Replication and translation of cowpea mosaic virus RNAs are tightly linked.

Abstract

The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules. B-RNA is able to replicate independently from M-RNA in cowpea protoplasts. Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans. Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only. Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis. This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA. In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors. Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA. Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay. These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked.