Inhibition Of Viral DNA Synthesis Research Articles

Anti-(herpes simplex virus) activity of 4′-thio-2′-deoxyuridines: a biochemical investigation for viral and cellular target enzymes

The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4'-thio-2'-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20-600 times) than that shown by the corresponding 4'-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4'-thio-2'-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4'-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4'-thio-2'-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4'-oxy- and 4'-thio-2'-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4'-thio-2'-deoxyuridines are better inhibitors than their 4'-oxy counterparts of [(3)H]thymidine incorporation in HSV-1-infected cells; (vi) 4'-thio-2'-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4'-thio-2'-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4'-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.

On the control of late gene expression in Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8).

Herpesvirus late genes require viral DNA replication for maximal expression. Although late gene expression appears to require DNA replication in cis in alphaherpesviruses, studies in Epstein-Barr virus (EBV) suggest that this cis-requirement might not pertain to the gammaherpesviruses. Based on these findings, a system was created to investigate the elements required for the regulation of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus-8) late gene expression. The transcript of a classic late gene encoding the viral assembly protein was characterized and reporter genes driven by the assembly protein promoter region were constructed. Unlike the EBV case, expression of a reporter gene under the control of the assembly protein promoter did not display authentic regulation when removed from the context of the viral genome. Although reporter expression rose in cells displaying lytic replication, this expression was not diminished by specific inhibitors of viral DNA synthesis. Minimal core promoters were similarly unable to reproduce late gene regulation. These results suggest that proper KSHV late gene expression is likely to be dependent upon virus lytic replication in cis and indicate that the regulation of KSHV late genes more closely resembles that observed in herpes simplex virus than that described for EBV.

An African swine fever virus ERV1-ALR homologue, 9GL, affects virion maturation and viral growth in macrophages and viral virulence in swine.

The African swine fever virus (ASFV) genome contains a gene, 9GL, with similarity to yeast ERV1 and ALR genes. ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity. 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined. Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase-9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL). Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein. A 9GL gene deletion mutant of MAL (Delta9GL), exhibited a growth defect in macrophages of approximately 2 log(10) units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus. 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in Delta9GL-infected macrophages. The Delta9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all Delta9GL-infected animals survived infection. With the exception of a transient fever response in some animals, Delta9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with Delta9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL. Thus, ASFV 9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.

Identification and characterization of the UL14 gene product of herpes simplex virus type 2.

The UL14 gene of herpes simplex virus type 2 (HSV-2) is predicted to encode a 219 amino acid protein with a molecular mass of 23 kDa. In this study, the HSV-2 UL14 gene product has been identified by using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL14 fusion protein expressed in E. coli. The antiserum reacted specifically with 34, 33 and 28 kDa proteins in HSV-2-infected cell lysates and also with a 34 kDa protein produced by in vitro transcription and translation reactions, suggesting that the 34 kDa protein is the primary translation product of the UL14 gene. The protein was synthesized at late times post-infection (p.i.) and was not detectable in the presence of the viral DNA synthesis inhibitor acycloguanosine. Indirect immunofluorescence studies localized the UL14 protein both to the nucleus and to perinuclear regions of the cytoplasm, and the nuclear UL14 protein was found to co-localize with the scaffolding protein ICP35 at 9 h p.i. However, the protein accumulated in a perinuclear region of the cytoplasm at 12 h p.i., while most of the ICP35 protein localized within assemblons in the nucleus. Although no detectable UL14 protein was associated with intracellular capsids isolated in the presence of 0.5 M NaCl, it was detected in purified virions. Furthermore, the UL14 protein expressed alone was detected both in the nucleus and in the cytoplasm at 24 h after transfection, but was mainly localized to the cytoplasm at later times.

Reverse transcriptase inhibitors can selectively block the synthesis of differently sized viral DNA transcripts in cells acutely infected with human immunodeficiency virus type 1.

We have recently reported that the in vitro inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcription by inhibitors of reverse transcriptase (RT) occurred most efficiently when the expected DNA products of RT reactions were long (Quan et al. , Nucleic Acids Res. 26:5692-5698, 1998). Here, we have used a quantitative PCR to analyze HIV-1 reverse transcription within acutely infected cells treated with RT inhibitors. We found that levels of minus-strand strong-stop DNA [(-)ssDNA] formed in acutely infected MT2 cells were only slightly reduced if cells were infected with viruses that had been generated in the presence of either azidothymidine or nevirapine (5 microM) and maintained in the presence of this drug throughout the viral adsorption period and thereafter. Control experiments in which virus inoculation of cells was performed at 4 degrees C, followed directly by cell extraction, showed that less than 1% of total (-)ssDNA within acutely infected cells was attributable to its presence within adsorbed virions. In contrast, synthesis of intermediate-length reverse-transcribed DNA products decreased gradually as viral DNA strand elongation took place in the presence of either of these inhibitors. This establishes that nucleoside and nonnucleoside RT inhibitors can exert similar temporal impacts in regard to inhibition of viral DNA synthesis. Generation of full-length viral DNA, as expected, was almost completely blocked in the presence of these antiviral drugs. These results provide insight into the fact that high concentrations of drugs are often needed to yield inhibitory effects in cell-free RT assays performed with short templates, whereas relatively low drug concentrations are often strongly inhibitory in cellular systems.

Expression of the open reading frame 74 (G-protein-coupled receptor) gene of Kaposi's sarcoma (KS)-associated herpesvirus: implications for KS pathogenesis.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) encodes a G-protein-coupled receptor (GCR) homolog. This protein is a potent, constitutively active signalling molecule that can influence both proliferation and angiogenesis when ectopically expressed in fibroblasts in vitro. Here we have examined the expression of the KSHV GCR gene in virus-infected lymphoid cells and in KS tumors. Our results show that in both situations the gene is expressed primarily during lytic replication; its transcription is unaffected by inhibition of viral DNA synthesis, indicating that it is expressed in the early phases of the lytic program. The major transcript bearing GCR sequences is bicistronic, harboring coding sequences for another viral gene, K14, at its 5' end. Extensive searches for monocistronic GCR mRNAs using nuclease mapping and reverse transcription-PCR failed to detect such species. The 5' end of K14/GCR mRNA maps to nucleotide (nt) 127848, and its poly(A) addition site maps to nt 130546; a 149-nt intron is present in the K14/GCR intergenic region. These results suggest that the KSHV GCR is translated by unconventional mechanisms involving either translational reinitiation, internal ribosomal entry, or leaky ribosomal scanning. The restriction of GCR expression to the lytic cycle has important implications for the potential role(s) of the GCR in KS pathogenesis.

Kaposi's sarcoma-associated herpesvirus encodes a bZIP protein with homology to BZLF1 of Epstein-Barr virus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a recently discovered human gamma herpesvirus strongly implicated in AIDS-related neoplasms. We report here the identification in the KSHV genome of a gene for a protein designated K-bZIP and belonging to the basic-leucine zipper (bZIP) family of transcription factors. K-bZIP shows significant homology to BZLF1, which plays a key role in the replication and reactivation of Epstein-Barr virus. K-bZIP is a homodimerizing protein of 237 amino acids with a prototypic bZIP domain at the C terminus. The N-terminal portion of K-bZIP is derived from the K8 open reading frame which, through in-frame splicing, adjoins the ZIP domain. This structure was revealed by rapid analysis of cDNA ends, followed by cloning of the entire cDNA. A 1.35-kb transcript encoding K-bZIP was detected in BCBL-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate. The synthesis of this transcript was blocked by the protein synthesis inhibitor cycloheximide but not by the viral DNA synthesis inhibitor phosphonoacetate, a result which classifies it as an early lytic gene. RNase protection analysis further mapped the major transcription start site for the 1.35-kb K-bZIP mRNA and identified two other splice variants which encode proteins with the N-terminal portion of K-bZIP but lacking the C-terminal ZIP domain. Full-length K-bZIP forms dimers with itself, and the C terminus encompassing the ZIP domain is required for this process. Our studies set the stage for understanding the role of K-bZIP in the replication and reactivation of the KSHV genome.

Human Cytomegalovirus Infection Downregulates Expression of the Cellular Aminopeptidases CD10 and CD13

During the course of a productive infection, human cytomegalovirus (HCMV) has a sophisticated relationship with its host cell. An increasing number of virus-encoded genes are being identified which act specifically to usurp or modulate functions in the host cell associated with transcriptional control, cell signalling, and protein synthesis. While HCMV infection is associated with a general upregulation of cellular gene expression, the expression a small subset of cellular proteins, including the MHC-1 heavy chain and fibronectin, is downregulated. This study now identifies two additional cellular proteins, aminopeptidase N (CD13) and neutral endopeptidase (CD10), that are downregulated during HCMV infection. While aminopeptidase N and neutral endopeptidase exhibit no significant sequence homology, both are expressed on the cell surface and have very similar enzymatic properties. HCMV infection was associated with reduced surface expression and enzyme activity of CD13 and CD10, an apparent decrease in the rate of synthesis of both proteins in metabolic-labelling experiments, and inhibited glycosylation of the nascent CD13 and CD10 polypeptide chains that were synthesized. Levels of CD10 poly A+RNA were suppressed efficiently at all stages of virus infection; however, the reduction in CD13 poly A+RNA levels was much less pronounced. This differential effect suggests that HCMV may be downregulating expression of CD10 and CD13 by independent mechanisms. Indeed, treatment of cells with an inhibitor of viral DNA synthesis blocks downregulation of CD13, whilst downregulation of CD10 is unaffected. While it is not yet clear what advantage is bestowed on the virus by downregulating expression of CD13 and CD10, aminopeptidases are known to have a role in peptide processing in both the MHC class I the MHC class II antigen presentation pathways.

Characterization of the UL55 gene product of herpes simplex virus type 2.

We have identified the herpes simplex virus type 2 (HSV-2) UL55 gene product using a rabbit polyclonal antiserum raised against a recombinant 6 x His-UL55 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 23 kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies localized the UL55 protein within and at the periphery of the nucleus as discrete granules at late times post-infection, and nuclear fractionation studies showed that the protein was associated with the nuclear matrix of infected cells. Moreover, these discrete regions containing the UL55 protein were found to be adjacent to compartments, designated assemblons, containing the capsid protein ICP35. However, the UL55 protein was not detected in purified virions. These results suggest that the UL55 protein of HSV-2 may play an accessory role in virion assembly or maturation.

Characterization of the UL4 gene product of herpes simplex virus type 2.

We have identified the herpes simplex virus type 2 (HSV-2) UL4 gene product using a rabbit polyclonal antiserum raised against a recombinant 6xHis-UL4 fusion protein expressed in Escherichia coli. The antiserum reacted specifically with a 27-kDa protein in HSV-2 186-infected cell lysates. The protein was not detectable in the presence of the viral DNA synthesis inhibitor, suggesting that the UL4 gene was expressed as a gamma 2 gene. Indirect immunofluorescence studies localized the UL4 protein within the nucleus as discrete punctate forms at late times postinfection. However, when expressed in the absence of other viral proteins, the UL4 protein was limited to the cytoplasm, indicating that an interaction with one or more other virus-induced proteins was responsible for the nuclear localization during infection. Subnuclear fractionation studies showed that the protein was released from the nuclear structure of infected cells by high salt treatment. Moreover, the UL4 protein was detected in purified virions and light particles.

Lamivudine Therapy of WHV-Infected Woodchucks

Hepatitis B viruses establish a chronic, productive, and noncytopathic infection of hepatocytes. Viral products are produced by transcription from multiple copies (5–50) of covalently closed circular (ccc) viral DNA. This cccDNA does not replicate, but can be replaced by DNA precursors that are synthesized in the cytoplasm. The present study was carried out to determine if long-term treatment with an inhibitor of viral DNA synthesis would lead to loss of virus products, including cccDNA, from the liver of woodchucks chronically infected with woodchuck hepatitis virus. Viral DNA synthesis was inhibited with the nucleoside analog, lamivudine (2′-deoxy-3′-thiacytidine). Lamivudine treatment produced a slow but progressive decline in viral titers in serum, to about 0.3% or less of the initial level. However, even after maintenance of drug therapy for 3–12 months, >95% of the hepatocytes in most animals were still infected. Significant declines in the percentage of infected hepatocytes and of intrahepatic cccDNA levels were observed in only three woodchucks, two in the group receiving lamivudine and one in the placebo control group. Moreover, virus titers eventually rose in woodchucks receiving lamivudine, suggesting that drug-resistant viruses began to spread through the liver starting at least as early as 9–12 months of treatment. Three types of mutation that may be associated with drug resistance were found at this time, in a region upstream of the YMDD motif in the active site of the viral reverse transcriptase. The YMDD motif itself remained unchanged. Not unexpectedly, the lamivudine therapy did not have a impact on development of liver cancer.

Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.

The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC. Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.

Infection of a human retinal pigment epithelial cell line with human herpesvirus 6 variant A

A retinal pigment epithelial (RPE) cell line (K-1034) was examined for its susceptibility to human herpesvirus 6 variant A (HHV-6A). Exposure of K-1034 cells to HHV-6A induced the formation of multinucleated giant cells, which was suppressed by an inhibitor of viral DNA synthesis. In the giant cells, herpesvirus nucleocapsids were demonstrated by electron microscopy and the viral glycoprotein B was detected by immunofluorescence assay. These results indicate that K-1034 cells are susceptible to HHV-6A and suggest that HHV-6A has an ability to directly destroy epithelial cells.

Characterization of the products of the U(L)43 gene of herpes simplex virus 1: potential implications for regulation of gene expression by antisense transcription.

The products, RNA or proteins, of the herpes simplex virus 1 open reading frame U(L)43 have not been previously identified. The expression of an open reading frame antisense to U(L)43, U(L)43.5 (P. L. Ward, D. E. Barker, and B. Roizman, J. Virol. 70:2684-2690, 1996), has been reported. We report the existence of a transcript corresponding to the domain of the U(L)43 open reading frame extending approximately 30 bp from the predicted TATA box to the predicted polyadenylation signal. The RNA of the U(L)43 open reading frame accumulates to higher levels in the presence of phosphonoacetic acid, an inhibitor of viral DNA synthesis, than in its absence, whereas the U(L)43.5 transcript accumulates in larger amounts in the absence of phosphonoacetic acid. The open reading frame tagged with a sequence encoding a 20-amino-acid epitope yielded a protein with an apparent Mr of 32,000, i.e., considerably lower than that predicted from the size of the open reading frame. The discovery of a pair of antisense genes expressed during productive infection raises the possibilities that additional antisense genes exist and that the antisense arrangement provides still another mechanism for regulation of gene expression.

Inhibition of topoisomerase II by ICRF-193 prevents efficient replication of herpes simplex virus type 1

Cellular topoisomerase II is specifically inactivated by the drug ICRF-193. This compound turns topoisomerase II into a closed clamp that is unable to cleave DNA. We have investigated the effects of this inhibitor on the replication of herpes simplex virus type 1. We show that ICRF-193 at low multiplicities of infection dramatically inhibits viral DNA synthesis and the production of infectious virus. The inhibition is less efficient at high multiplicities of infection. In addition, inhibition of viral DNA synthesis was observed only when ICRF-193 was present during the first 4 h of the infectious cycle. The transient replication of plasmids containing a herpes simplex virus type 1 origin of DNA replication, oriS, was affected by ICRF-193 in the same way. In contrast, neither cellular DNA synthesis nor replication of plasmids containing a simian virus 40 origin of DNA replication was inhibited. The observed effect on herpes simplex virus DNA replication was not caused by a decreased transcription of replication genes inasmuch as the levels of UL8, UL9, UL29, and UL30 rmRNAs were unaffected by the drug. These results suggest that topoisomerase II plays a vital role during the replication of herpes simplex virus type 1 DNA. We speculate that topoisomerase II is involved in the decatenation of newly synthesized daughter molecules.

Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein.

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

Inhibition of Varicella-Zoster Virus by Penciclovir in Cell Culture and Mechanism of Action

The effect of penciclovir (BRL 39123) on the replication of varicella-zoster virus (VZV) in human embryonic lung fibroblasts (MRC-5 cells) was similar to aciclovir when the compounds were present continuously. However, when the compounds were withdrawn the antiviral activity of penciclovir was maintained more effectively than that of aciclovir. In the plaque reduction assay, median 50% effective concentrations (EC50s) were 3.8 μg ml−1 for penciclovir and 4.2 μg ml−1 for aciclovir ( n = 29 clinical isolates). Similarly, penciclovir and aciclovir were equally effective in reducing the numbers of VZV-infected MRC-5 cells and in reducing VZV DNA synthesis within infected cells following continuous treatment. Within VZV-infected cells (S)-penciclovir-triphosphate was formed from penciclovir with >95% enantiomeric purity, and the concentration of penciclovir-triphosphate was 360-fold greater than aciclovir-triphosphate immediately after treatment. This phosphorylation ratio compensates for the lower affinity of VZV DNA polymerase for penciclovir-triphosphate compared with aciclovir-triphosphate (Kis = 7.5 μM and 0.2 μM, respectively). When VZV-infected cultures were treated for 3 days, followed by withdrawal of the compound, inhibition of viral DNA synthesis by penciclovir was maintained for 24 h, whereas viral DNA synthesis resumed more readily after removal of aciclovir. Furthermore, following 8 h daily pulse treatment for 5 days, penciclovir was significantly more active than aciclovir in reducing VZV DNA synthesis ( p = 0.006, n = 10 clinical isolates). The long intracellular half-life of penciclovir-triphosphate (9.1 h) compared with that of aciclovir-triphosphate (0.8 h) accounts for the sustained inhibition of virus replication by penciclovir. This property may contribute to the clinical efficacy of famciclovir, the oral form of penciclovir.

Activity of penciclovir in antiviral assays against herpes simplex virus.

The effect of penciclovir and acyclovir on the replication of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) strains was determined in MRC-5 cells infected with 0.01 pfu/cell and exposed to the drugs for 72 h to allow multiple cycles of replication. Penciclovir was significantly more active than acyclovir against three strains of HSV-1 and three strains of HSV-2 at 1 mg/L (P = 0.009), 3 mg/L (P < 0.001) and 10 mg/L (P = 0.001). Further comparisons between the compounds were made in MRC-5 cells infected with HSV-1 strain SC16 using four different antiviral assays namely, the 24 h virus yield reduction assay, plaque reduction assay, viral antigen inhibition assay, and a viral DNA inhibition assay, to determine the relative merits of each. Penciclovir and acyclovir shared similar activities in the plaque reduction assay (with 50% effective concentrations, EC50, being 0.8 and 0.6 mg/L, respectively) and in the viral antigen inhibition assay (EC50s. 0.6 and 0.7 mg/L, respectively). The EC50 of penciclovir in the 24 h viral DNA inhibition assay was 0.01 mg/L compared with 0.06 mg/L of acyclovir. In the 24 h virus yield reduction assay in which MRC-5 cells were infected with 0.3 pfu/cell, penciclovir was more active than acyclovir with 99% effective concentrations of 0.6 mg/L and 1.1 mg/L, respectively. The activity of penciclovir in the 24 h virus yield reduction and antigen inhibition assays was inversely related to the multiplicity of infection, whereas this had considerably less effect on the inhibition of viral DNA synthesis. These results suggest that famciclovir, which is the oral form of penciclovir, will be at least as effective as acyclovir in treating infections caused by HSV-1 and HSV-2.

Potent Inhibition of Herpes Simplex Virus by MDL 101028, a Novel Biphenyl Disulfonic Acid Urea Co-Polymer

A novel low-molecular-weight sulfonic acid polymer, MDL 101028, was investigated for antiviral activity against herpes simplex virus (HSV) type 1 and type 2 in human embryonic diploid fibroblasts (MRC-5) and in a line of monkey kidney cells (Vero). Potent antiviral activities were obtained when treatment was restricted to the adsorption phase of virus propagation as measured by plaque reduction assay, and this was confirmed by experiments at high multiplicity of infection. Inhibition of virus binding to host cells by MDL 101028 was compared to that of heparin, a known antagonist of virus attachment. Both compounds showed dose-dependent inhibition, with IC50 values of 1.1 and 0.43 μg ml−1, respectively. The effect on cell-to-cell fusion was investigated using a syncytial-positive (syn+) phenotypic mutant of HSV-1 (strain 17i). MDL 101028 (3-1 μm) dramatically halted the spread of syncytial lesions; this effect was accompanied by the destruction of the syncytium and regrowth into the lesion by uninfected Vero cells. In contrast, heparin (10 μg ml−1) only partially reduced the spread of syncytia. Protection was also observed with MDL 29797 (100 μg ml−1), an antagonist of glycoprotein maturation. Unexpectedly, only a marginal effect was observed with acyclovir (10 μg ml−1), a potent inhibitor of viral DNA synthesis and HSV growth. As an extension to the current therapy for herpes simplex infection, acyclovir, the potent anti-fusion activity of MDL 101028 may have a clinical use in tissues or organs in which cell-to-cell fusion contributes to the pathology.

Polymorphism within the herpes simplex virus (HSV) ribonucleotide reductase large subunit (ICP6) confers type specificity for recognition by HSV type 1-specific cytotoxic T lymphocytes.

A panel of herpes simplex virus type 1 (HSV-1)-specific, CD8+, major histocompatibility complex class I (H-2Kb)-restricted cytotoxic T-lymphocyte (CTL) clones was derived from HSV-1-immunized C57BL/6 (H-2b) mice in order to identify the HSV-1 CTL recognition epitope(s) which confers type specificity. HSV-1 x HSV-2 intertypic recombinants were used to narrow the region encoding potential CTL recognition epitopes to within 0.51 to 0.58 map units of the HSV-1 genome. Using an inhibitor of viral DNA synthesis and an ICP6 deletion mutant, the large subunit of ribonucleotide reductase (ICP6, RR1) was identified as a target protein for these type-specific CTL. Potential CTL recognition epitopes within RR1 were located on the basis of the peptide motif predicted to bind to the MHC class I H-2Kb molecule. A peptide corresponding to residues 822 to 829 of RR1 was shown to confer susceptibility on H-2Kb-expressing target cells to lysis by the type 1-specific CTL. On the basis of a comparison of the HSV-1 RR1 epitope (residues 822 to 829) with the homologous sequence of HSV-2 RR1 (residues 828 to 836) and by the use of amino acid substitutions within synthetic peptides, we identified HSV-1 residue 828 as being largely responsible for the type specificity exhibited by HSV-1-specific CTL. This HSV-1 RR1 epitope, when expressed in recombinant simian virus 40 large T antigen in primary C57BL/6 cells, was recognized by the HSV-1 RR1-specific CTL clones. These results indicate that an early HSV protein with enzymatic activity provides a target for HSV-specific CTL and that type specificity is dictated largely by a single amino acid.