In vitro transcription catalyzed by heat-treated human rotavirus

Abstract

The in-vitro characteristics of human rotavirus transcriptions have been examined. The virus has an associated RNA polymerase activity which was activated after a heat-shock treatment. The enzyme required the presence of the four ribonucleoside triphosphates and a divalent cation (Mg2+), and it required an optimum pH of 8.5. The polymerase was activated by monovalent salts and inhibited by Na PP(i). The addition of actinomycin D, α-amanitin, or rifampin did not inhibit the polymerase activity. After thermal shock of the virus, at least eight different RNA species were synthesized which may correspond to independent transcripts. Transcription also requires a hydrolyzable form of ATP. Analogs such as β,γ-methylene ATP were inhibitory, whereas others, such as the β,γ-imido or methylene analogs of CTP, UTP, or GTP, were not inhibitory. This suggests that ATP is related to reactions other than polymerization, probably to initiation or elongation of RNA molecules, as has been described for vesicular stomatitis virus or vaccinia virus