Studies on the mechanisms of cucumber mosaic virus RNA replication

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Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) belong to the alpha-like positive-strand RNA virus group. In this study, we compared the mechanisms of viral RNA replication between the two viruses. First, we compared the subcellular fractionation pattern of the replication proteins using the membrane-flotation method. Both for CMV and TMV, the replication proteins were fractionated into the tonoplast-rich fraction, endoplasmic reticulum-rich fraction and the soluble fraction. Second, we found that addition of purified vacuolar membranes enhanced the replication of TMV and CMV RNAs in cell-free extracts of evacuolated tobacco BY-2 protoplasts. These results suggest the involvement of vacuolar membranes in the replication of CMV and TMV RNAs. Lastly, we explored host proteins associated with the replication complex of CMV. To do this, we constructed a replication-competent CMV derivative that carried a FLAG-tagged replication protein. We purified membranes from BY-2 protoplasts infected with this CMV derivative, solubilized the membranes that contained the CMV replication complexes with a detergent, and purified the FLAG-tagged replication protein with anti-FLAG antibody-conjugated agarose beads. The co-purified proteins were separated with SDS-PAGE and detected by silver staining. Although the results are still preliminary, several specific bands were detected in the FLAG-purified fraction from protoplasts infected with the tagged CMV derivative. Interestingly, the pattern of co-purified host protein was different from those co-purified with FLAG-tagged TMV replication proteins. This result suggests CMV and TMV utilize different sets of host factors for RNA replication.