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Herpes simplex virus (HSV) persists in its human host and evades the immune response by undergoing a latent infection in sensory neurons, from which it can reactivate periodically. HSV expresses >80 gene products during productive ('lytic') infection, but only the latency-associated transcript (LAT) gene is expressed at abundant levels during latent infection. The LAT gene has been shown to repress lytic-gene expression in sensory neurons. In this study, we use chromatin immunoprecipitation to show that HSV lytic-gene promoters become complexed with modified histones associated with heterochromatin during the course of establishment of latent infection. Experiments comparing LAT-negative and LAT-positive viruses show that a function encoded by the LAT gene increases the amount of dimethyl lysine 9 form of histone H3 or heterochromatin and reduces the amount of dimethyl lysine 4 form of histone H3, a part of active chromatin, on viral lytic-gene promoters. Thus, HSV, and in particular the HSV LAT gene, may manipulate the cellular histone modification machinery to repress its lytic-gene expression and contribute to the persistence of its genome in a quiescent form in sensory neurons. chromatin modification | gene regulation | herpes simplex virus

After herpesviruses encapsidate their genomes in replication compartments (RCs) within the nuclear interior, capsids migrate to the inner nuclear membrane (INM) for nuclear egress. For human cytomegalovirus (HCMV), capsid migration depends at least in part on nuclear myosin Va. It has been reported for certain herpesviruses that the nucleoplasmic subunit of the viral nuclear egress complex (NEC) is important for this migration. To address whether this is true for HCMV, we used mass spectrometry and multiple other methods to investigate associations among the HCMV NEC nucleoplasmic subunit, UL53, myosin Va, major capsid protein, and/or capsids. We also generated complementing cells to derive and test HCMV mutants null for UL53 or the INM NEC subunit, UL50, for their importance for these associations and, using electron microscopy, for intranuclear distribution of capsids. We found modest associations among the proteins tested, which were enhanced in the absence of UL50. However, we found no role for UL53 in the interactions of myosin Va with capsids or the percentage of capsids outside RC-like inclusions in the nucleus. Thus, UL53 associates somewhat with myosin Va and capsids, but, contrary to reports regarding its homologs in other herpesviruses, is not important for migration of capsids towards the INM. [ABSTRACT FROM AUTHOR]

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among [Alpha]-like DNA polymerases. We measured [K.sub.m] and [k.sub.cat] values for dGTP and ACV triphosphate incorporation and [K.sub.i] values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced [k.sub.cat] values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered [k.sub.m] and [k.sub.cat] values. In most cases, the fold increase in [K.sub.i] of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in [K.sub.i]. The effects of mutations on enzyme kinetics could be explained by using a model of an [Alpha]-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.

Participatory sequences in the herpes simplex virus thymidine kinase (HSV tk) recoding are determined in a herpesvirus mutant. The findings demonstrate a novel mechanism that relies on the capacity of the G-rich sequences to generate unique inter- or intramolecular structures. Specifically, the sole influence of G-strings to activate a net +1 recoding in cultured cells is determined. On the other hand, mediation of recoding by the G-string structure inside the ribosome is proposed to proceed either by the Hoogsteen or non-Watson-Crick interactions.

A net + 1 frameshift facilitates the formation of full length thymidine kinase (TK) from a herpes simple virus mutant which is resistant to the drug acyclovir. A single base insertion in the tk gene is present in the acyclovir resistant clinical mutant which is examined. Translation of mutant tk genes and in vitro transcription generate full length and truncated polypeptides.

Changes in the catalytic subunit of herpes simplex virus DNA polymerase (HSV Pol) were investigated. The fluorescence spectroscopic and proteolytic analyses provided evidence for conformational changes in HSV Pol following binding to DNA. The locations of tryptic cleavage near sites implicated in substrate recognition and interaction suggest that the conformational changes are important for catalysis and processivity.