Quantification of newly synthesized virus RNA in Moloney murine leukaemia virus-infected cells

A technique for the isolation and characterization of newly transcribed murine leukaemia virus RNA in chronically infected cells has been developed. Cellular RNA was pulse labelled with ³H-uridine and virus-specific sequences were annealed with an excess of mercurated complementary DNA. Based on the affinity between mercurated cDNA and sulphydryl-Sepharose, the hybrid was specifically selected by affinity column chromatography. The specificity of this method was dependent on the purity of the cDNA and it was necessary to remove non-viral sequences from the cDNA in order to isolate virus-specific RNA. Between 0.5 and 0.8% of the labelled RNA in Moloney MuLV-infected rat cells and 1.5% of the labelled RNA in Moloney MuLV-infected NIH Swiss mouse cells were virus-specific. Using this methodology, the effect of the cell cycle on the transcriptional activity of proviral genes was investigated. Cultures of Moloney MuLV-infected rat cells arrested in G₀ phase of the cell cycle released reduced quantities of virus, but continued to synthesize virus RNA. The pools of virus RNA and p30 antigen in the G₀-arrested cells equalled the pools in actively dividing cells. These results suggested that post-transcriptional events controlled virus production in the G₀-arrested cells.