Stavudine resistance: an update on susceptibility following prolonged therapy.

The current report summarizes the available published and unpublished data from several investigators on resistance in clinical isolates following prolonged stavudine therapy. Results suggest that stavudine resistance is both modest in degree and infrequent in appearance. Phenotypic evaluation of 61 patients on stavudine therapy showed only modest changes in drug sensitivity following up to 29 months of treatment. The post-treatment isolates from 15 patients exhibited an increase in EC50 value > fourfold (level above variability of assay) when compared with the corresponding pretreatment isolates. However, the vast majority (11) of these pretreatment isolates either had unexpectedly low EC50 levels and/or had post-treatment isolates that lacked any amino acid changes within their reverse transcriptase (RT) gene to account for the observed change in sensitivity. Of the four remaining isolates, two appeared to have a multi-resistant phenotype to several nucleoside analogues and two had no detectable RT amino acid changes to account for the observed change in stavudine sensitivity. To date, clinical HIV-1 isolates displaying stavudine-specific resistance have yet to be reported. Furthermore, full or partial RT sequence analysis of 194 post-treatment isolates failed to identify any consistent amino acid changes. The strain-specific V75T mutation reported to confer stavudine resistance to the HXB2 HIV-1 strain in vitro, was found in only six isolates and did not correlate with stavudine resistance. This low incidence of stavudine resistance is in striking contrast to that observed with other nucleoside analogues and further supports the use of stavudine in first-line combination therapy for HIV patients.

Aminodiol HIV protease inhibitors. Synthesis and structure-activity relationships of P1/P1' compounds: correlation between lipophilicity and cytotoxicity.

A series of novel aminodiol inhibitors of HIV protease based on the lead compound 1 with structural modifications at P1' were synthesized in order to reduce the cytotoxicity of 1. We have observed a high degree of correlation between the lipophilicity and cytotoxicity of this series of inhibitors. It was found that appropriate substitution at the para position of the P1' phenyl group of 1 resulted in the identification of equipotent (both against the enzyme and in cell culture) compounds (10l, 10m, 10n, and 15c) which possess significantly decreased cytotoxicity.

Differential effect of modified capped RNA substrates on influenza virus transcription.

The RNA-dependent RNA polymerase of influenza virus transcribes messenger RNA through a unique cap scavenging mechanism. Viral enzyme binds to the cap structure of host mRNA, cleaves the molecule 9-15 bases downstream of the cap, and uses the short capped oligonucleotide as a primer for mRNA synthesis. Previously, we have shown that the viral polymerase can efficiently bind capped RNAs shorter than 9 nucleotides in length, but the viral enzyme can not utilize these RNAs as primers. For this reason, these short capped oligonucleotides are potent inhibitors of influenza virus transcription. In these studies, it is now shown that short capped oligomers inhibit capped-RNA dependent transcription at the initial step of cap binding. In contrast, low concentrations of these short capped RNAs can actually stimulate viral transcription primed with high concentrations of the dinucleotide ApG. Another capped RNA derivative containing phosphorothioate oligonucleotides was also investigated as a potential polymerase inhibitor. This longer capped RNA was able to bind to the polymerase, but could not be cleaved to primer length by the enzyme associated endonuclease. Thus, the capped phosphorothioate RNA inhibited cap-primed transcription at the step of cap binding. However, in contrast to the short capped oligonucleotide, it also inhibited ApG primed viral transcription.

Domains 1 and 2 of ICAM-1 are sufficient to bind human rhinoviruses.

The intercellular adhesion molecule-1 (ICAM-1) receptor was expressed in primary chicken embryo cells using a retroviral vector and shown to specifically bind major group human rhinoviruses (HRVs). A truncated, membrane-bound ICAM-1 protein containing N-terminal domains 1, 2, and 3 retained the ability to bind virus whereas proteins containing domains 1 and 2 or domain 1 were not expressed under these conditions. Soluble forms of ICAM-1 proteins were expressed to circumvent the reduced expression levels of shorter ICAM-1 truncations. Full-length and truncated ICAM-1 molecules containing only domains 1 and 2 were capable of neutralizing HRV binding to cells. Soluble receptors containing only domain 1 could not be recovered. Mutants of ICAM-1 lacking carbohydrate attachment sites were constructed and shown to have no effect on the ability of ICAM-1 to bind HRVs. In addition, ICAM-1 proteins expressed in the presence of tunicamycin also retained their virus binding capability. These data suggest that the N-terminal two domains of ICAM-1 are sufficient for virus interaction and that carbohydrates do not play a major role in virus binding.

Characterization of monoclonal antibodies recognizing amino- and carboxy-terminal epitopes of the herpes simplex virus UL42 protein.

A panel of monoclonal antibodies (MAbs) directed against the herpes simplex virus type 1 (HSV-1) DNA polymerase (Pol) accessory protein, UL42, was developed and characterized. Thirteen different MAbs were isolated which exhibited varied affinities for the protein. All MAbs reacted with UL42 in ELISA, Western blot and immunoprecipitation analyses. Competitive ELISA was used to show that 6 different epitopes within UL42 were recognized by the MAbs. Immunoprecipitation of amino- and carboxy-terminal truncations of UL42 mapped the epitopes to regions containing amino acids 1-10, 10-108, 338-402, 402-460, and 460-477. All but one of these epitopes were outside the minimal active portion of the protein previously mapped to amino acids 20-315. None of these MAbs, alone or in combination, specifically neutralized the ability of UL42 to stimulate Pol activity in vitro. These results are consistent with structure-function studies that showed that N- and C-terminal regions of the UL42 protein, those recognized by the MAbs, are not involved in UL42 function in vitro.

Modification of experimental rhinovirus colds by receptor blockade.

Human rhinovirus (HRV) infection can be inhibited in vitro by antibody directed against the cellular receptor for the major HRV group representing 90% of serotypes. We assessed the prophylactic effectiveness and safety of intranasally administered rhinovirus receptor murine monoclonal antibody (RRMA) in two double-blind, place-controlled, randomized studies of volunteers experimentally inoculated with HRV-39. In the first study, RRMA administration (135 micrograms/subject in 9 applications, -17 to +48 h) did not reduce infection (RRMA 12/15 vs. placebo 13/15) or illness (8/12 vs. 7/13) rates or modify the clinical course of experimental HRV-39 colds. In the second trial, a higher RRMA dosage (1 mg/subject in 10 applications, -3 to +36 h), similarly did not reduce overall infection (11/13 vs. 12/13) or illness (7/11 vs. 9/12) rates, but was associated with a 1-2 day delay in the onset of viral shedding and cold symptoms and with significant reductions in viral titers and nasal symptoms on the second day after challenge and in mucus weights on the third day after challenge. No toxicity related to RRMA was recognized. The results indicate that intranasal RRMA modified infection and illness after experimental HRV-39 challenge and suggest that blockade of host cell receptors offers a novel antiviral approach against HRV infections.

Antiviral efficacy of lobucavir (BMS-180194), a cyclobutyl-guanosine nucleoside analogue, in the woodchuck (Marmota monax) model of chronic hepatitis B virus (HBV) infection.

Lobucavir (BMS-180194), a cyclobutyl-guanosine nucleoside analogue, effectively reduced WHV-viremia in chronically infected carrier woodchucks (Marmota monax) by daily per os treatment. WHV-viremia in the animals was measured by the serum content of hybridizable WHV-genomic DNA. Lobucavir, given at daily doses of 10 and 20 mg/kg body weight, reduced WHV-viremia by a 10- to 200-fold range during therapy. Lobucavir, given at 5 mg/kg, suppressed WHV-viremia by a 10- to 30-fold range, whereas a 0.5 mg/kg dose had no significant effect. WHV-viremia was also measured by hepadnaviral endogenous polymerase activity (EPA) in sera of animals treated for 6 weeks at 5 and 0.5 mg/kg. Changes in EPA in sera of lobucavir treated animals were comparable to changes in WHV DNA levels. Viremia in treated carriers recrudesced to pretreatment levels by 2 weeks of therapy cessation. These results indicated that the minimally effective antiviral daily per os dose of lobucavir in WHV-carrier woodchucks was approximately 5 mg/kg.

Substituted naphthalenones as a new structural class of HIV-1 reverse transcriptase inhibitors.

A novel substituted naphthalenone (TGG-II-23A) has been found that inhibits HIV-1 infection of CEM-SS cells at concentrations that are not cytotoxic. Time of addition experiments indicate that TGG-II-23A functions at a stage of the HIV-1 life cycle at or near reverse transcription. Cell free assays confirmed that TGG-II-23A inhibits HIV-1 reverse transcriptase. Similar to other non-nucleoside inhibitors, TGG-II-23A was specific for HIV-1 and failed to inhibit the replication of HIV-2. The binding site of TGG-II-23A appears to be in close proximity to that of the TIBO-like inhibitors, since a TIBO-resistant HIV-1 was also resistant to TGG-II-23A treatment. TGG-II-23A is a mixed non-competitive inhibitor that exhibits the same template:primer selectivity as other non-nucleoside inhibitors. TGG-II-23A therefore represents a new structural entry into the TIBO/Nevirapine class of inhibitors of HIV-1 reverse transcriptase.

Accumulation of newly synthesized mRNAs in response to human fibroblast (beta) interferon.

Treatment of human fibroblast cells with human fibroblast (beta)interferon for up to 8 hr resulted in the accumulation of at least four mRNAs. The mRNAs were isolated from cellular polysomes and characterized by stimulation of translation in a wheat germ cell-free protein synthesis system. The mRNAs appear as early as 2 hr after exposure to interferon and can be translated in vitro into proteins having molecular weights of 61,000, 62,000, 64,000, and 68,000. The response is not elicited by mouse interferon or insulin and does not occur in the presence of actinomycin D. Chase experiments indicated that the induced mRNAs remain ribosome-associated for at least 3 hr after their synthesis. The appearance of the induced mRNAs correlated directly with the onset of an antiviral state. Velocity sedimentation of the induced mRNAs on sucrose gradients demonstrated that each of the four induced proteins are encoded by different-sized mRNAs.

Characterization of human rhinoviruses displaced by an anti-receptor monoclonal antibody.

The attachment of rhinoviruses to cellular receptors was studied by displacing bound virus particles with an anti-receptor monoclonal antibody. The two serotypes studied differed significantly with respect to the temperature dependence of displacement and the nature of the particles displaced. Binding was shown to be a two-step process, the first of which is reversible and is seen when viruses are bound either to isolated cell membranes or to cells at lower than physiological temperatures. Second-stage binding was seen with serotype 14 when bound to intact cells. Viral particles released from such cells by incubation at 37 degrees C or by anti-receptor antibody exhibited altered physical changes in the capsid and a loss of infectivity. In contrast, serotype 67 bound efficiently to cells at 37 degrees C and did not elute spontaneously but could be displaced by anti-receptor antibody to produce complete, infectious particles. Rhinoviruses labeled with [3H]myristic acid or with [35S]methionine were displaced similarly from cells or membranes by anti-receptor antibody, indicating that the majority of VP4 of rhinoviruses does not enter or remain attached to cells during either the first or second stage of virus binding. These data support the conclusion that the myristic acid moiety of VP4 is not involved in the initial viral interaction with cellular receptors.

Nucleotide sequence of the leader RNA of the New Jersey serotype of vesicular stomatitis virus.

Sequence for the leader RNA Synthesized by the New Jersey serotype of vesicular stomatitis virus is presented and its complementary sequence representing the 3'-terminal sequence of the genome RNA is deduced. Comparison with the leader RNA sequence of the serologically distinct Indiana strain reveals that the 3'-terminal region of the genomes of two viruses is highly conserved.

In vitro RNA transcription by the New Jersey serotype of vesicular stomatitis virus. II. Characterization of the leader RNA.

The New Jersey serotype of vesicular stomatitis virus (VSV) was able to synthesize a small RNA (leader RNA) approximately 70 bases in length similar to the leader RNA synthesized in vitro by the genetically distinct Indiana serotype of VSV. Also, the New Jersey leader RNA contained the same 5'-terminal sequence, ppA-C-G, as the Indiana leader RNA and had a very similar base composition, with 42% AMP, 16% CMP, 18.6% GMP, and 23.4% UMP. The 3'-terminal sequence of the VSV New Jersey genome RNA was detemined and found to contain the sequence- Py-G-UOH, again the same as that of the Indiana serotype of VSV. Evidence that the New Jersey leader RNA is transcribed from the 3' end of the genome RNA was obtained from the fact that it can protect the 3'-terminal base of [3H]borohydride-labeled New Jersey genome RNA from RNase digestion. Although the New Jersey and Indiana leader RNAs were similar in many respects, they were unable to form RNase-resistant hybrids when annealed to heterologous genome RNA.

Complete nucleotide sequence of the leader RNA synthesized in vitro by vesicular stomatitis virus.

The complete nucleotide sequence of the leader RNA synthesized in vitro by the Indiana serotype of vesicular stomatitis virus is presented. The sequence was determined by the technique described by Donis-Keller, Maxam and Gilbert (1977) in combination with the standard two-dimensional fingerprint techniques described by Barrell (1971). The leader RNA contains 48 nucleotides variably terminating at the 3' terminus with cytosine (68%) and adenosine at position 47 (32%). Since the leader RNA is complementary to the 3' terminal portion of the viral genome RNA, the first 48 nucleotides from the 3' end of the genome RNA can be decuded. The leader RNA contains repetitive and palindromic sequences with a polypurine sequence at its 3' terminus. The possible role of some of the sequences is discussed.

In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14.

Development of a novel infectious cDNA assay is described for human rhinovirus type 14. A full-length cDNA clone of the human rhinovirus type 14 genome RNA was assembled and transcribed in vitro by using the SP6 transcription system. Transfection of HeLa cells with the nascent RNA resulted in the production of rhinovirus indistinguishable from the parental virus by both immunological and polyacrylamide gel analysis.

Many rhinovirus serotypes share the same cellular receptor.

Twenty-four human rhinovirus serotypes were grown and purified by centrifugation in metrizamide density gradients. These preparations had a lower buoyant density (1.24 g/cm3) and higher specific infectivities (1:24 to 1:240) than did rhinoviruses described previously (E. J. Stott and R. J. Killington, Annu. Rev. Microbiol. 26:503-524, 1972). Binding conditions in which the unique cellular receptors for virus attachment were saturated were determined for each serotype. Competition binding assays between pairs of serotypes allowed 20 of the 24 serotypes to be assigned to the same cellular receptor. The remaining four serotypes appeared to attach to a different cellular receptor. Since most serotypes were chosen for study at random, it seems likely that many of the yet unstudied rhinoviruses will share this common cellular receptor.

Isolation of a receptor protein involved in attachment of human rhinoviruses.

Human rhinoviruses can be classified into major and minor groups on the basis of receptor specificity. Recently, a mouse monoclonal antibody was isolated which selectively blocked the attachment of the major group of human rhinoviruses to cells. Using this monoclonal antibody, the cellular receptor for the major group of human rhinoviruses was isolated. A radioimmunoassay was developed by using the receptor antibody to specifically detect rhinovirus receptor during isolation. Solubilized receptor from detergent-treated HeLa cell membrane extracts eluted from gel filtration columns with an apparent molecular weight of 440,000. A cellular receptor protein, which had a molecular weight of 90,000 when analyzed on sodium dodecyl sulfate-polyacrylamide gels, was purified from solubilized extracts on an immunoaffinity column containing receptor antibody. Polyclonal rabbit antiserum, resulting from immunization with the isolated receptor protein, specifically blocked the attachment of the major group of human rhinoviruses and indicated that the 90-kilodalton protein plays a functional role in attachment. Prolonged exposure of HeLa cell monolayers with the receptor antibody showed no inhibition of cell growth and division.

Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme.

Purified Newcastle disease virus contains an enzyme that incorporates the methyl group from S-adenosyl-L-methionine into RNA synthesized in vitro by the virion-associated RNA polymerase (RNA nucleotidyltransferase). Incorporation of radioactivity from S-adenosyl-L-[methyl-3H]methionine was totally dependent upon RNA synthesis. The methylation reaction was completely inhibited by S-adenosyl-L-homocysteine, suggesting the transfer of only the methyl group of S-adenosyl-methionine to RNA products. Velocity sedimentation and hybridization of the in vitro product RNA indicated that both [3H]methyl and [32P]GMP labels resided in single-stranded 18S RNA molecules which were virus specific. Approximately 1 to 2 methyl groups were incorporated per RNA molecule. DEAE-cellulose chromatography of product RNA after alkaline hydrolysis suggested that the 5' terminus was the site of methylation.