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

Abstract

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 Go 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 Go-arrested cells equalled the pools in actively dividing cells. These results suggested that post-transcriptional events controlled virus production in the Go-arrested cells.