5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83), a selective anti-human immunodeficiency virus agent with an improved metabolic and toxicological profile.

5-Chloro-2',3'-dideoxy-3'-fluorouridine (935U83) is a selective anti-human immunodeficiency virus (HIV) agent. When tested in phytohemagglutinin-stimulated normal human peripheral blood lymphocytes against fresh clinical isolates of HIV type 1 (HIV-1) obtained from patients naive to AZT (3'-azido-3'-deoxythymidine [zidovudine]), 935U83 inhibited virus growth with an average 50% inhibitory concentration (IC50) of 1.8 microM; corresponding IC50s were 0.10 microM for FLT (3'-deoxy-3'-fluorothymidine) and 0.23, 0.49, and 0.03 microM for the approved agents AZT, ddI (2',3'-dideoxyinosine), and ddC (2',3'-dideoxycytosine), respectively. Importantly, 935U83 retained activity against HIV strains that were resistant to AZT, ddI, or ddC. Of additional interest, we were unable to generate virus which was resistant to 935U83 by passaging either HXB2 (AZT-sensitive) or RTMC (AZT-resistant) strains in the presence of high concentrations of 935U83. The anabolic profile of 935U83 was similar to that of AZT, and 935U83 triphosphate was a potent inhibitor of HIV-1 reverse transcriptase. Pharmacokinetic evaluation showed good oral bioavailability (86% in mice and 60% in monkeys) and less extensive metabolism to the glucuronide relative to AZT. 935U83 showed low toxicity. In an in vitro assay for toxicity to a human erythrocyte progenitor, erythroid burst-forming unit (BFU-E), the IC50 for 935U83 (> 400 microM) was more than 1,000-fold those of FLT (0.07 microM) and AZT (0.30 microM). Mild reversible reductions in erythrocytes and associated parameters were seen in mice dosed orally with 2,000 mg of 935U83 per kg per day for 1 and 6 months. In monkeys dosed orally with up to 700 mg/kg/day for 1 and 6 months, the only possible treatment-related finding was cataracts in 1 of 12 animals given the intermediate dose of 225 mg/kg/day. At the highest doses in mice and monkeys, maximal concentrations in plasma were more than 100-fold the anti-HIV IC50s against clinical isolates. This safety profile in animals compares very favorably with that of any of the anti-HIV drugs approved to date and has led us to begin evaluation of 935U83 in patients with HIV infection.

Review of research leading to new anti-herpesvirus agents in clinical development: valaciclovir hydrochloride (256U, the L-valyl ester of acyclovir) and 882C, a specific agent for varicella zoster virus.

Research leading to the new anti-herpesvirus compounds discussed here has come from three approaches. The first approach was directed towards improving the bioavailability of acyclovir by examining the potential of a variety of prodrugs, leading to the new compound valaciclovir hydrochloride. The second approach was to examine a large number of 5-substituted pyrimidines for activity against those viruses which were not as potently inhibited by acyclovir as are herpes simplex viruses, i.e., varicella zoster virus (VZV) and human cytomegalovirus (HCMV). This research led to the new chemical entity 882C for VZV. A third approach has been to examine drug combinations with acyclovir. This research led to the compound 348U, an inhibitor of herpes simplex virus ribonucleotide reductase which acts synergistically in combination with acyclovir. This manuscript will focus on the first two approaches leading to new compounds valaciclovir hydrochloride and 882C since Dr. Safrin details such background for 348U/acyclovir. Attempts to improve the bioavailability of acyclovir began a decade ago. Early prodrugs were compounds with alterations in the 6-substituent of the purine ring of acyclovir. The 6-amino congener required the cellular enzyme adenosine deaminase for conversion to acyclovir and the 6-deoxycongener was dependent on cellular xanthine oxidase for conversion. Neither of these prodrugs had a chronic toxicity profile in laboratory animals as good as acyclovir. Efforts were directed towards simpler esters and 18 amino acid esters were made. The pharmacokinetic profile of each prodrug was determined in rats by measuring the recovery of acyclovir in urine after oral dosing.(ABSTRACT TRUNCATED AT 250 WORDS)

Random mutagenesis of the thymidine kinase gene of varicella-zoster virus.

To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.21), we established rapid screening and phenotypic selection of mutant VZV TK genes in TK-deficient Escherichia coli C600 by using a constitutive pKK223-3 expression plasmid. In this screening system, mutant TK genes generated by random mutagenesis were identified by the sensitivity of E. coli-expressing VZV TKs to 5-bromo-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil. Twenty-four mutant clones with amino acid substitutions were isolated, and their nucleotide sequence and enzymatic activities were determined. Of the 24 clones, 20 had single amino acid substitutions, 2 clones had double amino acid substitutions, and 1 clone had triple amino acid substitutions. In 17 cases of single amino acid substitution, six mutations led to lost enzyme activity, and four of these six mutations centered in the ATP-binding site. The other 11 mutations resulted in reduction of both TK and thymidylate kinase activities or only thymidylate kinase activity and were located in scattered positions in the VZV TK gene, although 5 mutations showed a tendency to cluster in the region between positions 251 and 260.

6-Methoxypurine arabinoside as a selective and potent inhibitor of varicella-zoster virus.

Seven 6-alkoxypurine arabinosides were synthesized and evaluated for in vitro activity against varicella-zoster virus (VZV). The simplest of the series, 6-methoxypurine arabinoside (ara-M), was the most potent, with 50% inhibitory concentrations ranging from 0.5 to 3 microM against eight strains of VZV. This activity was selective. The ability of ara-M to inhibit the growth of a variety of human cell lines was at least 30-fold less (50% effective concentration, greater than 100 microM) than its ability to inhibit the virus. Enzyme studies suggested the molecular basis for these results. Of the seven 6-alkoxypurine arabinosides, ara-M was the most efficient substrate for VZV-encoded thymidine kinase as well as the most potent antiviral agent. In contrast, it was not detectably phosphorylated by any of the three major mammalian nucleoside kinases. Upon direct comparison, ara-M was appreciably more potent against VZV than either acyclovir or adenine arabinoside (ara-A). However, in the presence of an adenosine deaminase inhibitor, the arabinosides of adenine and 6-methoxypurine were equipotent but not equally selective; the adenine congener had a much less favorable in vitro chemotherapeutic index. Again, this result correlated with data from enzyme studies in that ara-A, unlike ara-M, was a substrate for two mammalian nucleoside kinases. Unlike acyclovir and ara-A, ara-M had no appreciable activity against other viruses of the herpes group. The potency and selectivity of ara-M as an anti-VZV agent in vitro justify its further study.

Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction.

We have applied the polymerase chain reaction (PCR) technique to analyse mutations in the thymidine kinase (TK) gene of varicella-zoster virus (VZV) associated with resistance to the 5-bromovinyl (BVaraU) and 5-propynyl (PYaraU) analogues of arabinofuranosyl deoxyuridine. The results from this study allow three clear conclusions to be drawn. Firstly, the technique clearly shows that populations of VZV derived from plaque purification were truly clonal only when the plaques were initiated from cell-free virus (representing a tiny fraction of infectious virus) and plaques initiated by infected cells contained a mixture of variants. Secondly, despite the background mutations caused by errors of the Taq DNA polymerase, mutations relevant to drug resistance can easily be distinguished. The BVaraU-resistant mutant, 7-1, contained an aspartic acid to asparagine mutation at residue 18 and a single base deletion (position 65298 of the VZV DNA sequence), resulting in a frameshift and premature termination of the polypeptide chain, was found in the BVaraU-resistant mutant YSR. PYaraU-resistant virus populations contained viruses with one or more of three independent mutations, i.e. single base substitutions resulting in mutations from leucine to proline at residue 92, histidine to arginine at residue 97 and a deletion of 20bp (residues 65,135 to 65,154). Finally, the technique has uncovered novel sites in the virus TK associated with drug resistance. We conclude that in vitro amplification using the PCR combined with cloning and sequencing is a relatively rapid method for identifying mutations in small virus populations even when they are not homogeneous.

High-level expression of the Epstein-Barr virus alkaline deoxyribonuclease using a recombinant baculovirus: application to the diagnosis of nasopharyngeal carcinoma.

The Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV). Infection of the insect cell line Spodoptera frugiperda (SF9) with the recombinant virus led to the expression of an enzymatically active alkaline DNase. The recombinant EBV alkaline DNase was highly soluble, and the recombinant baculovirus produced approximately 10-20 mg of EBV DNase per 1 X 10(9) cells. The recombinant enzyme activity was neutralized by specific antisera to the EBV DNase and was recognized by these sera in Western blot analysis and immunofluorescence tests. The recombinant EBV DNase was neutralized by these sera from patients with nasopharyngeal carcinoma and chronic infectious mononucleosis. Western blot analysis using these patients' sera showed that IgG and IgA antibodies to the EBV DNase could be readily detected.

Immunological studies on the Epstein-Barr virus encoded alkaline deoxyribonuclease found in virus-producing lymphoblastoid cells.

Antisera were raised against a purified recombinant form of the Epstein-Barr virus (EBV) alkaline deoxyribonuclease (DNase) expressed in Escherichia coli. These sera were shown to be reactive with lymphoblastoid cells expressing EBV antigens (B95-8, P3HR-1 and Raji). Immunostaining studies of cells expressing EBV antigens revealed that the DNase was a component of the restricted early antigen complex of EBV. Western blot analysis of these chemically induced cells revealed that the polypeptide associated with the EBV DNase has an Mr of approximately 55,000, slightly greater than that of the recombinant form, suggesting that the protein undergoes some form of posttranslational modification during virus replication. The DNase enzymic activities observed in B95-8, P3HR-1 and Raji cells following chemical induction were neutralized using the specific antiserum. A detailed examination of protein extracts from the nude mouse-passaged nasopharyngeal carcinoma cell line C-15 failed to detect any antigenic or biochemical evidence for the presence of the DNase. Immunostaining of biopsies of oral 'hairy' leukoplakia with the antisera against EBV DNase revealed high level expression in the more differentiated spinous layers of the epithelium, a pattern of reactivity identical to that observed for other lytic cycle antigens.

The characterization of the EBV alkaline deoxyribonuclease cloned and expressed in E. coli.

Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus. We report here the expression of the BGLF5 open reading frame in E. coli and the expression of high levels of a novel alkaline DNase activity in induced cells. This alkaline DNase has been purified to apparent homogeneity as a single protein species. This is the first report of the expression of a herpesvirus coded DNase in a prokaryotic system and of the purification of the EBV DNase to demonstrable purity. It has the biochemical characteristics of a typical herpesvirus alkaline exonuclease showing a high pH optimum, an absolute requirement for Mg2+ for activity and sensitivity to high salt concentrations and polyamines. The enzyme activity was neutralized by sera from patients with nasopharyngeal carcinoma and was reactive with these sera in Western blot analysis. Thus the prokaryotic expression system described here provides an economical and efficient source of the EBV DNase for biochemical and seroepidemiological analysis.

Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity.

There is considerable interest in the potential of human immunodeficiency virus type 1 (HIV-1) to develop drug resistance, especially as 3'-azido-3'-deoxythymidine (Retrovir) is now in widespread clinical use to treat people with AIDS and AIDS-related complex (ARC). To address this possibility, mutations in the HIV reverse transcriptase [deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] gene have been introduced by site-directed mutagenesis of cloned constructs in Escherichia coli. Analysis of the recombinant mutant reverse transcriptase from a number of these constructs revealed enzymes that maintained enzyme activity but had a reduced ability to recognize inhibitors such as azidothymidine triphosphate. To assess the infectivity of these mutants, several constructs of proviral HIV clones with mutant reverse transcriptase genes have been made and used to transfect T cells. All five mutants tested have lower infectious potential, suggesting considerable levels of reverse transcriptase activity are required for efficient virus replication. Viable virus recovered from two clones showed decreased sensitivity to the antiviral compound phosphonoformate, thus demonstrating the potential for drug-resistant HIV to replicate. However, although the reverse transcriptase from these mutant viruses showed decreased sensitivity to azidothymidine triphosphate, paradoxically these viruses were hypersensitive to azidothymidine when tested in culture.

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of human immunodeficiency virus type 1 reverse transcriptase by using monoclonal antibodies: role of the C terminus in antibody reactivity and enzyme function.

We describe the production of eight monoclonal antibodies reactive with human immunodeficiency virus type 1 reverse transcriptase (RT) by immunization of mice with purified recombinant RT. These antibodies were found to react with one or the other of two regions of the enzyme and were found to be useful in immunodeficiency purification of large amounts of the enzyme. One epitope located at the C terminus of the enzyme was of particular interest, since it was present in only the larger, 66-kilodalton (kDa) RT species and not its smaller, 51-kDa counterpart. To define this epitope, a series of mutants was made which synthesized C-terminally truncated RT. These mutants indicated that the same region of the enzyme, when deleted, both removed the C-terminal epitope and drastically reduced RT activity, indicating the importance of this region in the function of the enzyme; however, even the 51-kDa enzyme component had demonstrable activity.

Structural studies of the acquired immunodeficiency syndrome virus reverse transcriptase.

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized. Crystallographic studies currently underway are aimed at elucidating the three-dimensional structure of HIV RT. The availability of a bacterial expression system has enabled structural/functional studies of the RT by site-directed mutagenesis. These studies have identified amino acid residues that are essential for activity of the enzyme and might be involved in substrate binding. It is hoped that structural information of this nature will allow the rational design of HIV RT inhibitors.

Production of antibodies of predetermined specificity against herpes simplex virus DNA polymerase and their use in characterization of the enzyme.

Peptides from preselected regions of the herpes simplex virus DNA polymerase were used to generate monospecific antisera to defined regions of the enzyme. The antisera were used to localize the polymerase within the infected cell and to determine the time of synthesis during productive infection. Comparison with a neutralizing polyclonal antiserum was used to show the specificity of the peptide antisera. By using the antisera the stabilities of the DNA polymerase, the alkaline nuclease, and the major DNA-binding protein were determined, and the state of phosphorylation of the DNA polymerase was compared with each of these proteins.

Site-specific mutagenesis of AIDS virus reverse transcriptase.

Human immunodeficiency virus (HIV) is the causative agent of AIDS (acquired immune deficiency syndrome) a disease which poses a serious challenge to modern medicine. If we are to conquer this disease we will need a protective vaccine or effective drugs able to block the life cycle of the virus. An early stage in the invasion of the host cell is the conversion of the RNA genome of the virus to a double-stranded DNA intermediate which subsequently becomes integrated into the host cell chromosome. The enzyme reverse transcriptase is crucial in this process and is thus an obvious chemotherapeutic target. In this study we have used site-directed mutagenesis of this enzyme expressed in Escherichia coli to reveal several important functional regions of the protein including putative components of the triphosphate binding site and pyrophosphate exchange sites.

Molecular basis of the glycoprotein C-negative phenotypes of herpes simplex virus type 1 mutants selected with a virus-neutralizing monoclonal antibody.

Previously (Holland et al., J. Virol. 52:566-574, 1984; Kikuchi et al., J. Virol. 52:806-815, 1984) we described the isolation and partial characterization of over 100 herpes simplex virus type 1 mutants which were resistant to neutralization by a pool of glycoprotein C- (gC) specific monoclonal antibodies. The genetic basis for the inability of several of these gC- mutants to express an immunoreactive envelope form of gC is reported here. Comparative nucleotide sequence analysis of the gC gene of the six mutants gC-3, gC-8, gC-49, gC-53, gC-85, and synLD70, which secrete truncated gC polypeptides, with that of the wild-type KOS 321 gC gene revealed that these mutant phenotypes were caused by frameshift or nonsense mutations, resulting in premature termination of gC translation. Secretion of the gC polypeptide from cells infected with these mutants was due to the lack of a functional transmembrane anchor sequence. The six secretor mutants were tested for suppression of amber mutations in mixed infection with a simian virus 40 amber suppressor vector. Mutant gC-85 was suppressed and produced a wild-type-sized membrane-bound gC. Nucleotide sequence analysis of the six gC deletion mutants gC-5, gC-13, gC-21, gC-39, gC-46, and gC-98 revealed that they carried identical deletions which removed 1,702 base pairs of the gC gene. The deletion, which was internal to the gC gene, removed the entire gC coding sequence and accounted for the novel 1.1-kilobase mRNA previously seen in infections with these mutants. The mutant gC-44 was previously shown to produce a membrane-bound gC protein indistinguishable in molecular weight from wild-type gC. This mutant differed from wild-type virus in that it had reduced reactivity with virus-neutralizing monoclonal antibodies. Nucleotide sequence analysis of the gC gene of mutant gC-44 demonstrated a point mutation which changed amino acid 329 of gC from a serine to a phenylalanine.

DNA-binding protein associated with herpes simplex virus DNA polymerase.

Purified preparations of herpes simplex virus type 2 DNA polymerase made by many different laboratories always contain at least two polypeptides. The major one, of about 150,000 molecular weight, has been associated with the polymerase activity. The second protein, of about 54,000 molecular weight, which we previously designated ICSP 34, 35, has now been purified. The purified protein has been used to prepare antisera (both polyclonal rabbit serum and monoclonal antibodies). These reagents have been used to characterize the protein, to demonstrate its quite distinct map location from that of the DNA polymerase on the herpes simplex virus genome, and to demonstrate the close association between the two polypeptides.

Studies on the herpes simplex virus alkaline nuclease: detection of type-common and type-specific epitopes on the enzyme.

Five monoclonal antibodies to the alkaline nuclease of herpes simplex virus (HSV) types 1 and 2 have been used in immunoperoxidase tests to demonstrate the nuclear localization of the enzyme within HSV-1- and HSV-2-infected cells and to purify the enzyme from cells infected with either virus by immunoadsorbant chromatography. Affinity chromatography with a 32P-labelled extract of HSV-2-infected cells has enabled us to demonstrate that the nuclease eluting from the immunoadsorbant is a phosphoprotein, hence confirming the nuclease to be identical to the phosphorylated polypeptide previously referred to as ICSP 22 (HSV-2) or ICP 19 (HSV-1). In addition, the results clearly demonstrate that monoclonal antibodies Q1, CC1 and CH2 are directed against HSV type-common epitopes while V1 and T2T1 antibodies are against HSV-2-specific epitopes on the enzyme. Using the type-specific monoclonal antibodies in an immunoperoxidase test, the enzyme specified in cells infected with intertypic recombinants has been typed; correlation of these data with restriction endonuclease maps of the recombinants has enabled us to map the position of the active site of the nuclease gene to map units 0.168 to 0.184 on the genomes of both HSV-1 and HSV-2. Taken with the data mapping the mRNA encoding this enzyme, the nuclease active site can be mapped to 0.168 to 0.175 on the genome. Finally, the use of monoclonal antibodies in immunofluorescence tests on infected cells has demonstrated that the nuclease is synthesized within 2 h post-infection.

Interactions between herpes simplex virus DNA-binding proteins.

Monoclonal antibodies directed against several herpes simplex virus (HSV)-induced DNA-binding proteins were used to investigate protein interactions in HSV-infected cells. Q1 monoclonal antibody, which is specific for the HSV-induced alkaline nuclease, when used in an immunoadsorbant column resulted in the purification of the alkaline nuclease, to which large quantities of the major DNA-binding protein were bound. Conversely, when a monoclonal antibody to the major DNA-binding protein was used in affinity chromatography other polypeptides (including the DNA polymerase and alkaline nuclease) were eluted in addition to the major DNA-binding protein. Similar results were obtained when the experiment was performed using a monoclonal antibody to another HSV-2 DNA-binding protein. These results suggest the possibility that these polypeptides interact as part of the HSV DNA replication complex, and this hypothesis is discussed.