Antiviral efficacy in vivo of the anti-human immunodeficiency virus bicyclam SDZ SID 791 (JM 3100), an inhibitor of infectious cell entry.

SID 791, a bicyclam inhibiting human immunodeficiency virus (HIV) replication in vitro by blocking virus entry into cells, is an effective inhibitor of virus production and of depletion of human CD4+ T cells in HIV type 1-infected SCID-hu Thy/Liv mice. Steady levels of 100 ng of SID 791 or higher per ml in plasma resulted in statistically significant inhibition of p24 antigen formation. Daily injections of SID 791 caused a dose-dependent decrease in viremia, and this inhibition could be potentiated by coadministration of zidovudine or didanose. The present study suggests that SID 791 alone or in combination with licensed antiviral agents may decrease the virus load in HIV-infected patients and, by extension, that the infectious cell entry step is a valid target for antiviral chemotherapy of HIV disease. The SCID-hu Thy/Liv model in effect provides a rapid means of assessing the potential of compounds with novel modes of antiviral action, as well as the potential of antiviral drug combinations.

Use of standardized SCID-hu Thy/Liv mouse model for preclinical efficacy testing of anti-human immunodeficiency virus type 1 compounds.

We have developed standardized procedures and practices for infection of SCID-hu Thy/Liv mice with human immunodeficiency virus type 1 for the prophylactic administration of antiviral compounds and for evaluation of the antiviral effect in vivo. Endpoint analyses included quantitation of viral load by intracellular p24 enzyme-linked immunosorbent assay, DNA PCR for the presence of proviral genomes, flow cytometry to measure the representation of CD4+ and CD8+ cells, and cocultivation for the isolation of virus. Efficacy tests in this model are demonstrated with the nucleoside analogs zidovudine and dideoxyinosine and with the nonnucleoside reverse transcriptase inhibitor nevirapine. This small-animal model should be particularly useful in the preclinical prioritization of lead compounds within a common chemical class, in the evaluation of alternative in vivo dosing regimens, and in the determination of appropriate combination therapy in vivo.

The molecular target of bicyclams, potent inhibitors of human immunodeficiency virus replication.

Bicyclams are a novel class of antiviral compounds which act as potent and selective inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2. They block an early step in the viral life cycle following adsorption to the CD4 receptor and preceding reverse transcription. To identify the molecular target of these compounds, we genetically analyzed variants of the HIV-1 molecular clone NL4-3, which developed resistance against two structurally related bicyclams, JM2763 and the more potent SID791. The resistant strains were obtained after long-term passaging in MT-4 cells in the presence of progressively increasing compound concentrations. Recombinants between selected genes of the resistant strains and the parental NL4-3 provirus were generated by adapting the marker rescue technique to MT-4 cells. The bicyclam-resistant phenotype was rescued by transferring the envelope gp120 gene of bicyclam-resistant virus into the NL4-3 parental genetic background. In the gp120 genes of the resistant strains, we identified several mutations leading to amino acid substitutions in the V3 loop. Furthermore, two substitutions of highly conserved amino acids in close proximity to the disulfide bridges of the V3 and V4 loops were found in both SID791- and JM2763-resistant strains. Additional mutations in regions encoding V3, C4, V5, and C5 were present in SID791-resistant viruses. Recombination experiments with overlapping parts of the envelope gene indicated that most, if not all, of the mutations were necessary to develop the fully SID791 resistant phenotype. The mutations in the C-terminal part of gp120 downstream of the V3 loop sequence conferred partial resistance to JM2763 but did not significantly decrease susceptibility to SID791. The genetic data and the biological properties of the resistant viruses point to inhibition of entry and fusion as the mode of action of the HIV-inhibitory bicyclams. A possible mechanism of binding of bicyclams to gp120 leading to inhibition of unfolding of gp120 and its shedding from the gp41 fusion domain is discussed.

Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonimmunosuppressive cyclosporine analog.

(Me-Ile-4)cyclosporin (SDZ NIM 811) is a 4-substituted cyclosporin which is devoid of immunosuppressive activity but retains full capacity for binding to cyclophilin and exhibits potent anti-human immunodeficiency virus type 1 (HIV-1) activity. SDZ NIM 811 selectively inhibits HIV-1 replication in T4 lymphocyte cell lines, in a monocytic cell line, and in HeLa T4 cells. Furthermore, its antiviral activity against laboratory strains and against clinical isolates from geographically distinct regions in primary T4 lymphocytes and in primary monocytes (50% inhibitory concentration = 0.011 to 0.057 micrograms/ml) was demonstrated. SDZ NIM 811 does not inhibit proviral gene expression or virus-specific enzyme functions, either free or bound to cyclophilin. The compound does not influence CD4 expression or inhibit fusion between virus-infected and uninfected cells. SDZ NIM 811 was, however, found to block formation of infectious particles from chronically infected cells. Oral administration to mice, rats, dogs, and monkeys resulted in levels in blood considerably exceeding the drug concentration, which completely blocked virus replication in primary cells. SDZ NIM 811 caused changes of toxicity parameters in rats to a smaller degree than cyclosporine (formerly cyclosporin A). Thus, the potent and selective anti-HIV-1 activity of SDZ NIM 811 and its favorable pharmacokinetic behavior together with its lower nephrotoxicity than that of cyclosporine make this compound a promising candidate for development as an anti-HIV drug.

Highly potent and selective inhibition of human immunodeficiency virus by the bicyclam derivative JM3100.

Bicyclams, in which the cyclam (1,4,8,11-tetraazacyclotetradecane) moieties are tethered via an aliphatic bridge (i.e., propylene, as in JM2763) are potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) (E. De Clercq, N. Yamamoto, R. Pauwels, M. Baba, D. Schols, H. Nakashima, J. Balzarini, Z. Debyser, B. A. Murrer, D. Schwartz, D. Thornton, G. Bridger, S. Fricker, G. Henson, M. Abrams, and D. Picker, Proc. Natl. Acad. Sci. USA 89:5286-5290, 1992). We have now found that the bicyclam JM3100, in which the cyclam moieties are tethered by an aromatic bridge [i.e., phenylenebis(methylene)], inhibits the replication of various HIV-1 and HIV-2 strains in various cell lines at a 50% effective concentration (EC50) of 1 to 10 ng/ml, which is about 100-fold lower than the concentration required for JM2763 to inhibit HIV replication and at least 100,000-fold lower than the cytotoxic concentration (> 500 micrograms/ml). In primary T4 lymphocytes or primary monocytes, JM3100 proved inhibitory to HIV-1(IIIB) and several clinical HIV-1 isolates at an EC50 of less than 1 ng/ml. On the basis of time-of-addition experiments, JM3100 appeared to interact with a viral uncoating event, and this was further corroborated by an uncoating assay in which RNase sensitivity of [5-3H]uridine-labeled virions was monitored. In addition, but possibly mechanistically related, JM3100 blocks formation of infectious particles. JM3100 was also found to interfere directly with virus-induced syncytium formation, albeit at a higher concentration (1 to 2 microgram/ml) than that required for inhibition of viral replication. Following subcutaneous injection of 10 mg of JM3100 per kg of body weight to rabbits, anti-HIV activity was detected in serum corresponding to serum drug levels exceeding for at least 6 h by >100-fold the EC(50) required to inhibit HIV replication in vitro. When combined with either 3'-azido-2',3' -dideoxythymidine or 2',3' -dideoxyinosine, JM3100 achieved a additive inhibition of HIV replication, and when repeatedly subcultivated in the presence of JM3100, the virus remained sensitive to the compound for at least 30 passages (120 days) in cell culture.

Synthesis and antiviral activity of 2'-substituted 9-[2-(phosphonomethoxy)ethyl]guanine analogues.

A series of 2'-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and evaluated in vitro for anti-human immunodeficiency virus (HIV) activity in the XTT assay and for anti-herpes activity in the plaque reduction assay. It has been observed that the anti-HIV activity of these derivatives depends on the size and the nature of the substituent as well as the chirality at the 2'-position of PMEG. In addition, these compounds generally demonstrated greater activity against HIV than herpes viruses. The most interesting analogues which emerged from these studies are (R)-2'-(azidomethyl)-PMEG [(R)-5] and (R)-2'-vinyl-PMEG [(R)-11]. The former showed anti-HIV activity with an IC50 of 5 microM and a cytotoxicity (CC50) greater than 1.4 mM in CEM cells. The latter has an IC50 of 13 microM for anti-HIV activity and a CC50 of greater than 1.6 mM. Furthermore, we have demonstrated that replacement of the guanine base of these 2'-substituted PMEG analogues with cytosine drastically reduces anti-HIV and anti-herpes activity.

Synthesis and antiviral activity of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine.

A number of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and tested in vitro for anti-herpes and anti-human immunodeficiency virus (HIV) activity. Among these analogues, (R)-2'-methyl-PMEG [(R)-3] and 2',2'-dimethyl-PMEG (7) demonstrated potent anti-HIV activity in the XTT assay with EC50 values of 1.0 and 2.6 microM, respectively. The corresponding (S)-2'-methyl-PMEG [(S)-3] was found to be less potent against HIV. In addition, the (R) and (S) enantiomers of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG, 8) were prepared for comparison of biological activity, and shown to be active and equipotent against herpesviruses, but inactive against HIV.

Prolonged and potent therapeutic and prophylactic effects of (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine against herpes simplex virus type 2 infections in mice.

The acyclic nucleotide analog (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) is a potent and selective inhibitor of herpesviruses. Cells preincubated with HPMPC are refractory to herpes simplex virus type 2 (HSV-2) infection for several days after removal of the drug from the medium. A single administration of 30 mg of HPMPC per kg of body weight 4 days prior to virus infection intraperitoneally with HSV-2 (strain G) completely protected mice from death, and the protective effect was dose dependent. HPMPC was equally efficacious in protecting mice when the same total amount of the drug was administered as a single dose as when it was given daily in several smaller doses (5 mg/kg with treatment initiation at 3 h postinfection [p.i.], 90 versus 80% survival, respectively). In contrast, ganciclovir [9(1,3-dihydroxy-2-propoxymethyl)guanine] was more efficacious when it was given daily than it was when it was given less than daily in a late stage of HSV-2 infection (100 mg/kg; when mice were treated 96 h p.i., 80 versus 50% survival, respectively; when mice were treated 120 h p.i., 60 versus 20% survival, respectively). Therefore, single doses of HPMPC were more effective than ganciclovir in protecting mice from death (80 versus 20% survival, respectively; P less than 0.05), whereas there was no difference when the drugs were given daily (50 versus 60% survival, respectively). Our studies suggest a potential of HPMPC for conventional and prophylactic treatments of herpesvirus infections with infrequent drug administration.

Selective induction of discrete epitopes of herpes simplex virus type 1-specified glycoprotein C by interference with terminal steps in glycosylation.

We have described two types of oligosaccharide modification influencing the antigenicity of the herpes simplex virus type 1 (HSV-1)-specified glycoprotein C (gC-1). First, the expression of several epitopes belonging to antigenic site II of gC-1 is dependent on the peripheral galactose of N-linked oligosaccharides. We have also shown that treatment of HSV-1-infected cells with 5-n-propyl-2'-deoxyuridine (PdU) under certain circumstances results in other modifications of peripheral carbohydrate determinants, which are associated with increased antigenic activity of gC-1. In the present study we have mapped and characterized the epitopes susceptible to PdU induction by analysing the reactivity to a number of monoclonal antibodies defining several epitopes of antigenic sites I and II. The results indicate that the strict galactose dependence of epitopes and the PdU-induced increase of antigenic activity are independent and unrelated phenomena. Thus, we identified galactose-dependent epitopes that were not PdU-inducible and vice versa, and some epitopes were both galactose-dependent and PdU-inducible. The results support a model where PdU treatment blocks synthesis of an antigen-masking carbohydrate determinant. In addition, PdU treatment of HSV-1-infected cells seemed to increase the antigenic activity of other HSV-1 glycoproteins.

Inhibition of HIV-1 reverse transcriptase by 5'-triphosphates of 5-substituted uridine analogs.

The 5'-triphosphates of some 5-substituted 2'-deoxyuridine analogs were investigated for their effects on purified recombinant reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) as well as cellular DNA polymerase alpha. The triphosphates were competitive inhibitors of the viral enzyme with dTTP as the variable substrate and poly(rA)oligo(dT) as template, and preferentially inhibited the viral polymerase. Ordering the compounds according to their decreasing binding affinities, as reflected by their increasing inhibition constants for the reverse transcriptase, gave nPrearaUTP greater than nPrdUTP greater than EtdUTP greater than nPredUTP greater than HMdUTP greater than CEdUTP. Although nPredUTP was less inhibitory than nPrearaUTP under conditions of competitive inhibition, nPredUTP caused a time- and concentration-dependent inactivation of reverse transcriptase activity when preincubated with template. This inactivation was not reversed by excess dTTP. The decrease in template-primer activity did not occur with nPrearaUTP, but was shown with the chain-terminating 5'-triphosphates of 3'-fluoro- and 3'-azidothymidine. As nPredUTP, but not nPrearaUTP, was an alternative substrate, shown by the ability to support DNA synthesis in absence of competing substrate, the incorporation of nPredUTP into the primer-template apparently leads to increased inhibition of the enzyme.

Comparison of foscarnet and foscarnet esters as anti-influenza virus agents.

Foscarnet is shown to inhibit influenza A virus replication by inhibiting viral RNA synthesis in infected cells. Viral RNA synthesis by isolated nuclei from infected cells was as sensitive to foscarnet as viral RNA synthesis by enzymes from isolated virions or viral cores. It is, therefore, unlikely that the mere association of the polymerase in a replication complex, as in isolated nuclei and infected cells, is the reason for the fact that foscarnet is 10-20 times less active in inhibiting virus replication than cell-free RNA synthesis. We, therefore, tested 44 esters of foscarnet for improved antiviral effect. Of these only a few phenyl esters were more potent than foscarnet itself. These esters did not inhibit the viral RNA polymerase activity and may be hydrolyzed intracellularly to foscarnet. The increased antiviral potency of the phenyl esters was, however, accompanied by increased cellular toxicity, and these compounds, therefore, were less selective antiviral agents than foscarnet. The results suggest that it is not possible to increase the anti-influenza activity of foscarnet by converting it to an ester.

An analysis of the inhibition of replication of HIV and MuLV by some 3'-blocked pyrimidine analogs.

Some 3'-blocked pyrimidine analogs were synthesized and tested as inhibitors of replication of human immunodeficiency virus (HIV) and Moloney-murine leukemia virus (MuLV). The analogs were of 3 kinds: (1) analogs of 3'-azido-3'-deoxythymidine (AZT) in which the C-5 CH3 of the base was exchanged for H (AZU) or C2H5 (AZEU); (2) 3'-fluoro-3'-deoxythymidine (FLT) and analogs thereof, in which the C-5 CH3 of the base was exchanged for H (FLU), C2H5 (FLEU) or nC3H7 (FLPU); (3) the threo analogs of AZT (AZT increases) and AZU (AZU increases). All analogs were less active inhibitors of HIV replication than AZT, except FLT, which was as active as AZT. The 3'-fluoro analogs and AZEU did not inhibit MuLV replication at non-cytotoxic concentrations. Oral administration of FLT to MuLV-infected mice result in antiviral effects only at toxic drug levels. AZU and FLU were less potent inhibitors of HIV replication than AZT or FLT, but the 2'-deoxy uridine analogs were less cytotoxic to human embryonic fibroblasts than the thymidine analogs. The 5'-triphosphates of AZU, AZT, AZEU, FLT and FLEU were tested as inhibitors of the HIV- and MuLV-reverse transcriptases. Ranking of the Ki/Km values for HIV-RT resulted in the following order of potency of the 5'-triphosphates AZT = FLT greater than AZU greater than AZEU greater than FLEU. The 5'-triphosphates of AZEU, FLT and FLEU did not inhibit the MuLV-RT, which explains, in part, the lack of effect of these analogs against MuLV replication. The threo forms (azido "up") of AZU and AZT were less active inhibitors of HIV replication than the erythro forms (azido "down"). A 15N-NMR and 1H-NMR study showed that the furanose moieties of analogs with the azido function "up" assume a conformation distinct from that of the analogs with azido "down". This is due to intramolecular stabilisation of the "N" conformer in the threo ("up") diastereomer, due to interaction of the azido functions with the nucleobase and possibly the OH group of C-5' of the furanose. As discussed, this conformation might explain the decreased biological activity of threo forms compared with the erythro forms.

Improvement of the absorption of oral (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, an anti-varicella-zoster virus drug, in rats and monkeys.

In an effort to improve the gastrointestinal absorption of (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine [(+/-)2HM-HBG], various salts and esters of the compound were synthesized and pharmacokinetic experiments were performed in rats and monkeys. The sodium or hydrochloride salts and short-chain esters of (+/-)2HM-HBG showed bioavailability characteristics that were equally as poor as those of (+/-)2HM-HBG. However, the esters given as salts tended to be better absorbed than the parent compound. The 6-deoxy and 6-deoxydiacetate analogs were extensively oxidized in vivo and represent prodrugs with considerable potential in improving the absorption of oral (+/-)2HM-HBG.

Inhibition of human hepatitis B virus DNA polymerase and duck hepatitis B virus DNA polymerase by triphosphates of thymidine analogs and pharmacokinetic properties of the corresponding nucleosides.

Replication of hepadnaviruses involves a viral DNA polymerase containing both a DNA-dependent and an RNA dependent activity. This polymerase is a potential target for chemotherapy against hepatitis B. We have used human hepatitis B virus DNA-dependent DNA polymerase from human serum and duck hepatitis B virus DNA-dependent DNA polymerase from duck serum as well as RNA-dependent DNA polymerase activity from duck hepatitis B-infected duck liver. Triphosphates of thymidine analogs have been synthesized and tested for their inhibitory activities against these enzymes with the intention both to explore differences between these enzymes and structural requirements for inhibitors. The results showed that with the inhibitors tested, hepatitis B virus DNA-dependent DNA polymerase was the most sensitive enzyme and the triphosphate of 5-propenyl-2'-deoxyuridine was the most active inhibitor. In addition, the 5'-triphosphate of 5-propenyl-arabinofuranosyluracil also inhibited the hepadnavirus DNA-dependent DNA polymerases, and was a competitive inhibitor with respect to 2'-deoxythymidine triphosphate as showed by kinetic studies with duck hepatitis B virus DNA-dependent DNA polymerase from serum. Pharmacokinetic analysis showed 5-propenyl-2'-deoxyuridine to be well absorbed orally, but rapidly cleared from plasma. The arabinofuranosyl analog was also well absorbed but cleared less rapidly. Hence, these results indicate the potential of 5-propenyl-2'-deoxyuridine and 5-propenyl-arabinofuransyluracil for chemotherapy of hepatitis B.

Pharmacokinetics and antiviral activity in simian varicella virus-infected monkeys of (R,S)-9-[4-hydroxy-2-(hydroxymethyl) butyl]guanine, an anti-varicella-zoster virus drug.

The acyclic guanosine analog (R,S)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, (+/-)2HM-HBG, is an effective inhibitor of herpes simplex virus and varicella-zoster virus infections in vitro. This report is concerned with the pharmacokinetic evaluation of the drug in rats and monkeys and its antiviral activity in African green monkeys infected with simian varicella virus (SVV), a virus closely related to varicella-zoster virus that is also susceptible to inhibition by (+/-)2HM-HBG. Elimination half-lives in plasma following intravenous administration to monkeys (100 mumol/kg of body weight) ranged from 1.8 to 2.2 h, and total body clearance was 9.0 +/- 0.4 ml/min per kg (mean +/- standard error). After oral administration, levels in plasma were low, with a maximum concentration of the drug of only 3.1 +/- 0.8 microM, a time to reach maximum concentration of drug of 2.7 +/- 0.4 h, and an oral bioavailability of 10.6 +/- 1.4%. Because of the low oral bioavailability, SVV-infected monkeys were treated intramuscularly with (+/-)2HM-HBG. (+/-)2HM-HBG at a dosage of 10 mg/kg of body weight per day allowed moderate viremia, whereas a dosage of 30 mg/kg of body weight per day strongly suppressed viremia with minimal numbers of virus plaques from blood specimens collected at days 3, 5, and 7 postinfection and complete clearance at day 9 postinfection. Titers of antibody to SVV were also low. Treatment three times daily was somewhat more efficacious than treatment twice daily. Thus, (+/-)2HM-HBG is an effective inhibitor of SVV replication in vivo, despite the fact that leves of (+/-)2HM-HBG in plasma were low at extended periods of time and below the concentration of drug giving 50% inhibition of plaque formation obtained in vitro.

Inhibition of terminal N- and O-glycosylation specific for herpesvirus-infected cells: mechanism of an inhibitor of sugar nucleotide transport across Golgi membranes.

The nucleoside analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) inhibited the Golgi-associated terminal glycosylation in herpes simplex virus type 1- and type 2-infected cells, specifically incorporation of galactose and sialic acid into N-linked oligosaccharides, and incorporation of sialic acid and, to a lesser extent, of galactose into O-linked oligo saccharides. This resulted in formation of viral glycoproteins with terminal GlcNAc and Fuc in N-linked oligosaccharides and terminal O-linked GalNAc. Inhibition of formation of UDP-hexoses and of acceptor glycoprotein synthesis and inhibition of cellular transport of viral glycoproteins were not observed. No evidence for the formation of a sugar nucleotide analog of BVdU was obtained. Inhibition required phosphorylation of BVdU to its 5' monophosphate (BVdUMP) by the virus-coded thymidine kinase. In a cell-free system, this monophosphate inhibited the transport of pyrimidine sugar nucleotides across Golgi membranes and, as a consequence, the incorporation of sugars into glycoproteins. Inhibition of galactosyltransferase by BVdUMP was insignificant. BVdUMP did not inhibit translocation across the Golgi membrane of purine sugar nucleotides. Inhibition of sugar nucleotide translocation represents the first target for design of virus-specific glycosylation inhibitors.

Limited efficacy of inhibitors of herpes simplex virus DNA synthesis in murine models of recrudescent disease.

The herpesvirus DNA polymerase inhibitor foscarnet, applied topically, and the anti-herpesvirus guanosine analogue buciclovir, given orally, decreased virus replication and disease development in primary skin infections of mice caused by herpes simplex virus type 1 (HSV-1). If the same tissues were infected via sensory nerves, following zosteriform spread of the virus the same treatments showed strongly decreased efficacy, or were inefficacious, when started before development of clinical signs in the infected tissues. These results were obtained in murine models of zosteriform spread of HSV-1 to the ear (following inoculation of the ventral side of the neck) or to the lower flank (following inoculation of the upper flank). In these models the immune system played a dominant role in virus clearance. The topically applied foscarnet could not prevent disease development in these models of recrudescent disease even when applied before the virus was detected in the skin, but a decrease in virus titre was obtained. Orally administered buciclovir lost efficacy when administered at the time of virus entry into the skin, i.e. 1 or 2 days before development of clinical signs. In the flank model, measuring lesion development, orally administered acyclovir also had a strongly decreased efficacy, when compared with its effect during infections in which lesion development did not involve translocation of virus through nerves. In the presence of developing immunity the inhibitors could not accelerate the clearance of virus from infected tissues. Furthermore, all treatments (topical foscarnet and oral buciclovir or acyclovir) were without effect on disease development when treatment was initiated on appearance of the first clinical signs of disease. As disease development following zosteriform spread of HSV resembles that in recurrent herpes in humans, and as the limited efficacy of the inhibitors observed resembles the poor results obtained with inhibitors of herpesvirus DNA synthesis in clinical studies on the treatment of symptomatic recurrent herpes, we suggest the use of animal models of zosteriform spread for pre-clinical evaluation of new antiherpes drugs.

Incorporation into nucleic acids of the antiherpes guanosine analog buciclovir, and effects on DNA and protein synthesis.

Using cells expressing herpes simplex virus (HSV) thymidine kinase, we investigated the metabolism of the acyclic antiherpes guanosine analog buciclovir, in relation to the effects of the drug on viral DNA and protein synthesis. In these cells the predominant metabolite of buciclovir was its triphosphate, as in the HSV-1 infected Vero cells investigated in parallel. Further metabolism of buciclovir led to incorporation into RNA and DNA. Buciclovir inhibited DNA synthesis, not RNA synthesis, and prevented an increase in the size of newly synthesized DNA. To study the relative effects of BCV on cellular and viral DNA synthesis, human TK-cells transformed to a TK+ phenotype with HSV-2 DNA, were infected with HSV-1. In these HSV-1 infected cells buciclovir-triphosphate caused a preferential inhibition of viral DNA synthesis. Despite incorporation of buciclovir into RNA, and the presence of buciclovir-triphosphate from the time of infection onwards, no effect was observed on the synthesis of the beta proteins ICP-6 and ICP-8. Presumably as a consequence of inhibition of viral DNA synthesis, the synthesis of a beta gamma protein (gD) and a gamma protein (gC) were inhibited, and synthesis of the beta proteins (ICP-6 and ICP-8) was not shut-off. Glycosylation of gC that was still synthesized, was not inhibited. Thus, the biological effects of buciclovir can be explained by its inhibition of DNA synthesis.

Host cell-induced differences in O-glycosylation of herpes simplex virus gC-1. I. Structures of nonsialylated HPA- and PNA-binding carbohydrates.

Lectins with narrow oligosaccharide specificities were established as probes to study the host cell influence on the biosynthesis of O-linked oligosaccharides of the herpes simplex virus type 1 (HSV-1)-specified glycoprotein C (gC-1). We found that only gC-1 and no other glycoprotein bound to the peanut lectin (PNA), with main specificity for Gal(beta 1-3)GalNAc. Previously, we have shown that only gC-1 binds to the Helix pomatia lectin (HPA), with main specificity for terminal GalNAc. The O-linked oligosaccharides binding to PNA and HPA were released by alkaline borohydride treatment and characterized. A structural determination of these oligosaccharides showed that the HPA-binding carbohydrates were monosaccharides (GalNAc), and that the PNA-binding oligosaccharides were disaccharides with the structure Gal-GalNAc. The PNA- and HPA-binding oligosaccharides were arranged as Pronase-resistant clusters on gC-1, consisting of about seven individual, adjacent oligosaccharides. In addition to these disaccharides, Pronase-resistant PNA-binding glycopeptides of gC-1 also contained neutral trisaccharides. Larger O-linked oligosaccharides, binding to the wheat germ lectin, were found in gC-1, but not in proximity to the PNA-binding ones. It was concluded that the lectins mentioned should be useful probes in screening HSV-infected cells of different lineages for differences in processing of O-linked oligosaccharides.