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Several different families of DNA viruses encode proteins that inactivate the cellular retinoblastoma tumor suppressor protein (pRb), which normally functions to bind E2F transcription factors and restrict expression of genes necessary for cellular processes including DNA replication. Human cytomegalovirus (HCMV) UL97, a protein kinase functionally orthologous to cellular cyclin-dependent kinases, phosphorylates pRb on inactivating residues during HCMV infection. To assess if such phosphorylation is biologically relevant, we tested whether the human papillomavirus type 16 E7 protein, which inactivates pRb family proteins by direct binding and destabilization, could substitute for UL97 during HCMV infection. In the absence of UL97, expression of wild-type E7 protein, but not a mutant E7 unable to bind pRb family proteins, restored E2F-responsive cellular gene expression, late viral gene expression, and viral DNA synthesis to levels normally observed during wild-type virus infection of quiescent cells. UL97-null mutants exhibited more pronounced defects in virus production and DNA synthesis in quiescent cells as compared to serum-fed, cycling cells. E7 expression substantially enhanced infectious virus production in quiescent cells, but did not complement the defects observed during UL97-null virus infection of cycling cells. Thus, a primary role of UL97 is to inactivate pRb family proteins during infection of quiescent cells, and this inactivation likely abets virus replication by induction of cellular E2F-responsive genes. Our findings have implications for human cytomegalovirus disease and for drugs that target UL97. E2F | cyclin-dependent kinase | cell cycle | antiviral drugs | retinoblastoma protein

Many DNA-interacting proteins diffuse on DNA to perform their biochemical functions. Processivity factors diffuse on DNA to permit unimpeded elongation by their associated DNA polymerases, but little is known regarding their rates and mechanisms of diffusion. The processivity factor of herpes simplex virus DNA polymerase, UL42, unlike 'sliding clamp' processivity factors that normally form rings around DNA, binds DNA directly and tightly as a monomer, but can still diffuse on DNA. To investigate the mechanism of UL42 diffusion on DNA, we examined the effects of salt concentration on diffusion coefficient. Ensemble studies, employing electrophoretic mobility shift assays on relatively short DNAs, showed that off-rates of UL42 from DNA depended on DNA length at higher but not lower salt concentrations, consistent with the diffusion coefficient being salt-dependent. Direct assays of the motion of single fluorescently labeled UL42 molecules along DNA revealed increased diffusion at higher salt concentrations. Remarkably, the diffusion coefficients observed in these assays were [approximately equal to] [10.sup.4]-fold higher than those calculated from ensemble experiments. Discrepancies between the single-molecule and ensemble results were resolved by the observation, in single-molecule experiments, that UL42 releases relatively slowly from the ends of DNA in a salt-dependent manner. The results indicate that UL42 'hops' rather than 'slides,' i.e., it microscopically dissociates from and reassociates with DNA as it diffuses rather than remaining so intimately associated with DNA that cation condensation on the phosphate backbone does not affect its motion. These findings may be relevant to mechanisms of other processivity factors and DNA-binding proteins. herpes simplex virus | linear diffusion

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

We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion in the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5' cap or had a stable stem loop 5' of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-elF2-GTP/Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the single-base deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs. drug resistance | pathogenesis | proteomics | translation | acyclovir

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.

Circular dichroism spectroscopy and analytical centrifugation techniques help model polymerase heterodimerization-inhibiting antiviral drugs and indicate the necessity of a hairpin-like structure formed by the interaction of two distinct alpha-helical regions for inhibitory activity. The inhibitors block DNA synthesis that depends UL42, an accessory subunit of herpes simplex virus DNA polymerase. The catalytic subunit-dependent synthesis is unaffected by these antiviral drugs.

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.

Herpesviruses require a nuclear egress complex (NEC) for efficient transit of nucleocapsids from the nucleus to the cytoplasm. The NEC orchestrates multiple steps during herpesvirus nuclear egress, including disruption of nuclear lamina and particle budding through the inner nuclear membrane. In the important human pathogen human cytomegalovirus (HCMV), this complex consists of nuclear membrane protein UL50, and nucleoplasmic protein UL53, which is recruited to the nuclear membrane through its interaction with UL50. Here, we present an NMR-determined solution-state structure of the murine CMV homolog of UL50 (M50; residues 1-168) with a strikingly intricate protein fold that is matched by no other known protein folds in its entirety. Using NMR methods, we mapped the interaction of M50 with a highly conserved UL53-derived peptide, corresponding to a segment that is required for heterodimerization. The UL53 peptide binding site mapped onto an M50 surface groove, which harbors a large cavity. Point mutations of UL50 residues corresponding to surface residues in the characterized M50 heterodimerization interface substantially decreased UL50-UL53 binding in vitro, eliminated UL50-UL53 colocalization, prevented disruption of nuclear lamina, and halted productive virus replication in HCMV-infected cells. Our results provide detailed structural information on a key protein-protein interaction involved in nuclear egress and suggest that NEC subunit interactions can be an attractive drug target. [ABSTRACT FROM AUTHOR]

Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3'-hydroxyl group. Certain HCMV GCV resistance (GCVr) mutations, including ones associated with treatment failures, result in substitutions in the 3'-5' exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCVr Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides ("idling"). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance. [ABSTRACT FROM AUTHOR]

Ribosomal frameshifting entails slippage of the translational machinery during elongation. Frameshifting permits expression of more than one polypeptide from an otherwise monocistronic mRNA, and can restore expression of polypeptides in the face of frameshift mutations. A common mutation conferring acyclovir resistance in patients with herpes simplex virus disease deletes one cytosine from a run of six cytosines (C-chord) in the viral thymidine kinase (tk) gene. However, this mutation does not abolish TK activity, which is important for pathogenicity. To investigate how this mutant retains TK activity, we engineered and analyzed viruses expressing epitope-tagged TK. We found that the mutant's TK activity can be accounted for by low levels of full-length TK polypeptide produced by net-1 frameshifting during translation. The efficiency of frameshifting was relatively high, 3-5%, as the polypeptide from the reading frame generated by the deletion, which lacks stop codons (nonstop), was poorly expressed mainly because of inefficient protein synthesis. Stop codons introduced into this reading frame greatly increased its expression, but greatly decreased the level of full-length TK, indicating that frameshifting is strongly stimulated by a new mechanism, nonstop mRNA, which we hypothesize involves stalling of ribosomes on the polyA tail. Mutational studies indicated that frameshifting occurs on or near the C-chord, a region lacking a canonical slippery sequence. Nonstop stimulation of frameshifting also occurred when the C-chord was replaced with a canonical slippery sequence from HIV. This mechanism thus permits biologically and clinically relevant TK synthesis, and may occur more generally. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many antiviral and anticancer drugs are nucleoside analogs that target polymerases and cause DNA chain termination. Interestingly, ganciclovir (GCV), the first line of therapy for human cytomegalovirus (HCMV) infections, induces chain termination despite containing the equivalent of a 3'-hydroxyl group. Certain HCMV GCV resistance (GCV(r)) mutations, including ones associated with treatment failures, result in substitutions in the 3'-5' exonuclease (Exo) domain of the catalytic subunit of the viral DNA polymerase (Pol). To investigate how these mutations confer resistance, we overexpressed and purified wild-type (WT) HCMV Pol and three GCV(r) Exo mutants. Kinetic studies provided little support for resistance being due to effects on Pol binding or incorporation of GCV-triphosphate. The mutants were defective for Exo activity on all primer templates tested, including those with primers terminating with GCV, arguing against the mutations increasing excision of the incorporated drug. However, although the WT enzyme terminated DNA synthesis after incorporation of GCV-triphosphate and an additional nucleotide (N+1), the Exo mutants could efficiently synthesize DNA to the end of such primer templates. Notably, the Exo activity of WT Pol rapidly and efficiently degraded N+2 primer templates to N+1 products that were not further degraded. On N+1 primer templates, WT Pol, much more than the Exo mutants, converted the incoming deoxynucleoside triphosphate to its monophosphate, indicative of rapid addition and removal of incorporated nucleotides ("idling"). These results explain how GCV induces chain termination and elucidate a previously unidentified mechanism of antiviral drug resistance.