In vitro transcription by cytoplasmic extracts from cells infected with African swine fever virus

Abstract

A cell-free system for the study of transcription of African swine fever virus (ASFV) mRNA was developed from cytoplasmic extracts of infected cells permeabilized with lysolecithin. Extracts prepared from infected cells early and late after infection incorporated [α- 32P]UTP into acid-insoluble material that was resistant to DNase and sensitive to RNase. The incorporation was inhibited by actinomycin D but not by a-amanitin. The presence of the nuclei was not required. In vitro transcription was optimal at pH 7.9 and at concentrations of 100 m M NH 4Ck, 5 m M magnesium acetate, and 250 μ M MnCl 2. Early infected cell extracts transcribed from endogenous viral DNA a set of RNAs similar in electrophoretic migration to that observed in intact infected cells. Late infected cell extracts seemed to be unable to transcribe new RNA species besides those transcribed early after infection. The activity of the extracts could be made dependent on exogenous templates by digestion with micrococcal nuclease. RNAs transcribed after addition of native or denatured viral DNA to nuclease-treated extracts were indistinguishable from those transcribed from endogenous viral DNA. Late infected cell extracts digested with micrococcal nuclease were also active in transcribing virus-specific RNA from p2SB21, a recombinant plasmid containing the Sall B fragment of ASFV DNA.