cis-acting sequences required for in vivo amplification of genomic RNA3 are organized differently in related bromoviruses

Abstract

Cowpea chlorotic mottle virus (CCMV) is a positive-strand RNA virus that infects dicotyledonous plants. The genome comprises three capped RNAs: RNA1 (3.2 kb), RNA2 (2.9 kb), and RNA3 (2.1 kb). cis-Acting sequences required for amplification in vivo were explored for RNA3, which does not contribute trans-acting factors to viral RNA replication. Using a CCMV cDNA expression system, deletions throughout RNA3 were constructed and tested for successful replication in barley protoplasts coinoculated with RNAs 1 and 2. As previously found for RNA3 of the related brome mosaic virus (BMV) (R. French and P. Ahlquist, 1987, J. Virol. 61, 1457-1465), either of the two coding regions can be individually deleted without blocking RNA3 amplification. However, in striking contrast to BMV, the entire intercistronic noncoding region separating these genes is also dispensable for CCMV RNA3 amplification. Moreover, although simultaneous deletions of the 3a and coat protein genes were deleterious for BMV RNA3 accumulation, CCMV RNA3 derivatives bearing larger deletions encompassing the 3a gene, intercistronic region, and coat protein gene amplify to high levels. Thus, unlike BMV RNA3, cis-acting sequences required for CCMV RNA3 amplification map solely in the 5' and 3' noncoding regions. Normal levels of CCMV RNA3 accumulation require over 125 but no more than 220 bases from the 3' noncoding region, and no more than the first 89 bases of the 238-base-long 5' noncoding region.