Replicase and replication of red clover necrotic mosaic virus Photographs and illustrations can be viewed by clicking the highlited texts

Replicase and Replication of Red Clover Necrotic Mosaic Virus

Zhongguo Xiong
Department of Plant Pathology, University of Arizona

Red clover necrotic mosaic virus has small, icosahedral particles. It infects mainly solanaceous and legume plants, producing necrotic and mosaic type symptoms. RCNMV is a member of the Dianthovirus genus.

The genome of RCNMV is composed of positive-sense, single-stranded RNA-1 (3.9 kb) and RNA-2 (1.5 kb) . RCNMV genome organization and gene expression strategies have been well characterized by in vitro translation studies , sequencing analysis, and mutagenesis. RCNMV RNA-2 encodes a single p35 movement protein that is required for RCNMV cell-to-cell movement through plasmodesmata. RCNMV RNA-1 contains three open reading frames. They encode, from 5' to 3', p27, p57, and the p37 capsid protein. The first two ORFs (p27 and p57) slightly overlap with each other. A translational ribosomal frameshifting event, similar to that observed in retroviruses, occurs in the overlapping region to generate a p88 fusion protein. This fusion protein consists of the complete p27 as the N-terminal half and p57 as the C-terminal half.

As with other RNA viruses, RCNMV is replicated by an RNA-depedent RNA polymerase (replicase). An active RCNMV replicase has been partially purified. The purified replicase is capable of producing double-stranded RNA products corresponding to RCNMV RNA-1 and RNA-2 in the absence of exogenous templates. The endogenous templates appear tightly bound to the replicase. The double-stranded nature of the replicase products was confirmed by RNase A digestion. The replicase products are double-stranded and are susceptible to RNase A in a low salt solution but are resistant to RNase A in high salt solution. In a denaturing agarose gel, the replicase products behave as full-length genomic RNA and co-migrate with RCNMV RNAs. Northern blot analysis shows that the replicase products are exclusively (+) stranded, suggesting that the replicase-bound template are (-)-strand.

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