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  • Title: Quiescent viral genomes in human fibroblasts after infection with herpes simplex virus type 1 Vmw65 mutants.
    Author: Jamieson DR, Robinson LH, Daksis JI, Nicholl MJ, Preston CM.
    Journal: J Gen Virol; 1995 Jun; 76 ( Pt 6)():1417-31. PubMed ID: 7782770.
    Abstract:
    The development and utilization of a tissue culture system for the analysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 Vmw65 mutant in1814, which is impaired for immediate early (IE) transcription, was retained for many days in human fetal lung (HFL) fibroblasts in a quiescent 'latent' state. Molecular analysis of the viral genome was not possible, however, due to residual expression of IE proteins and consequent cytotoxicity at high m.o.i. In the study reported here, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the use of mutant in1820, a derivative of in1814 in which the Vmw110 promoter was replaced by the Moloney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer was not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture system to be developed in which all cells stably retained at least one quiescent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene controlled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of beta-galactosidase was not detected after infection of IFN-alpha-pretreated cells with in1883 or in1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-derived viruses were retained for at least 9 days and were not reactivated by subculture of cells. A regular arrangement of nucleosomes, as found in cellular chromatin, was not detected on the viral genome at the thymidine kinase locus. The non-linear genome was a template for reactivation with no requirement for prior conversion to a linear form. A small number of remaining linear genomes resulted from incomplete uncoating of input virus.
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