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Structure of the murine cytomegalovirus genome and its expression in productive and non-productive infections

University of British Columbia

The purpose of this investigation was to examine the structure of the murine cytomegalovirus (MCMV) genome and to study its expression during productive and non-productive infections caused by the virus. The kinetic complexity of MCMV DNA was not less than its molecular weight implying the absence of major reiterations. The restriction endonuclease EcoR^ cleaved this molecule into twenty-five fragments, which were present in the digest in equimolar amounts and ranged in molecular weights from 20 to 1 million. The sum of the molecular weights of the fragments was 136 million. The genomes of the 'K 181' and 'Smith' strains of MCMV appeared to share more than 99 percent of their sequences, although the DNAs exhibited slightly different fragmentation patterns when treated with EcoR^ and Hind III endonucleases. Control was exerted on the transcription of the MCMV genome at temporal, quantitative, and processing levels. During productive infections, approximately 25 percent of the genome was represented as stable transcripts in the cell at 6 hours post infection, i.e., before the onset of viral DNA synthesis, whereas RNA transcribed from 35 to 40 percent of the DNA was present in the cells in the later stages of infection. RNA sequences corresponding to 6 h (early) transcripts would be detected in the cell throughout the infectious cycle. Both 'early' and 'late' RNA comprised two RNA classes differing about 7 to 10 fold in concentration. Viral DNA synthesis in the host cell was required for the expression of 'late' genes since in the presence of inhibitors of protein and DNA synthesis only 'early' transcription occurred. Control was also exerted on the transport of transcripts from the nucleus to the cytoplasm of infected cells. Although RNA extracted from the nuclei of infected cells arose from 25 (early) and 35 (late) percent of the viral genome, transcripts from only 11 (early) and 15 (late) percent of the DNA were detected in the cytoplasm. Cells of mouse origin (3T3.cells), arrested in the G^ phase of the cell cycle, retained the viral genome in a non-replicating state, but could be induced to enter the lytic cycle by serum activation. Transcripts from 19 percent of the genome were observed in G^ arrested, MCMV-infected cells. Viral RNA in these cells comprised only one abundance class, which was similar to the scarce class in 'early' RNA from infected exponentially growing cells. Some evidence was also obtained for the transmission of latent MCMV genomes from mother to progeny. Cells cultured from embryos of infected mice did not normally produce infectious virus. However, the presence of the virus, at least in some of these cells, could be demonstrated by immunofluorescence, and by in-situ hybridization, using iodinated MCMV DNA as probe.

Text

2010-02-19

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http://hdl.handle.net/2429/20555

Microbiology and Immunology

University of British Columbia

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