Symptom Attenuation

Abstract

Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus (family Virgaviridae) that causes serious economic losses in cucurbit crops. A possibility for CGMMV control is the use of cross-protection, for which stable attenuated isolates are required. In this study, an infectious clone was constructed for the hn isolate of CGMMV. Unexpectedly, this clone carried a nonconserved mutation involving a single nucleotide change resulting in the replacement of Arg by Cys at residue 284 of the replicase protein; this mutation correlated with delayed symptom induction and RNA accumulation, as shown in time-course experiments. Sequencing of the viral progeny showed that restoration of wild-type symptoms and increased RNA accumulation correlated with reversion of the mutation to the wild-type sequence, a phenomenon that occurred at approximately 7 to 10 days postinoculation. Thus, Arg284 seems to be crucial but not strictly necessary for virus infection. Subsequently, four other mutants in the triplet encoding Arg284 were constructed and assayed. Results showed that symptoms and their timing were diverse for the different mutants, with enhanced pathogenicity and RNA accumulation always correlating with reversion to Arg284. Therefore, the nature of the mutation strongly influenced the genetic stability of the mutant. At least two mutants were identified for which reversion did not occur by 30 days postinoculation, and these were defined as good candidates to attain stable symptom attenuation that could be useful in cross-protection.