1887

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 1814, 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-α (IFN-α) and by the the use of mutant 1820, a derivative of 1814 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 1820 was more impaired for replication than 1814 and behaved as if deficient for both Vmw65 and Vmw110. In cells pretreated with IFN-α and subsequently infected with 1820 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, 1820 was further modified by insertion of the gene controlled by the human cytomegalovirus enhancer (mutant 1883) or the HSV-1 immediate early Vmw110 promoter (1884). Expression of -galactosidase was not detected after infection of IFN--pretreated cells with 1883 or 1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. 1820-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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1995-06-01
2021-09-24
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