ANALYSIS OF CIS-ACTING ELEMENTS REQUIRED FOR REPLICATION OF BARLEY STRIPE MOSAIC-VIRUS RNAS

The replicative abilities of mutant RNA transcripts derived from barle y stripe mosaic virus cDNA clones were investigated in barley protopla sts that had been coinoculated with wild-type RNA alpha and -gamma tra nscripts. The 5' and 3' noncoding regions were required for replicatio n, and lack of a 5' cap structure (GpppG) reduced the replicative abil ity substantially. All internal deletions within RNA alpha abrogated r eplication in trans. A 2-base change that produced a truncated alpha a protein lacking the first 16 amino acids also compromised the ability of RNA alpha to be replicated. In contrast, RNA beta transcripts cont aining deletions involving each ORF and the downstream poly(A) tract w ere effectively amplified by RNAs alpha and gamma, but collective dele tion of all four ORFs drastically reduced accumulation. The intergenic region between beta a and beta b was not absolutely required for repl ication, but small deletions within this region reduced the abundance of RNA beta by at least 10-fold. Deletions within the first 507 nt of the gamma a ORF abrogated replication. However, transcripts containing deletions within the central and 3' regions of the gamma a ORF, the g amma a-gamma b intergenic region, and the gamma b ORF could be amplifi ed in trans. Two mutants containing extensive deletions encompassing t he central region of the gamma a ORF and most of gamma b behaved like defective interfering RNAs because they multiplied to high levels in t rans and caused a pronounced reduction in accumulation of the coinocul ated wild-type RNAs alpha and gamma. (C) 1996 Academic Press, Inc.