New overlapping gene encoded by the cucumber mosaic virus genome

Cucumber mosaic virus (CMV) contains three genomic RNAs (RNAs 1, 2, and 3) and a subgenomic RNA (RNA 4), a shared feature of the Bromoviridae family which includes cucumoviruses, bromoviruses, alfalfa mosaic virus, and ilarviruses. We describe in this paper the molecular characterization of a novel subgenomic RNA of the Q strain of CMV (Q-CMV), RNA 4A, which was first reported in 1973 (K. W. C. Peden and R. H. Symons, Virology 53, 487-492, 1973). RNA 4A is 682 nucleotides and is identical in sequence to the 3'-terminal 682 nucleotides of RNA 2. RNA 4A encodes a small open reading frame (ORF) of 100 codons, which, in RNA 2, overlaps the C-terminal portion of the major 2a gene; thus it is likely that RNA 4A functions as the mRNA for the in vivo expression of the ORF, called ORF 2b. Polyclonal antibodies raised against a 2b fusion protein expressed in Escherichia coli specifically detected the 2b gene product in Q-CMV-infected cucumber plants by Western immunoblotting. Examination of published viral RNA sequences revealed the conservation of ORF 2b in all of the four other cucumoviruses sequenced to date; however, it is absent from the rest of the Bromoviridae. We suggest that the proposed ORF 2b may be expressed in other cucumoviruses, most likely via 4A-like subgenomic RNAs, and that the predicted gene product may have a unique functional role in the infection process of cucumoviruses.