ANALYSIS OF RNA REPLICASE COMPONENTS OF DIANTHOVIRUS

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Infectious cDNA clones of genomic RNAs 1 and 2 of Red clover necrotic mosaic virus Canadian strain (RCNMV-Can) with temperature sensitive (ts) phenotype were constructed and the nucleotide sequences were determined. Experiments with reassortants and chimeric RNA1s between ts-insensitive RCNMV-Aus and RCNMV-Can indicated that the temperature sensitivity of RCNMV-Can is in an early stage of RNA replication and that viral determinants for the ts phenotype are located in the 3'-untranslated region of RCNMV-Can RNA1.RCNMV infection-specific RNA dependent RNA polymerase (RdRp) was effectively prepared from virus-infected protoplasts. Results from in vitro RNA synthesis using the membrane bound RdRp and its solubilized fraction suggested that the ts phenotype of RCNMV-Can was not due to defect in the elongation steps of RNA synthesis but was due to initiation steps including negative-strand RNA.Polyclonal antibody that specifically reacts with both 27 kD and 88 kD replicase component proteins of RCNMV was obtained by using a Histidine tag-fusion polypeptides of 27 kD produced in E.coli. We failed to produce other antibodies reacting with other regions of 88 kD replicase component because of unstable expression of virus fusion proteins in E.coli.