Abstract
Red clover necrotic mosaic virus (RCNMV) is a member of the genus Dianthovirus and has a bipartite positive-sense genomic RNA with 3' ends that are not polyadenylated. In this study, we show that both genomic RNA1 and RNA2 lack a 5' cap structure and that uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to that for capped transcripts in cowpea protoplasts. Because the 5' cap and 3' poly(A) tail play important roles in the translation of many eukaryotic mRNAs, genomic RNAs of RCNMV should contain an element(s) responsible for 5' cap- and poly(A) tail-independent translation of viral protein. By using a luciferase reporter assay system in vivo, we showed that the 3' untranslated region (UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5' cap structure. Deletion studies revealed that the middle region (between nucleotides 3596 and 3732) in the 3' UTR, designated the 3' translation element of Dianthovirus RNA1 (3'TE-DR1), plays an important role in cap-independent translation. This region contained a stem-loop structure conserved among members of the genera Dianthovirus and LUTEOVIRUS: A five-base substitution in the loop abolished cap-independent translational activity, as reported for a luteovirus, indicating that this stem-loop is one of the functional structures in the 3'TE-DR1 involved in cap-independent translation. Finally, we suggest that cap-independent translational activity is required for RCNMV RNA1 replication in protoplasts.