Virus-specified Thymidine Kinase Research Articles

Acyclovir

Acyclovir is one of the oldest antiviral drugs, having been in use for three decades now. It is an acyclic guanosine derivative and has activity mainly against herpes virus-1 (HSV-1), herpes virus-2 (HSV-2), and varicella zoster virus (VZV). It is only weakly active against Epstein–Barr virus (EBV), cytomegalovirus (CMV), and human herpes virus-6 (HHV-6), and not used for these infections. It acts by inhibition of viral DNA synthesis. Before it can act, it has to be phosphorylated by virus-specific thymidine kinase. This step gives it a high therapeutic index, but is also a major mechanism of viral resistance.

Pharmacokinetics of a single oral dose of acyclovir and valacyclovir in North American box turtles (Terrapenesp.)

Eastern box turtle (Terrapene carolina) populations have significantly declined throughout their range (Currylow et al., 2011). While a combination of factors are likely playing a role in the decline of the box turtle, disease outbreaks have been emerging across the Eastern United States in chelonians and may be important (De Voe et al., 2004; Johnson et al., 2008; Allender et al., 2011). Herpesviruses and iridoviruses are two common virus families that lead to clinical signs of upper respiratory tract disease in affected chelonians (Harper et al., 1982; Brown et al., 1999; Johnson et al., 2008; Allender et al., 2011). Acyclovir has been used anecdotally in the treatment of both viral agents in chelonians (Marschang et al., 1997; De Voe et al., 2004; Funk & Diethelm, 2006); however, to date, only a single pharmacokinetic study has been performed in a single species (Gaio et al., 2007). Acyclovir and its pro-drug valacyclovir are guanine analogue antiviral drugs (Elion, 1993). They are activated by phosphorylation of a virus-specific thymidine kinase (TK). Acyclovir uptake has been shown to be enhanced in herpesvirus-infected cells, with a 10to 30-fold greater affinity for infected cells than uninfected cells (Beutner, 1995). Once incorporated into the viral genome, relatively low levels of the drug are needed to achieve viral inhibition, and adequate intracellular concentrations can be maintained for several hours (Beutner, 1995). Thymidine kinase genes or functional TK enzymes have similarly been identified in iridoviruses (Scholz et al., 1988; Jakob et al., 2001; Coupar et al., 2005; Tsai et al., 2005). This study evaluated the pharmacokinetics of acyclovir and valacyclovir after a single oral dose in twelve North American box turtles. Animals were housed individually in Vision cages (Model #332; Vision Products Plus Inc., Canoga Park, CA, USA) that maintained a thermal gradient of 72 F to 87 F. A varied diet of fruits and vegetables was provided every other day. All activities were approved by the University of Illinois IACUC (protocol 11003). A pilot study using oral acyclovir was initially performed at doses of 40 and 80 mg ⁄kg in one animal each. After the results demonstrated low maximum observed plasma concentration (Cmax) at the doses used, a decision was made to evaluate the pharmacokinetics of oral valacyclovir at 20 and 40 mg ⁄kg in one animal each. Based on these results, eight animals were administered a single oral dose of 40 mg ⁄kg valacyclovir. Venipuncture was performed via the subcarapacial sinus for all phases of the study. For the final study, blood (up to 0.3 mL) was collected in a 3-mL syringe with a 22-ga needle at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h following oral valacyclovir. Samples were placed in a lithium heparin microtainer (Becton Dickinson, Franklin Lakes, NJ, USA), centrifuged immediately, the plasma placed in a separate cryovial, and stored at )20 C. Samples were transported on dry ice to the Pharmacology Laboratory at the University of Tennessee for analysis. Plasma samples were analyzed using a reverse-phase highperformance liquid chromatography method (HPLC). The system consisted of a 2695 separations module, a 2475 fluorescence detector, and a computer equipped with Empower software (Waters, Milford, MA, USA). Valacyclovir and acyclovir were extracted from plasma samples using 1 cc HLB solid-phase extraction (SPE) cartridges. The compounds were separated on an Atlantis T3 (4.6 · 100 mm, 5 lm) column with a guard column. The mobile phase was a mixture of (A) 10 mM ammonium phosphate pH 2.9 and (B) acetonitrile (97:3). The flow rate was 1.2 mL ⁄min, and the column temperature was ambient. Fluorescence was measured at an excitation of 260 nm and an emission of 375 nm with a gain of ·100. Standard curves for plasma analysis were prepared by fortifying untreated, pooled turtle plasma with valacyclovir and acyclovir to produce a linear concentration range of 10–1500 ng ⁄mL. Average recovery for both drugs was 87%, while intraand inter-assay variabilities were <10%. The lower limit of quantification was 10 ng ⁄mL. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365-2885.2012.01418.x SHORT COMMUNICATION

ChemInform Abstract: Synthesis and Antiviral Activity of 5-Heteroaryl-Substituted 2′- Deoxyuridines.

The synthesis of 5-heteroaryl-substituted 2'-deoxyuridines is described. The heteroaromatics were obtained from three different 5-substituted 2'-deoxyuridines. Cycloaddition reaction of nitrile oxides on the 5-ethynyl derivative 1 gave the isoxazoles 4a-e. The thiazole derivatives 14a-c were obtained from the 5-thiocarboxamide 11, while 5-pyrrol-1-yl-2'-deoxyuridine (17) could be synthesized directly from 5-amino-2'-deoxyuridine. The compounds were evaluated for antiviral activity. Selective activity against herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) was noted for 5-(3-bromoisoxazol-5-yl)-2'-deoxyuridine (4c). The compound was inactive against herpes simplex virus type 2, cytomegalovirus, and thymidine kinase (TK)-deficient mutants of HSV-1 and VZV, which indicates that, most likely, its antiviral activity depends on phosphorylation by the virus-specified TK.

Therapeutical effect of modified adamantane copolymer compounds: study of molecular mechanisms.

Copolymers of N-polyvinylpyrrolidone-acrylic acid (AB-1) and adamantane derivatives are known to possess marked antiviral activity in in vitro and in ovo models. Among the constructed preparations of AB-1 modified by adamantane derivatives some, especially AB-4 (modified by deitiforin), were found to show more extended antiviral activity and to inhibit markedly virus reproduction in susceptible permissive cell cultures and chicken embryos. In AB-4 treated cells and allantoic sacs, virus titers (influenza virus, herpes virus, and HIV) and virus antigen concentration were decreased. On the other hand, herpes virus-specific thymidine kinase and of DNA-polymerases isolated from Escherichia coli, Plectonema boryanum, and herpes virus type 1 infected murine brain tissue retained their activity after incubation with AB-4 or AB-2. The compounds investigated, in view of their effect on virus reproduction, are thought to be prospective as antiviral agents.

Acyclovir‐resistant herpes simplex virus infection in HIV‐infected patients: a report of three cases and a review of management

AbstractCase reports Three cases of acyclovir‐resistant ano‐genital HSV infection.Management review Acyclovir‐resistant mucocutaneous herpes simplex virus (HSV) infections are increasingly seen in patients with advanced HIV disease. The primary mechanism of resistance is deficiency of virus specific thymidine kinase. Foscarnet, a pyrophosphate analogue, which directly inhibits HSV DNA polymerase is an effective therapeutic alternative to acyclovir, and is commonly used. Topical trifluridine recently has been useful as a non‐toxic alternative to foscarnet. Management of recusrrent episodes of acyclovir‐resistant HSV infection following successful inital or induction therapy with foscarnet requires development of a clear strategy for effective suppressive therapy.

Synthesis and antiviral activity of 5-heteroaryl-substituted 2'-deoxyuridines

The synthesis of 5-heteroaryl-substituted 2'-deoxyuridines is described. The heteroaromatics were obtained from three different 5-substituted 2'-deoxyuridines. Cycloaddition reaction of nitrile oxides on the 5-ethynyl derivative 1 gave the isoxazoles 4a-e. The thiazole derivatives 14a-c were obtained from the 5-thiocarboxamide 11, while 5-pyrrol-1-yl-2'-deoxyuridine (17) could be synthesized directly from 5-amino-2'-deoxyuridine. The compounds were evaluated for antiviral activity. Selective activity against herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) was noted for 5-(3-bromoisoxazol-5-yl)-2'-deoxyuridine (4c). The compound was inactive against herpes simplex virus type 2, cytomegalovirus, and thymidine kinase (TK)-deficient mutants of HSV-1 and VZV, which indicates that, most likely, its antiviral activity depends on phosphorylation by the virus-specified TK.

Anti-herpes simplex virus activity of 5-substituted 2-pyrimidinone nucleosides

Several 5-substituted 2-pyrimidinone 2'-deoxyribonucleoside (PdR) analogs were examined for their anti-herpes simplex virus (HSV) activity in cell culture. The order of potency of their antiviral activities against HSV type 1 (HSV-1) and HSV-2 was iodo PdR approximately ethynyl PdR approximately propynyl PdR. The antiviral action of iodo PdR is dependent on the ability of HSV to induce virus-specified thymidine kinase in infected cells. Several HSV-1 variants with altered thymidine kinase changed their sensitivity to iodo PdR, whereas HSV-1 variants with altered DNA polymerase were as sensitive as the parental virus to iodo PdR. Continuous presence of iodo PdR for more than one virus replication cycle was required for optimal antiviral activity. Iodo PdR (100 microM) had no activity against Epstein-Barr virus DNA replication in P3HR-1 cells. With an oral, an intraperitoneal, or a subcutaneous route of injection, iodo PdR administered twice a day for 2.5 days could prevent the death of mice infected with HSV-2. This in vivo activity is unlikely to be related to the potential conversion of iodo PdR to iododeoxyuridine, since iodo PdR is not a substrate of xanthine oxidase.

Synthesis and antiherpes virus activity of phosphate and phosphonate derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

A series of phosphate esters of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) were synthesized and evaluated for antiherpes virus activity. The cyclic phosphate esters were made by a new, efficient method utilizing stannic chloride as a solubilizing agent. Monophosphate 2 and bisphosphate 4 showed comparable activity to DHPG and probably acted as prodrugs of DHPG. On the other hand, the cyclic phosphate of DHPG 3 was taken up by cells and bypassed the virus-specified thymidine kinase. As a result, 3 was active against DHPG-resistant HSV mutants that lacked the viral-specified thymidine kinase and was more toxic than DHPG to uninfected cells. The phosphonate 5, the least toxic of the derivatives tested, was only marginally active against HSV but showed substantial activity against human cytomegalovirus in vitro.

Utilization of internal AUG codons for initiation of protein synthesis directed by mRNAs from normal and mutant genes encoding herpes simplex virus-specified thymidine kinase.

Previous studies (H.S. Marsden, L. Haarr, and C.M. Preston, J. Virol. 46:434-445, 1983) have shown that at least three polypeptides, with molecular weights of 43,000, 39,000, and 38,000, are encoded by the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene. It has been suggested that the 39,000- and 38,000-molecular-weight polypeptides arise from preinitiation complexes bypassing the first and second AUG codons before commencement of translation since, according to previous work (M. Kozak, Nucleic Acids Res. 9:5233-5252, 1981), these codons are not of the most efficient structure for initiation. This possibility was investigated by using specific herpes simplex virus mutants with alterations in the TK gene. Mutant TK4 has an amber mutation between the first and second AUG codons, whereas mutant delta 1 has a deletion which removes the first AUG codon but leaves other AUG codons, as well as transcriptional promoter sequences, intact. Both mutants synthesized only the 39,000- and 38,000-molecular-weight polypeptides, and the amounts produced were normal in TK4-infected cells but increased in delta 1-infected cells. Furthermore, the levels of TK produced after infection with the mutant viruses correlated with the amounts of the 39,000- and 38,000-molecular-weight polypeptides synthesized. The 43,000-, 39,000-, and 38,000-molecular-weight polypeptides were shown to be related by their positive reaction with anti-TK serum in both immunoprecipitation and immunoblotting experiments. The production of the 39,000- and 38,000-molecular-weight polypeptides through bypassing of the first AUG codon was examined by hybrid arrest experiments with a DNA fragment complementary to only 50 bases at the 5' terminus of TK mRNA. This fragment arrested the synthesis of the 30,000- and 38,000-molecular-weight polypeptides when annealed to mRNA from wild-type HSV-1- or TK4-infected cells, showing that those polypeptides arise from an mRNA initiated upstream from the first AUG codon. mRNA from cells infected with mutant delta 1, which lacks DNA sequences upstream from the first AUG, was not affected by the 50-base-pair fragment. The data therefore confirm that three polypeptides encoded by the HSV-1 TK gene arise by differential use of in-phase AUG codons for the initiation of protein synthesis. This mechanism for the production of related but distinct polypeptides has not previously been demonstrated in a eucaryotic system, and the implications for the regulation of TK enzyme activities are discussed.

Synthesis and biological activities of 2-pyrimidinone nucleosides. 2. 5-Halo-2-pyrimidinone 2'-deoxyribonucleosides.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-bromo-2-pyrimidinone (BrPdR) and 1-(2-deoxy-beta-D-ribofuranosyl)-5-iodo-2-pyrimidinone (IPdR) have been synthesized by condensation of the appropriate silylated bases 2a and 2b, respectively, with 3,5-bis-O-(p-chlorobenzoyl)-2-deoxy-alpha-D-ribofuranosyl chloride (8) in 1,2-dichloroethane, in the presence of SnCl4, followed by separation of the anomeric blocked nucleosides via column chromatography and subsequent deprotection with methanolic ammonia. Both BrPdR and IPdR exhibited significant antiherpes activities against various strains of HSV-1 and HSV-2, the latter compound (IPdR) showing the higher activity as well as the stronger binding to the virus-specific thymidine kinase.

2′-nor-cGMP: A seco-cyclic nucleotide with powerful anti-DNA-viral activity

As part of our study of antiherpetic acyclonucleosides, we synthesized a cyclic GMP analog, 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide, sodium salt (2′-nor-cGMP), and discovered its potent and broad spectrum anti-DNA-viral activities. 2′-Nor-cGMP inhibits the replication of many DNA viruses, including herpes simplex virus, human cytomegalovirus, vaccinia, SV 40, and adenovirus, but does not inhibit RNA viruses. In plaque reduction studies this potent antiviral agent is also approximately 10-fold more potent than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2′NDG) against varicella-zoster virus and inhibits cell transformation by bovine papilloma virus. Unlike 2′NDG, the potent activity of 2′-nor-cGMP against herpes virus is not dependent upon the action of virus-specified thymidine kinase. Intercellular metabolism of 2′-nor-cGMP produced small amounts of 2′NDG triphosphate which were insufficient to account for the antiviral activity observed, implying that this potent anti-DNA-viral agent operates by a mechanism different from that of known acyclonucleosides.

Acyclic analogues of 2'-deoxynucleosides related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine as potential antiviral agents.

A series of acyclic analogues of 2'-deoxynucleosides related in structure to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) have been synthesized and evaluated for antiviral activity against herpes simplex virus type 1 (F strain). Additionally, the ability of these analogues to function as substrates for the virus-specified thymidine kinase was examined. Phosphorylation by this kinase is essential for antiviral activity. Although the acyclic 4-oxopyrimidine nucleosides were substrates for the kinase, they were devoid of antiviral activity. In the purine series, most analogues similar in structure to DHPG did exhibit significantly lower antiviral activity, indicating that even small modifications in the purine substituents substantially reduce the antiviral potency. The most active agent, 2,6-diaminopurine 27, was only poorly phosphorylated by the viral kinase; therefore, its activity was most likely due to a prior enzymatic deamination to give DHPG. Evaluation of 27 in a mouse encephalitis model has shown it to be nearly as potent as DHPG (1).

Cytomegalovirus DNA polymerase inhibition and kinetics

Cytomegalovirus (CMV) DNA polymerase has immunologic specificity in relation to the virus specific polymerases of other herpesviruses. During the early phase of CMV infection in vitro, the virus DNA polymerase is rapidly induced. Due to the lack of virus specific thymidine kinase, cytomegalovirus is resistant to nucleosides which require herpesvirus thymidine kinases for activation. Cytomegalovirus DNA polymerase is therefore the known possible target for antiviral drugs. Several pyrophosphate analogs have been assayed for their inhibitory effects on this enzyme. The most active compound is phosphonoformate (PFA). PFA also effectively inhibits other partially purified herpesvirus DNA polymerases as well as the multiplication of all human herpesviruses, at concentrations which do not affect cellular DNA polymerases or normal cell growth. The mechanism of inhibition is a non-competitive inhibition of CMV DNA polymerase activity with respect to the four deoxyribonucleoside triphosphates, and an uncompetitive inhibition with respect to the template used. In cell culture, PFA inhibits the formation of late CMV polypeptides, but not the synthesis of early CMV polypeptides. The CMV specific polymerase persists in the presence of PFA, as measured by immunological methods, and enzyme activity can be demonstrated after removal of PFA. The inhibition of CMV replication is reversible even after long exposure to PFA. Our interpretation is that the CMV genome is highly resistant to the cellular metabolism also in non-producing cells. A new rapid CMV neutralization test was established, based on the appearance of early CMV-induced antigens.

Interaction of DNA polymerase and nucleotide analog triphosphates

The properties of virus and host DNA polymerases are important factors in determining the selectivity of deoxynucleotide analogs used in antiviral chemotherapy. The high affinity of herpes DNA polymerase for nucleotide analogs may be particularly important in CMV and EBV-infected cells, since these viruses do not induce the synthesis of a virus-specified thymidine kinase. In general, the effect of nucleotide analog incorporated into DNA may be summarized as follows: analogs with modifications at the base moiety do not affect the rate of DNA chain elongation whereas those modified at the sugar moiety will inhibit the rate of chain elongation. ACGTP and DHPGTP competitively inhibit incorporation of dGTP into DNA; however, steric freedom of the acyclic phosphate may allow these nucleotides to bind virus enzyme in a conformation similar to that assumed by dGTP only at the transitional stage of the enzyme reaction. This may explain the high affinity of virus enzyme for these inhibitors. The interaction of aphidicolin with virus enzyme differs from that with host enzyme. These differences suggest new strategies for antiviral chemotherapy using aphidicolin derivatives.

The biochemistry and mechanism of action of acyclovir.

Acyclovir, 9-(2-hydroxyethoxymethyl)guanine, is an acyclic nucleoside analogue which has a high activity and selectivity for herpes viruses, particularly herpes simplex viruses types 1 and 2 and varicella zoster virus. This selectivity is due to the initial activation of the drug by phosphorylation by a herpes virus-specified thymidine kinase. Normal cellular enzymes do not phosphorylate acyclovir to any significant degree. Acyclovir monophosphate is subsequently converted to a triphosphate which is a more potent inhibitor of herpes virus DNA polymerases than of cellular DNA polymerases. The relationship between the amount of acyclovir triphosphate formed and its inhibition constant (Ki) for the particular viral or cellular DNA polymerase is predictive of the inhibitory activity of acyclovir on DNA replication.

Perspectives on interactions of acyclovir with Epstein-Barr and other herpes viruses

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] inhibits Epstein-Barr virus (EBV) replication in lymphoblastoid cells at concentrations nontoxic to cellular growth. The mode of action of the drug against EBV differs from the mechanism described in herpes simplex virus systems. Due to the absence of virus-specified thymidine kinase, the drug is poorly phosphorylated in EBV-infected cells. The extent of monophosphorylation is similar both in mock-infected and EBVinfected cells. Despite weak phosphorylation of the drug, the replication of linear EBV DNA is inhibited due to exquisite sensitivity of the viral DNA polymerase. Activation of acyclovir does not require phosphorylation by virus-specified thymidine kinase. Inhibition of different herpes-group viruses depends on three variable factors: degree of phosphorylation, cellular metabolism of the drug, and degree of sensitivity of the viral polymerase. Interaction of acyclo-vir-triphosphate with EBV DNA polymerase is reversible. Cells infected with EBV and treated with acyclovir resume virus replication following removal of the drug even after long exposure. Acyclovir inhibits replication of linear genomes and stops production of virus, but has no effect on latent cellular infection. These results lead us to predict that acyclovir will suppress, but not cure, EBV infection.

Acyclovir in mouse cytomegalovirus infections

A virus-specified thymidine kinase appears to be a general requirement for herpes virus susceptibility to the antiviral effect of acyclovir. Surprisingly, mouse cytomegalovirus (MCMV), which does not encode for a thymidine kinase, is exquisitely sensitive to the drug both in vitro and in vivo. The drug is active against the virus in the absence of a cellular thymidine kinase and the antiviral activity is not diminished in the presence of excess thymidine or a variety of nucleosides and deoxynucleosides. Thus, a thymidine phosphorylation pathway is not required for the drug's activation in this infection. The enzyme system responsible for phosphorylation of the drug has not been identified. Mouse cytomegalovirus mutants resistant to the drug have been isolated, indicating that the antiMCMV effect results from selective inhibition of viral replication rather than indirectly through toxicity to the host cell. Eight resistant mutants appear to be in the same complementation group and seven of the mutants demonstrate coresistance to phosphonoacetic acid, a marker for the DNA polymerase locus of herpes viruses. The evidence to date indicates that the MCMV DNA polymerase is the final site of action of the drug. Investigations of the antiMCMV activity of acyclovir should provide insights into the antiviral effects of this drug and other nucleoside analogs in other herpes virus infections in which the virus does not code for a thymidine kinase (for example, human cytomegalovirus and Epstein-Barr virus).

Selection and preliminary characterization of acyclovir-resistant mutants of varicella zoster virus

A series of acyclovir-resistant mutants of varicella zoster virus (VZV) were selected in vitro by serial passage of VZV-infected human fibroblasts in increasing drug concentrations, or by continuous exposure of cultures infected at high multiplicity to 100 μ M acyclovir. The in vitro susceptibility of these mutants to several antiherpetic agents was measured by the plaque-reduction assay, the capacity of extracts of cells infected with these mutants to phosphorylate acyclovir was examined and compared with that of their acyclovirsensitive parent strains. Based on these studies, VZV could be shown to acquire resistance to acyclovir through diminished acyclovir phosphorylation. This was presumably due to loss of viral specific thymidine kinase (TK) function. Two acyclovir-resistant mutants remained TK competent but demonstrated phenotypic changes in sensitivity to antiviral agents known to act at the herpes simplex virus (HSV)-specific DNA polymerase level. These results suggest that the resistance of VZV to acyclovir results from qualitative or quantitative alterations in the virus-specified TK or DNA polymerase.

Thymidine kinase not required for antiviral activity of acyclovir against mouse cytomegalovirus.

Previous studies of herpesvirus infections have indicated that a virus-specified thymidine kinase is required for the initial phosphorylation of acyclovir [acycloguanosine or 9-(2-hydroxyethoxymethyl)guanine] in the formation of acycloguanosine triphosphate. The latter compound accumulates in infected cells and competitively inhibits the viral DNA polymerase. We found that mouse cytomegalovirus, which does not express a thymidine kinase, was sensitive to the antiviral effects of acyclovir at a 50% inhibitory dose of approximately 0.23 microM. Acyclovir was equally effective against mouse cytomegalovirus in normal 3T3 cells and in 3T3 cells deficient in cellular thymidine kinase. Furthermore, the activity of acyclovir could not be reversed by excess thymidine, which easily reversed the antiviral activity of acyclovir against herpes simplex virus. Using a high-pressure liquid chromatography technique that easily detected acycloguanosine triphosphate in cells infected with herpes simplex virus, we could not detect acycloguanosine triphosphate in mouse cytomegalovirus-infected cells. These experiments demonstrated that the activity of acyclovir against mouse cytomegalovirus is not dependent on a thymidine phosphorylation pathway. Additional experiments are underway to determine whether acycloguanosine triphosphate is produced by another pathway in concentrations sufficient to inhibit mouse cytomegalovirus DNA polymerase.

Synthesis and antiviral activity of 1-(2-deoxy-beta-D-ribofuranosyl)-5-(methylmercapto)-2-pyrimidinone.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-(methylmercapto)-2-pyrimidinone (1b) was synthesized via modification of the silyl method. 1b inhibits the Herpes simplex virus type 1 (98%) and type 2 (97%) at a concentration which is nontoxic to human HeLa cells. The compound shows 50 times greater binding affinity (lower Ki) to the virus-specific thymidine kinase than to the thymidine kinase of uninfected HeLa cells.