Characterization of Kunjin virus RNA-dependent RNA polymerase: Reinitiation of synthesis in Vitro

Abstract

RNA-dependent RNA polymerase (RDRP) activity was characterized in a cytoplasmic extract of Kunjin virus-infected Vero cells at 24 hr. The activity was influenced, possibly indirectly, by the length of prior treatment of infected cells with actinomycin D; however, 6 μg/ml actinomycin D and 10 −5 M α-amanitin in the RDRP assay had no effect. The replication complex was membrane-bound and Mg 2+ was essential for RDRP activity. Incorporation was more dependent on exogenous UTP and GTP than ATP or CTP. The specific activity was low, and rate of incorporation of GMP decreased as the period of assay was increased; however, incorporation of label lasted for at least 60 min. RNA products were fractionated by LiCl precipitation, and kinetic studies showed that the sequence of accumulation of label was the same as that observed in vivo, viz., RI → RF → 44 S RNA; limited reinitiation was also observed. This sequence of labeling also indicated that the in vitro RDRP activity was due to an enzyme capable of elongation, release, and reinitiation of Kunjin RNA synthesis and not merely end labeling or elongating preexisting RNA molecules. No labeled bands in urea-polyacrylamide gels were observed using extracts from mock-infected cells and hence the three RNA products of assays were readily identified in a single gel. The replication complex was still active after treatment with nonionic detergent, but no labeled 44 S RNA was detected in gels, even in the presence of RNasin in the assay which inhibited some nuclease activity. Antibodies to flavivirus-specific nonstructural proteins were preincubated with infected cell extracts in the presence and absence of detergent but no inhibition of RDRP activity was observed. However, anti-dsRNA plus detergent blocked activity by as much as 78% and label was found only in RF.