QUIESCENT VIRAL GENOMES IN HUMAN FIBROBLASTS AFTER INFECTION WITH HERPES-SIMPLEX VIRUS TYPE-1 VMW65 MUTANTS

The development and utilization of a tissue culture system for the ana lysis of quiescent, nonreplicating herpes simplex virus type 1 (HSV-1) genomes is described. It was demonstrated previously that the HSV-1 V mw65 mutant in1814, which is impaired for immediate early (IE) transcr iption, was retained for many days in human fetal lung (HFL) fibroblas ts in a quiescent 'latent' state. Molecular analysis of the viral geno me was not possible, however, due to residual expression of IE protein s and consequent cytotoxicity at high m.o.i. In the study reported her e, IE transcription was reduced further by pretreatment of cells with interferon-alpha (IFN-alpha) and by the the use of mutant in1820, a de rivative of in1814 in which the Vmw110 promoter was replaced by the Mo loney murine leukaemia virus (Momulv) enhancer. The Momulv enhancer wa s not expressed under IE conditions; thus in1820 was more impaired for replication than in1814 and behaved as if deficient for both Vmw65 an d Vmw110. In cells pretreated with IFN-alpha and subsequently infected with in1820 cytotoxicity was overcome, enabling a tissue culture syst em to be developed in which all cells stably retained at least one qui escent viral genome. To assist the analysis of gene expression, in1820 was further modified by insertion of the Escherichia coli lacZ gene c ontrolled by the human cytomegalovirus enhancer (mutant in1883) or the HSV-1 immediate early Vmw110 promoter (in1884). Expression of beta-ga lactosidase was not detected after infection of IFN-alpha-pretreated c ells with in 1883 or in 1884 but could be induced in almost all cells containing a viral genome, by superinfection of cultures. In1820-deriv ed 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 th ymidine kinase locus. The non-linear genome was a template for reactiv ation with no requirement for prior conversion to a linear form. A sma ll number of remaining linear genomes resulted from incomplete uncoati ng of input virus.