Analysis of cis-acting elements required for replication of barley stripe mosaic virus RNAs

Abstract

The replicative abilities of mutant RNA transcripts derived from barley stripe mosaic virus cDNA clones were investigated in barley protoplasts that had been coinoculated with wild-type RNAα and -γ transcripts. The 5' and 3' noncoding regions were required for replication, and lack of a 5' cap structure (GpppG) reduced the replicative ability substantially. All internal deletions within RNAα abrogated replication in trans. A 2-base change that produced a truncated αa protein lacking the first 16 amino acids also compromised the ability of RNAα to be replicated. In contrast, RNAβ transcripts containing deletions involving each ORF and the downstream poly(A) tract were effectively amplified by RNAs α and γ, but collective deletion of all four ORFs drastically reduced accumulation. The intergenic region between βa and βb was not absolutely required for replication, but small deletions within this region reduced the abundance of RNAβ by at least 10-fold. Deletions within the first 507 nt of the γa ORF abrogated replication. However, transcripts containing deletions within the central and 3' regions of the γa ORF, the γa-γb intergenic region, and the γb ORF could be amplified in trans. Two mutants containing extensive deletions encompassing the central region of the γa ORF and most of γb behaved like defective interfering RNAs because they multiplied to high levels in trans and caused a pronounced reduction in accumulation of the coinoculated wild-type RNAs α and γ.