brivudine and fialuridine

When employed as a transgene reporter, the herpes simplex type 1 virus (HSV1) thymidine kinase gene (tk) is ectopically expressed in mouse testis. The principal testicular mRNA lacks the 5'-end of the tk reading frame. As a result the principal translation products, P2 and P3, are N-terminally truncated. These co-migrate in SDS-PAGE with polypeptides synthesised during HSV1 infection that were previously thought to be initiated at methionine codons ATG46 and ATG60. Prompted by these observations we generated modified tk genes each carrying only one of the first three ATG codons. Transfected cells expressed both full-length enzyme (P1) and P2 when only ATG1 was unmodified, P2 and P3 when only ATG46 was unmodified or P2 and a fourth polypeptide (P4) when only ATG60 was unmodified. Our observations indicate that P3 is initiated at ATG46 rather than ATG60, while P2 is initiated at a non-ATG codon rather than ATG46 and P4 is initiated at ATG60. When either of two putative non-ATG initiation codons was modified P2 was no longer produced. Cells mainly expressing either P1 or P3 exhibited the same sensitivity to Ganciclovir as cells transfected with the unaltered tk gene. P1 and P3 both have TK activity while P4 probably has none.

Topics: Animals; Antiviral Agents; Arabinofuranosyluracil; Base Sequence; Bromodeoxyuridine; Codon; Electrophoresis, Polyacrylamide Gel; Ganciclovir; Gene Expression; Herpesvirus 1, Human; Male; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; RNA, Messenger; Testis; Thymidine Kinase; Transfection

The 2'-fluoropyrimidine nucleoside analogs 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC). 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), and 1(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) showed higher in vitro activity against herpes simplex virus type 1 (HSV-1), than equine herpesvirus 1 (EHV-1) or pseudorabies virus (PRV). Comparison of the 50% plaque inhibitory doses for HSV-1 and its mutant MMdUr-20 in cell cultures with inhibition constants (Ki's) for the viral deoxythymidine kinases (dTKs) suggests that in the infected cell FMAU is phosphorylated by host enzymes. As compared to HSV-1, EHV-1 and PRV were more resistant to E-5-(2-bromovinyl-2'-deoxyuridine (BVdU) and to the 2'-fluoropyrimidine analogs, as are HSV-2 and the HSV-1 mutants MMdUr-20 and S1. Because the dTKs of the latter lack deoxythymidylate kinase (dTMPK) activity, there appears to be a correlation between resistance to these analogs and BVdU on the one hand, and lack of dTMPK activity on the other. We predict that EHV-1 and PRV dTKs will be shown to lack significant dTMPK activity.

Topics: Animals; Antiviral Agents; Arabinofuranosyluracil; Bromodeoxyuridine; Cytarabine; Drug Resistance, Microbial; Herpesviridae; Herpesvirus 1, Suid; Horses; Nucleoside-Phosphate Kinase; Pseudorabies; Pyrimidine Nucleosides; Substrate Specificity

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. All mutants exhibited resistance to acyclovir and arabinosylthymine, as well as marginal resistance to iododeoxyuridine, whereas all but one exhibited resistance to phosphonoformic acid. The mutants exhibited either sensitivity or hypersensitivity to other drugs tested--2'-nor-deoxyguanosine, 5-methyl-2'-fluoroarauracil, 5-iodo-2'-fluoroarauracil, and bromovinyldeoxyuridine--some of which differed only slightly from drugs to which the mutants were resistant. These results suggest ways to detect and treat arabinosyladenine-resistant isolates in the clinic. Antiviral hypersensitivity was a common phenotype. Mutations conferring hypersensitivity to 2'-nor-deoxyguanosine in mutant PAAr5 and to bromovinyldeoxyridine in mutant tsD9 were mapped to nonoverlapping regions of 1.1 and 0.8 kilobase pairs, respectively, within the herpes simplex virus DNA polymerase locus. Thus, viral DNA polymerase mediates sensitivity to these two drugs. However, we could not confirm reports of mutations in the DNA polymerase locus conferring resistance to these two drugs. All of the mutants exhibited altered sensitivity to two or more types of drugs, suggesting that single mutations affect recognition of the base, sugar, and triphosphate moieties of nucleoside triphosphates by viral polymerase.

Topics: Acyclovir; Antiviral Agents; Arabinofuranosyluracil; Arabinonucleosides; Bromodeoxyuridine; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Foscarnet; Ganciclovir; Genes, Viral; Idoxuridine; Mutation; Phosphonoacetic Acid; Simplexvirus; Thymidine; Vidarabine

The effect of several antiviral drugs on the reactivation of herpes simplex virus type 1 in explant cultures of latently infected mouse trigeminal ganglia was investigated. Phosphonoacetate and phosphonoformate, which act directly on the virus-induced DNA polymerase, require a drug concentration of 400 micrograms/ml for the inhibition of virus reactivation in latently infected ganglia. Arabinosyladenine and arabinosyladenine monophosphate, which are phosphorylated to triphosphates by cellular enzymes and inhibit virus synthesis either by blocking the DNA polymerase or by incorporation into viral DNA, require a concentration of only 100 micrograms/ml for the inhibition of the reactivation process. Drugs that are phosphorylated by the virus-induced thymidine kinase, such as acyclovir, arabinosylthymine, bromovinyldeoxyuridine, and three fluorinated pyrimidine nucleosides require the lowest drug concentrations for complete inhibition of virus reactivation in latently infected ganglia explant cultures. Our data suggest that the inhibition of virus reactivation is dependent not only on drug concentration, but also on the number of latently infected neurons in the ganglia.

Topics: Acyclovir; Animals; Antibodies, Viral; Antiviral Agents; Arabinofuranosyluracil; Arabinonucleosides; Bromodeoxyuridine; Culture Media; Culture Techniques; Cytarabine; Foscarnet; Mice; Mice, Hairless; Neutralization Tests; Phosphonoacetic Acid; Simplexvirus; Thymidine; Trigeminal Nerve; Vidarabine Phosphate; Virus Activation