A herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells.

Herpes simplex virus encodes a ribonucleotide reductase that is not essential for virus growth in dividing cells at 37 degrees. This enzyme has been proposed as a target for antiviral drugs; its utility in this regard could depend upon its importance in vivo. To test the requirement of viral ribonucleotide reductase in a mammalian host, we tested a mutant virus, lacking most of the gene encoding the ribonucleotide reductase large subunit, in a mouse eye model of pathogenesis and latency where the wild-type virus establishes reactivatable latent infections in trigeminal ganglia following corneal inoculation.

Examination of the roles of transcription factor Sp1-binding sites and an octamer motif in trans induction of the herpes simplex virus thymidine kinase gene.

Herpes simplex virus mutants with both Sp1-binding sites in the thymidine kinase (tk) promoter inactivated or an octamer motif deleted were at most modestly impaired for tk expression. Thus, no cellular transcription factor that binds upstream of the tk TATA box is solely required for trans induction of this gene.

Herpes simplex virus ribonucleotide reductase mutants are hypersensitive to acyclovir.

Two mutants defective in herpes simplex virus-encoded ribonucleotide reductase activity exhibited the novel phenotype of hypersensitivity to acyclovir, aphidicolin, and to a lesser extent, phosphonoacetic acid. These results have implications for acyclovir resistance and the development of drugs that potentiate acyclovir action by inhibition of viral ribonucleotide reductase.

A deletion mutant of the latency-associated transcript of herpes simplex virus type 1 reactivates from the latent state with reduced frequency.

We have generated and characterized a deletion mutant of herpes simplex virus type-1, dlLAT1.8, which lacks the putative promoter region, transcriptional start site, and 1,015 base pairs of the DNA sequences specifying the latency-associated transcripts (LATs). When tested in a CD-1 mouse ocular model, dlLAT1.

Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate.

Herpes simplex virus infection of mammalian hosts involves lytic replication at a primary site, such as the cornea, translocation by axonal transport to sensory ganglia and replication, and latent infection at a secondary site, ganglionic neurons. The virus-encoded thymidine kinase, which is a target for antiviral drugs such as acyclovir, is not essential for lytic replication yet evidently is required at the secondary site for replication and some phase of latent infection. To determine the specific stage in viral pathogenesis at which this enzyme is required, we constructed virus deletion mutants that were acyclovir resistant and exhibited no detectable thymidine kinase activity.

A conserved open reading frame that overlaps the herpes simplex virus thymidine kinase gene is important for viral growth in cell culture.

Of 18 mutants containing clustered point mutations within UL24 (an open reading frame that overlaps the herpes simplex virus thymidine kinase gene on the opposite strand), 15 formed small plaques and were substantially impaired for virus growth in cell culture. Mutations conferring the small plaque phenotype disrupt regions of UL24 that share considerable sequence similarity with open reading frames common to herpesviruses of mammals and birds. We infer that UL24 is expressed and important for virus growth in cell culture and suggest that possible effects on UL24 should be considered in studies of thymidine kinase-deficient mutants.

Low levels of herpes simplex virus thymidine- thymidylate kinase are not limiting for sensitivity to certain antiviral drugs or for latency in a mouse model.

Herpes simplex virus mutant KG111 contains a nonsense mutation at codon 44 of the viral thymidine kinase (tk) gene and produces low amounts of a truncated tk polypeptide. We tested mutant KG111 and related viruses that specify varying amounts of similar truncated tk polypeptides for their sensitivities to antiviral nucleoside analogs at different temperatures using plaque reduction assays. The results of these assays showed that the nonsense mutation confers high resistance to bromovinyldeoxyuridine (BVdU) at any temperature and temperature-dependent resistance to acyclovir (ACV), buciclovir (BCV), ganciclovir (DHPG), and fluoroiodoarabinouracil (FIAU).

Effect of an amber mutation in the herpes simplex virus thymidine kinase gene on polypeptide synthesis and stability.

KG111 is a mutant of herpes simplex virus (HSV), strain KOS, that exhibits temperature-dependent drug resistance. For example, it is almost as resistant as a thymidine kinase (tk)-deficient virus at 39 degrees, but is relatively sensitive to acyclovir at 34 degrees, Using marker transfer techniques, we have mapped the mutation conferring temperature-dependent drug resistance in KG111 to the 5' portion of the tk gene. Sequencing of this region revealed an amber mutation at codon 44, which lies between the first and second methionine codons of the tk polypeptide.

Immediate-early regulatory gene mutants define different stages in the establishment and reactivation of herpes simplex virus latency.

Using nonsense and deletion mutants of herpes simplex virus type 1, we investigated the roles of three immediate-early proteins (ICP4, ICP27 and ICP0) in the establishment and reactivation of ganglionic latency in a mouse ocular model. DNA hybridization, superinfection-rescue, and cocultivation techniques provided quantitative data that distinguished between the failure of a virus to establish latency in the ganglion and its failure to reactivate. Null mutants with lesions in the genes for ICP4 and ICP27 did not replicate in the eye or in ganglia and failed to establish reactivatable latent infections.

Identification of amino acids in herpes simplex virus DNA polymerase involved in substrate and drug recognition.

Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase alpha. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases.

Analysis of the transcript of the herpes simplex virus DNA polymerase gene provides evidence that polymerase expression is inefficient at the level of translation.

We have mapped the termini and determined the relative abundance and ribosome density of the major cytoplasmic transcript of the DNA polymerase (pol) gene of herpes simplex virus type 1. Nuclease protection and primer extension analyses located the 5' end of the major pol transcript at two closely spaced sites 51 and 57 nucleotides to the left of a BamHI site at map position 0.413.

Are "routine" clinical and laboratory examinations of any help in the treatment of chronic hemodialysis patients?

The relevance of routine physical examinations, laboratory tests, and x-rays in guiding therapeutic decisions was investigated in 54 patients on hemodialysis. Patients were observed for 1 year, while recording all therapeutic interventions and tracing the procedures that had determined them. In no case did a variation in treatment follow the routine physical examination of a patient who was not symptomatic or already signaled for BP or dialytic problems by the hemodialysis nurses.

A human cytomegalovirus mutant resistant to the nucleoside analog 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U) induces reduced levels of BW B759U triphosphate.

We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-([2-hydroxy-1-(hydroxymethyl)ethoxy]methyl)guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate approximately equal to 1/10th the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed.

A genetic approach to promoter recognition during trans induction of viral gene expression.

Viral infection of mammalian cells entails the regulated induction of viral gene expression. The induction of many viral genes, including the herpes simplex virus gene encoding thymidine kinase (tk), depends on viral regulatory proteins that act in trans. Because recognition of the tk promoter by cellular transcription factors is well understood, its trans induction by viral regulatory proteins may serve as a useful model for the regulation of eukaryotic gene expression.

General aspects of virus drug resistance with special reference to herpes simplex virus.

The features of virus drug resistance are reviewed with examples from studies of herpes simplex virus drug-resistant mutants. Virus drug resistance, compared with drug resistance of bacteria or eukaryotes, is distinguished by its ability to provide information on drug selectivity. Identification of genes in which mutations arise to confer drug resistance defines gene products which contribute to antiviral selectivity.

Pathogenicity of herpes simplex virus mutants containing drug resistance mutations in the viral DNA polymerase gene.

Three herpes simplex virus mutants that contain drug resistance mutations in the DNA polymerase gene exhibited no significant reduction in replication in the ears of mice compared with the wild type after inoculation at that site but were attenuated for pathogenicity after intracerebral inoculation. Cataracts were common sequelae in mice that survived mutant infections.

Mutation within the herpes simplex virus DNA polymerase gene conferring resistance to (R)-9-(3,4-dihydroxybutyl)guanine.

Five herpes simplex virus mutants known or presumed to contain mutations in their DNA polymerase genes conferring resistance to acyclovir and arabinosyladenine also proved to exhibit some degree of resistance to (R)-9-(3,4-dihydroxybutyl)guanine (buciclovir). For one mutant, a buciclovir resistance mutation was mapped to a region of the viral DNA polymerase gene proposed to encode the deoxynucleoside 5'-triphosphate binding domain. These data implicate the viral polymerase as a target of buciclovir action that contributes to its antiviral selectivity.

Structural organization and unusual codon usage in the DNA polymerase gene from herpes simplex virus type 1.

We have analyzed the protein and nucleic acid sequences of the DNA polymerase from herpes simplex virus type 1 (HSV-1) to provide insight into the expression and possible structure of this enzyme. Extensive similarity between the amino acid sequence and that of the Epstein Barr virus DNA polymerase is reported. We describe probable structural similarities between these proteins and the use of these similarities to define structural and functional domains within the polymerase.

Sequence and mapping analyses of the herpes simplex virus DNA polymerase gene predict a C-terminal substrate binding domain.

The herpes simplex virus DNA polymerase provides an excellent model for studies of eukaryotic replicative polymerases. We report here the nucleotide sequence of the gene which encodes this enzyme. The gene includes a 3705-base-pair major open reading frame capable of encoding a Mr 136,519 polypeptide, in rough agreement with previous estimates of the size of the major polypeptide found in partially purified viral polymerase preparations.

Mutations in the herpes simplex virus major DNA-binding protein gene leading to altered sensitivity to DNA polymerase inhibitors.

Five herpes simplex virus mutants containing temperature-sensitive mutations in the gene for the major DNA-binding protein were assayed for their sensitivities to the DNA polymerase inhibitors aphidicolin and phosphonoacetic acid (PAA). Four of the mutants (tsA1, tsA15, tsA24, and tsA42) exhibited altered sensitivity to one or both of the inhibitors relative to the wild-type parent. In tsA1, a mutation or mutations conferring aphidicolin and PAA hypersensitivity were mapped by corescue with the temperature-sensitivity marker of tsA1 to a region of the DNA-binding protein locus, between map coordinates 0.

Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat.

The Moloney murine sarcoma virus long terminal repeat (LTR) harbors two distinct positive activators of transcription, namely, a distal signal and an enhancer. In this report we demonstrate that infection by herpes simplex virus (HSV) can markedly affect the utilization of these two Moloney murine sarcoma virus transcription signals. We investigated the HSV-mediated trans-acting effects with two goals in mind: first, to gain insight into LTR function, and second, to probe the mechanisms used by HSV to establish its own transcription cascade.

Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes.

A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene.

Sensitivity of arabinosyladenine-resistant mutants of herpes simplex virus to other antiviral drugs and mapping of drug hypersensitivity mutations to the DNA polymerase locus.

Seven herpes simplex virus mutants which have been previously shown to be resistant to arabinosyladenine were examined for their sensitivities to four types of antiviral drugs. These drugs were a pyrophosphate analog, four nucleoside analogs altered in their sugar moieties, two nucleoside analogs altered in their base moieties, and one altered in both. The seven mutants exhibited five distinct phenotypes based on their sensitivities to the drugs relative to wild-type strain KOS.