Inhibition of cellular DNA polymerase alpha and human cytomegalovirus-induced DNA polymerase by the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

The triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were examined for their inhibitory effect on highly purified cellular DNA polymerase alpha and human cytomegalovirus (Towne strain)-induced DNA polymerase. These two nucleoside triphosphates competitively inhibited the incorporation of dGMP into DNA catalyzed by the DNA polymerases. The virus-induced DNA polymerase had greater binding affinity for the triphosphate of 9-(2-hydroxyethoxymethyl)guanine (Ki, 8 nM) than for the triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (Ki, 22 nM), although the nucleoside of the latter compound was strikingly more effective against human cytomegalovirus replication in cell cultures than the nucleoside of the former. The Ki values of these two nucleoside triphosphates for alpha polymerase were 96 and 146 nM, respectively, and were 7- to 12-fold higher than those for the virus-induced enzyme. These data indicated that virus-induced DNA polymerase was more sensitive to inhibition by these two nucleoside triphosphates than was the cellular alpha enzyme.     

Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.

The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.     

Metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine, a new anti-herpes virus compound, in herpes simplex virus-infected cells

The metabolism of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), one of the most promising new anti-herpes virus compounds, in HeLa cells infected with herpes simplex virus type 1 was compared with that in the uninfected HeLa cells. In the virus-infected cells, the uptake of DHPG was enhanced and the major metabolites were found to be the mono-, di-, and triphosphate derivatives. The formation of these metabolites was dependent on the extracellular concentration of DHPG (0.5 to 5.0 microM). Virus-induced thymidine kinase was capable of phosphorylating DHPG to its monophosphate which could be further phosphorylated to the di- and triphosphate derivatives by the host cellular enzymes. Incorporation of the DHPG into DNA was observed in virus-infected cells. In contrast with 9-(2-hydroxyethoxymethyl)guanine, DHPG seemed not to serve as a chain terminator, but to be incorporated internally into DNA strands.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Properties of purified enzymes induced by pathogenic drug-resistant mutants of herpes simplex virus. Evidence for virus variants expressing normal DNA polymerase and altered thymidine kinase

The DNA polymerases and thymidine kinases induced by three drug-resistant mutants of herpes simplex virus type 1 (S1, Tr7, and B3) and their common parent strain, SC16, have been purified and their properties compared. No significant differences were seen in the affinities of the polymerases for TTP and dGTP, or for the triphosphates of 9-(2-hydroxyethyloxymethyl)guanine (acyclovir) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) (drugs used in their isolation). In contrast all three mutants induced abnormal thymidine kinases. Those induced by the acyclovir-resistant mutants, S1 and Tr7, showed reduced affinities for thymidine, acyclovir, and also BVdU. Thymidine kinase induced by the BVdU-resistant mutant B3 showed reduced affinity for BVdU, but its affinities for thymidine and acyclovir were similar to those of the wild type enzyme. Thus, it appears that these variants of herpes simplex virus express altered thymidine kinases with impaired ability to phosphorylate particular nucleoside analogue drugs and these characteristics probably account for the drug resistance of the viruses. This strategy for resistance is important as it may result in variants with undiminished pathogenicity.     

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.     

Properties of cells carrying the herpes simplex virus type 2 thymidine kinase gene: mechanisms of reversion to a thymidine kinase-negative phenotype

We have isolated cells with a thymidine kinase-negative (tk⁻) phenotype from cells which carry the herpes simplex virus type 2 tk gene by selection in 5-bromodeoxyuridine or 9-(2-hydroxyethoxymethyl)guanine. Both selection routines generated revertants with a frequency of 10⁻³ to 10⁻⁴, and resistance to either compound conferred simultaneous resistance to the other. tk⁻ revertants fell into three classes: (i) cells that arose by deletion of all virus sequences, (ii) cells that had lost the virus tk gene but retained a nonselected virus-specific function and arose by deletion of part of the virus-specific sequence, and (iii) cells that retained the potential to express all of the virus-specific functions of the parental cells and retained all of the virus-specific DNA sequences.     

Physical mapping of the mutation in an antigenic variant of herpes simplex virus type 1 by use of an immunoreactive plaque assay.

Two mutations affecting herpes simplex virus type 1 glycoprotein B were mapped by marker rescue using cloned sequences of wild-type herpes simplex virus type 1 strain KOS DNA. One mutant, tsB5, is a temperature-sensitive mutant which does not express mature, functional glycoprotein B at the nonpermissive temperature. The other mutant, marB1.1, expresses an antigenic variant of glycoprotein B and was selected for resistance to neutralization by a monoclonal antibody. The mutation in tsB5 mapped to a 1.2-kilobase segment of the herpes simplex virus type 1 genome between coordinates 0.361 and 0.368, whereas the mutation in marB1.1 mapped to a 1.6-kilobase segment between coordinates 0.350 and 0.361. An in situ enzyme immunoassay was used to detect plaques of recombinant wild-type virus among the progeny of transfections with mutant marB1.1 DNA and wild-type DNA fragments.     

Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate

The ability of human alpha and beta DNA polymerases and herpes simplex virus type 2 (HSV-2) and human cytomegalovirus (HCMV) DNA polymerases to insert and extend several nucleotide analogs has been investigated using a variation of Sanger-Coulson DNA sequencing technology. The analogs included the triphosphates of two antiviral nucleosides with incomplete sugar rings: 9-(1,3-dihydroxy-2-propoxymethyl)guanine (dhpG) and 9-(2-hydroxyethoxymethyl)guanine (acyG or acyclovir), as well as dideoxy and arabinosyl nucleoside triphosphates. Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertion/extension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP versus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP greater than (acyGTP and araNTP) greater than ddGTP, whereas by human alpha polymerase it was araATP greater than ddGTP much greater than (acyGTP and dhpGTP) and by human beta polymerase it was (araATP and ddGTP) much greater than (acyGTP and dhpGTP). Evidence is presented for three mechanisms of inhibition by extendible nucleotides (of dhp and ara types) exhibiting frequent internalization: araATP acted as a simple pseudoterminator of alpha and beta polymerases, but was easily extended past singlet sites by Herpesviridae polymerases and only stalled at sites requiring two or more araATP insertions in a row. Herpesviridae polymerases stalled after adding dhpGMP and one additional nucleotide, suggesting that polymerase translocation problems may be a factor in polymerase inhibition by modified sugar nucleotide analogs. The amino acid sequence of the human alpha DNA polymerase, which is acyGTP resistant, was found to vary by one amino acid from the amino sequences of the Herpesviridae polymerases in a region of significant similarity and probable functional homology. Amino acid differences at that same site differentiate acyclovir-resistant HSV-1 mutants from the acyclovir-sensitive HSV-1 wild type.     

1,2,4-Triazoloazine derivatives as a new type of herpes simplex virus inhibitors

A new class of inhibitors of herpes simplex virus replication was found. The compounds under study are derived from condensed 1,2,4-triazolo[5,1-c][1,2,4]triazines and 1,2,4-triazolo[1,5-a]pyrimidines, structural analogues of natural nucleic bases. Antiherpetic activity and cytotoxicity of the compounds were studied. The corresponding triphosphates of several active compounds were prepared and tested as inhibitors of DNA synthesis catalyzed by herpes simplex virus polymerase. The potential mechanism of their action is blocking of DNA dependent DNA polymerase, a key enzyme of viral replication.     

Phosphorylation of acyclovir [9-(2-hydroxyethoxymethyl)guanine] in Epstein-Barr virus-infected lymphoblastoid cell lines.

The extent of phosphorylation of 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]) in fresh peripheral leukocytes, in Epstein-Barr virus (EBV)-infected lymphoblastoid cell lines, and in herpes simplex virus type 1-infected lymphoblastoid (P3HR-1) and monkey kidney (Vero) cells was determined by high-pressure liquid chromatography, Mono-, di-, and triphosphorylated derivatives of [8-14C]ACV were detected at low levels at various times after superinfection of Raji cells with EBV. The extent of phosphorylation appeared to be related to the concentration of ACV in the medium. Small amounts of ACV mono-, di-, and triphosphates were formed in fresh peripheral leukocyte preparations from EBV- seropositive and -seronegative donors. Comparable ACV monophosphate levels were detected in EBV-negative BJAB and the EBV-positive BJAB/GC cell lines; however, no di- or triphosphate derivatives were detected. Comparable ACV-monophosphate levels were detected in both P3HR-1 and HSV-infected P3HR-1 cell lines; however, larger amounts of ACV di- and triphosphorylated derivatives were detected in the HSV-infected P3HR-1 cells. ACV was converted to the triphosphate to a greater extent in HSV-infected Vero cells than in mock-infected Vero cells or in HSV-infected P3HR-1 cells. ACV or its phosphorylated derivatives were converted to guanine nucleotides to a greater extent in lymphoblastoid cells than in fibroblasts (Vero). In conclusion, neither the productive replication of EBV nor the presence of latent viral DNA is required for ACV monophosphate formation in B lymphoblastoid cells. ACV triphosphate, however, was detected only in cells infected productively with EBV.     

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.     

Differential effect of nucleoside analog triphosphates on ribonucleotide reductases from uninfected and herpes simplex virus-infected HeLa cells.

Effects of the triphosphates of eight pyrimidine nucleoside analogs (5-substituted, 2'-fluoroara-, and acyclonucleosides) and acycloguanosine were examined on the ribonucleotide reductases prepared from uninfected and herpes simplex virus (types 1 and 2)-infected HeLa cells. Of the analogs tested, E-5-propenyl- and E-5-(2-bromovinyl)-dUTP were more potent inhibitors than dTTP of the enzymes from virus-infected cells, whereas only the former compound showed this effect on the uninfected HeLa enzyme.     

Physical mapping of drug resistance mutations defines an active center of the herpes simplex virus DNA polymerase enzyme.

The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.     

Herpes simplex virus type 1 and human DNA polymerase interactions with 2'-deoxyguanosine 5'-triphosphate analogues. Kinetics of incorporation into DNA and induction of inhibition

The ability of herpes simplex virus type 1 (HSV-1) DNA polymerase, HeLa polymerase alpha, and HeLa polymerase beta to utilize several dGTP analogues has been investigated using a defined synthetic template primer. The relative efficiencies of the triphosphates of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir triphosphate, ACVTP), 9-[(1,3-dihydroxy-2-propoxy)methyl] guanine (ganciclovir triphosphate, DHPGTP), and 2',3'-dideoxyguanosine (ddGTP) as substrates for the three polymerases were: HSV-1 polymerase, dGTP greater than ACVTP approximately equal to DHPGTP greater than ddGTP; polymerase alpha, dGTP greater than ACVTP approximately equal to DHPGTP much greater than ddGTP; polymerase beta, ddGTP greater than dGTP much greater than ACVTP approximately equal to DHPGTP. The potent inhibition of HSV-1 polymerase by ACVTP has been shown previously to be due to the formation of a dead-end complex upon binding of the next 2'-deoxynucleoside 5'-triphosphate encoded by the template after incorporation of acyclovir monophosphate into the 3' end of the primer (Reardon, J. E., and Spector, T. (1989) J. Biol. Chem. 264, 7405-7411). This mechanism was shown here to be a general mechanism for inhibition of polymerases by the obligate chain terminators, ACVTP and ddGTP. The ACVTP-induced inhibition was 30-fold more potent with HSV-1 polymerase than with polymerase alpha. This difference may contribute to the antiviral selectivity of this nucleotide analogue. The effect of ganciclovir monophosphate incorporation (a nonobligate chain terminator) on subsequent primer extension was also evaluated. With HSV-1 polymerase and polymerase alpha, although there was a considerable reduction in the efficiency of utilization of the 3'-DHPGMP-terminal primer, contrasting kinetic behavior was observed. With HSV-1 polymerase, insertion of DHPGTP resulted in a significant reduction in Vmax for subsequent nucleotide incorporations. In contrast, with polymerase alpha, a relatively small decrease in Vmax was accompanied by increased Km values for subsequent nucleotide incorporations.     

A point mutation within a distinct conserved region of the herpes simplex virus DNA polymerase gene confers drug resistance.

We have shown that a drug-resistant mutant from a clinical isolate of herpes simplex virus contains a single point mutation in the DNA polymerase gene that confers resistance to both acyclovir and foscarnet. The mutated amino acid is located within a distinct conserved region shared among alpha-like DNA polymerases which we designate region VII. We infer that these conserved sequences are directly or indirectly involved in the recognition and binding of nucleotide and PPi substrates.     

Inhibition of herpes simplex virus type 1 DNA polymerase activity by peptides from the UL42 accessory protein is largely nonspecific.

To identify regions in the UL42 protein of herpes simplex virus type 1 which affect viral DNA polymerase activity, a series of 96 overlapping pentadecapeptides spanning the entire 488 amino acids of the UL42 protein were synthesized and tested for their ability to inhibit polymerase activity on a primed single-stranded M13 DNA template. Two assays were used: formation of full-length double-stranded M13 molecules and rate of incorporation of deoxyribonucleoside triphosphates. Peptides from five noncontiguous regions of the UL42 protein were found to inhibit herpes simplex virus type 1 polymerase activity in both the presence and absence of UL42 protein. The most active peptides from each region correspond to amino acids 23 to 38 (peptide 6), 64 to 78 (peptide 14), 89 to 102 (peptide 19), 229 to 243 (peptide 47), and 279 to 293 (peptide 57). By two different methods (DNA mobility shift and DNA precipitation), peptides 14, 19, 47, and 57 were found to bind DNA; they most probably inhibit enzyme activity by this mechanism. Peptide 6 did not bind DNA and must act by some mechanism other than competing for DNA. The inhibitory peptides were also tested for activity against mammalian polymerase alpha and the Klenow fragment of Escherichia coli polymerase. Although some limited specificity was demonstrated (up to 10-fold for peptide 6), all the peptides showed significant activity against both polymerase alpha and E. coli polymerase.     

Deoxycytidine kinase from rabbit kidney cells infected with herpes simplex virus type 1 or 2.

When rabbit kidney cells were infected with herpes simplex virus type 1 (strain Seibert) or herpes simplex virus type 2 (strain 316D), deoxycytidine kinase (CdR kinase) activity, assayed at 38 degrees, increased 5- to 15-fold relative to controls. The CdR kinase activity induced by type 2 virus was more thermolabile than the enzyme activity induced by type 1 virus. When CdR kinase activity was assayed at various temperatures between 0.5 and 38 degrees, maximum activity for type 1 enzyme was obtained at 16 degrees while maximum activities for host and type 2 enzymes were obtained at 38 degrees. Both type 1 and type 2 induced CdR kinase activities eluted at the same positions as deoxythymidine kinase activities on a Sephadex G-100 column. The estimated mol wt for HSV-1 (Seibert) and HSV-2 (316D) induced CdR kinases are 67,000 and 60,000, respectively.     

Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.