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1.
BJOG ; 128(4): 756-767, 2021 03.
Article in English | MEDLINE | ID: mdl-33480468

ABSTRACT

OBJECTIVE: Determine the associations between factors and sexual practices and the composition of the vaginal microbiome (VM) of women treated for bacterial vaginosis (BV). DESIGN: Prospective cohort study. SETTING: The Melbourne Sexual Health Centre, Melbourne, Australia. POPULATION: Seventy-five reproductive-age women diagnosed with clinical BV, treated with first-line antibiotics and followed for up to 6 months. METHODS: Women self-collected vaginal swabs and completed questionnaires at enrolment, the day following antibiotics and monthly for up to 6months until BV recurrence or no BV recurrence (n = 430 specimens). Bacterial composition was determined using 16S rRNA gene amplicon sequencing. The effects of ongoing factors on VM composition (utilising 291 monthly specimens) were assessed using generalised estimating equations population-averaged models, which accounted for repeated measures within individuals. MAIN OUTCOME MEASURES: The relative abundance of vaginal bacterial taxa. RESULTS: Women who reported ongoing sex with a regular sexual partner (RSP) had a VM comprised of increased relative abundance of non-optimal BV-associated bacteria (Adjusted co-efficient [Adjusted co-eff] = 11.91, 95% CI 3.39to20.43, P = 0.006) and a decreased relative abundance of optimal, Lactobacillus species (Adjusted co-eff = -12.76, 95% CI -23.03 to -2.49, P = 0.015). A history of BV was also associated with a decreased relative abundance of Lactobacillus spp. (Adjusted co-eff = -12.35, 95% CI -22.68, P = 0.019). The relative abundance of Gardnerella, Atopobium and Sneathia spp. increased following sex with an RSP. CONCLUSIONS: Sex with an untreated RSP after BV treatment was associated with a VM comprised of non-optimal BV-associated bacteria. BV treatment approaches may need to include partner treatment if they are to achieve a sustained optimal VM associated with improved health outcomes. TWEETABLE ABSTRACT: Sex drives a return to a 'non-optimal' vaginal microbiota after antibiotics for bacterial vaginosis.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Coitus , Microbiota , Vagina/microbiology , Vaginosis, Bacterial/drug therapy , Vaginosis, Bacterial/microbiology , Adolescent , Adult , Female , Follow-Up Studies , Humans , Middle Aged , Prospective Studies , Recurrence , Treatment Outcome , Young Adult
2.
BMC Microbiol ; 19(1): 168, 2019 07 25.
Article in English | MEDLINE | ID: mdl-31345159

ABSTRACT

BACKGROUND: Over-the-counter intra-vaginal lactic-acid containing douches are marketed as vaginal hygiene products that support optimal vaginal pH balance. We report the effect of a commercially available douche (Etos®) on the vaginal microbiota (VM) in a prospective study. RESULTS: Twenty-five healthy women were recruited through advertisements in 2015-2017 (ethical approval: METC-2014_413) and followed over three menstrual cycles. The participants had a median age of 24 years [IQR: 22-29], were mostly Dutch-Caucasian (88%), and 60% used combined oral contraceptives. All participants douched three times a week during the second cycle, starting on the first day of that cycle. Participants completed a questionnaire at baseline, kept a daily diary to report douching, menses, and sexual activity, self-collected vaginal swabs every other day during the first and third cycle and daily during the second cycle, and measured vaginal pH mid-cycle. A median of 44 vaginal swabs [inter-quartile range (IQR): 41-50] were assessed per participant by 16S rRNA gene (V3-V4 region) sequencing and a Candida albicans PCR was done at four time-points. At baseline, 21 participants (84%) had Lactobacillus-dominated VM (Lactobacillus crispatus (n = 14), L. iners (n = 6), or diverse Lactobacillus species (n = 1) and 4 participants (16%) had VM consisting of diverse anaerobes. In multinomial logistic regression models, a trend towards increased odds were observed for having diverse anaerobic VM in the second and third cycle, compared to the first cycle, after adjusting for menses [odds ratio (OR) = 1.4 (95% CI: 0.9-2.1) and OR = 1.7 (95% CI: 0.9-3.1), respectively] (p = 0.376). Douching did not affect vaginal pH. Menses increased the odds for having VM consisting of diverse anaerobes almost two-fold (OR = 1.7; 95% CI: 1.0-2.8), while douching during menses increased the odds 2.6 fold (OR = 2.6; 95% CI: 1.0-6.5), compared to not menstruating (p = 0.099). Participants were more likely to test positive for C. albicans after cycle 2, compared to cycle 1 [OR = 3.0 (95% CI: 1.2-7.2); p = 0.017]. CONCLUSION: The Etos® douche did not significantly affect the vaginal pH or VM composition, although increased odds for having diverse anaerobic VM was observed, especially when douching during menses. Furthermore, douching may promote C. albicans infections.


Subject(s)
Lactic Acid/administration & dosage , Vagina/microbiology , Vaginal Douching , Adolescent , Adult , Candida albicans/genetics , Candida albicans/growth & development , Female , Humans , Lactobacillus/genetics , Lactobacillus/growth & development , Microbiota/genetics , Prospective Studies , RNA, Ribosomal, 16S/genetics , Young Adult
3.
Mucosal Immunol ; 10(6): 1480-1490, 2017 11.
Article in English | MEDLINE | ID: mdl-28401934

ABSTRACT

Inflammation in the female reproductive tract (FRT) is associated with increased HIV transmission. Lactobacillus spp. dominate the vaginal microbiota of many women and their presence is associated with reduced HIV acquisition. Here we demonstrate that lactic acid (LA), a major organic acid metabolite produced by lactobacilli, mediates anti-inflammatory effects on human cervicovaginal epithelial cells. Treatment of human vaginal and cervical epithelial cell lines with LA (pH 3.9) elicited significant increases in the production of the anti-inflammatory cytokine IL-1RA. When added simultaneously or prior to stimulation, LA inhibited the Toll-like receptor agonist-elicited production of inflammatory mediators IL-6, IL-8, TNFα, RANTES, and MIP3α from epithelial cell lines and prevented IL-6 and IL-8 production by seminal plasma. The anti-inflammatory effect of LA was mediated by the protonated form present at pH≤3.86 and was observed with both L- and D-isomers. A similar anti-inflammatory effect of LA was observed in primary cervicovaginal cells and in an organotypic epithelial tissue model. These findings identify a novel property of LA that acts directly on epithelial cells to inhibit FRT inflammation and highlights the potential use of LA-containing agents in the lower FRT as adjuncts to female-initiated strategies to reduce HIV acquisition.


Subject(s)
Anti-Inflammatory Agents/metabolism , Epithelial Cells/physiology , HIV Infections/immunology , HIV/immunology , Inflammation/immunology , Lactic Acid/metabolism , Vagina/metabolism , Cell Line , Cytokines/metabolism , Epithelial Cells/virology , Female , Humans , Inflammation Mediators/metabolism , Lactobacillus , Microbiota , Vagina/immunology , Vagina/pathology
4.
J Viral Hepat ; 19(3): 189-98, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22329373

ABSTRACT

The NS2 protein of hepatitis C virus (HCV) plays a critical role in virus morphogenesis and infectivity. The crystal structure of the C-terminus of the NS2 protein (NS2(Pro)) from the H77 strain indicates that NS2(Pro) forms a homodimer. In this study, using computational modelling, we identified residues at the NS2(Pro) dimer interface that have a role in dimerization and confirmed their capacity to influence dimerization by expression studies. Our modelling analysis identified 22 residues at the NS2(Pro) dimer interface that may be important for dimer formation. Based on the free binding energy, we selected the top five ranked mutations (V162A, M170A, I175A, D186A and I201A) for further study. Western blot analysis revealed that M170A, I175A, I201A, D186A and V162A resulted in a 4.0-, 3.2-, 3.0-, 2.8- and 1.5-fold increase, respectively, in the monomer/dimer ratio compared to wild type, confirming a role in homodimer formation or stability. Japanese Fulminant Hepatitis type 1 mutants expressing M170A, I175A, D186A and I201A demonstrated increasing defects in both RNA replication and the production of infectious virus compared to wild type. This study identified residues at the NS2(Pro) dimer interface that modulate NS2(Pro) homodimerization and demonstrated that abrogation of NS2(Pro) homodimerization results in defects in HCV replication and release of infectious virus.


Subject(s)
Hepacivirus/physiology , Protein Multimerization , Viral Nonstructural Proteins/metabolism , Amino Acid Sequence , HEK293 Cells , Hepacivirus/genetics , Hepacivirus/metabolism , Humans , Mutation , Sequence Analysis, Protein , Viral Nonstructural Proteins/genetics , Virus Assembly/genetics
5.
Curr Pharm Des ; 12(15): 1879-94, 2006.
Article in English | MEDLINE | ID: mdl-16724954

ABSTRACT

Emergence of drug resistant strains of human immunodeficiency virus type 1 (HIV-1) is a major hindrance in the long-term treatment of HIV-1 infected individuals. Alternative strategies, including those directed to structural elements of viral targets, are needed to combat the growing acquired immune deficiency syndrome (AIDS) pandemic. The HIV-1 reverse transcriptase (RT) dimer interface, critical for dimer stability and catalytic function, is a novel target for designing new anti-HIV-1 drugs. Several existing RT inhibitors are known to impair polymerase function by destabilizing RT dimer stability and can serve as useful leads in this direction. Conversely, studies have shown that potent nonnucleoside reverse transcriptase inhibitors (NNRTIs) can enhance RT subunit interaction, which may contribute in part to the inhibitory effect of these drugs. Interface peptides are reported to suppress enzyme activity by interfering with active RT heterodimer formation. This review focuses on small molecule and peptide inhibitors that interfere with the formation of the active RT heterodimer and also discusses regions in the RT that are critical for RT dimerization that can be considered as potential targets for chemotherapeutic intervention.


Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , Reverse Transcriptase Inhibitors/pharmacology , Binding Sites , Dimerization , Enzyme Stability/drug effects , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , Humans , Models, Molecular , Molecular Structure , Protein Structure, Quaternary , Reverse Transcriptase Inhibitors/chemistry
6.
Proteins ; 60(1): 5-13, 2005 Jul 01.
Article in English | MEDLINE | ID: mdl-15852304

ABSTRACT

The multifunctional enzyme human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a heterodimer composed of a 66-kDa (p66) subunit and a p66-derived 51-kDa (p51) subunit. p66/p51 HIV-1 RT contains 1 functional DNA polymerase and 1 ribonuclease H (RNase H) active site, which both reside in the p66 subunit at spatially distinct regions. In this study, we have investigated the relationship between the heterodimeric structure of HIV-1 RT and its enzymatic properties by introducing mutations at RT codon W401 that inhibit the formation of p66/p51 heterodimers. We demonstrate a striking correlation between abrogation of both HIV-1 RT dimerization and DNA polymerase activity. In contrast, the p66 monomers exhibited only moderately slowed catalytic rates of DNA polymerase-dependent and DNA polymerase-independent RNase H cleavage activity compared with the wild-type (WT) enzyme. Furthermore, no major changes in the unique cleavage patterns were observed between the WT and mutant enzymes for the different substrates used in the RNase H cleavage assays. Based on these results, and on our current understanding of HIV-1 RT structure, we propose that the p66 monomer can adopt an open tertiary conformation that is similar to that observed for the subunit in the heterodimeric enzyme. We also propose that the formation of intersubunit interactions in HIV-1 RT regulates the establishment of a functional DNA polymerase active site.


Subject(s)
Enzyme Activation/genetics , HIV Reverse Transcriptase/metabolism , Binding Sites/genetics , Circular Dichroism , DNA-Directed DNA Polymerase/metabolism , Dimerization , HIV Reverse Transcriptase/genetics , Humans , Kinetics , Mutation , Protein Conformation , Protein Folding , Protein Subunits/genetics , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Ribonuclease H/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Structure-Activity Relationship
7.
Proc Natl Acad Sci U S A ; 98(13): 7188-93, 2001 Jun 19.
Article in English | MEDLINE | ID: mdl-11416202

ABSTRACT

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV type 1 (HIV-1) reverse transcriptase (RT). Yeast grown in the presence of many of these drugs exhibited dramatically increased association of the p66 and p51 subunits of the HIV-1 RT as reported by a yeast two-hybrid assay. The enhancement required drug binding by RT; introduction of a drug-resistance mutation into the p66 construct negated the enhancement effect. The drugs could also induce heterodimerization of dimerization defective mutants. Coimmunoprecipitation of RT subunits from yeast lysates confirmed the induction of heterodimer formation by the drugs. In vitro-binding studies indicate that NNRTIs can bind tightly to p66 but not p51 and then mediate subsequent heterodimerization. This study demonstrates an unexpected effect of NNRTIs on the assembly of RT subunits.


Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Amino Acid Substitution , Binding Sites , Dimerization , Drug Resistance, Microbial/genetics , HIV Reverse Transcriptase/genetics , HIV-1/enzymology , HIV-1/genetics , Humans , Kinetics , Models, Molecular , Mutation , Protein Conformation , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Structure-Activity Relationship
8.
Proc Natl Acad Sci U S A ; 97(12): 6334-9, 2000 Jun 06.
Article in English | MEDLINE | ID: mdl-10841542

ABSTRACT

HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity. To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system. Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction. However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66. A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system. Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization. In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits. The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.


Subject(s)
HIV Reverse Transcriptase/chemistry , Dimerization , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/chemistry
9.
Antimicrob Agents Chemother ; 42(11): 3038-43, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9797252

ABSTRACT

Human immunodeficiency virus type 1 (HIV-1) isolates obtained from a patient with AIDS were assessed for coresistance to foscarnet and zidovudine. An HIV-1 strain (AP20) coresistant to foscarnet and zidovudine was isolated after 20 months of continuous combination therapy. The reverse transcriptase (RT) gene of AP20 had 41 substitutions which were different from the HXB2-D sequence and 9 that were different from the sequence of its foscarnet-sensitive, zidovudine-resistant progenitor virus (AP6). Six of these mutations were nonpolymorphic (T39A, V108I, K166R, K219R, K223Q, and L228R). Both strains had the conventional mutations mediating zidovudine resistance. In vivo selection may result in HIV-1 strains that are coresistant to foscarnet and zidovudine, but coresistance appears to require a complex evolutionary path and multiple RT mutations.


Subject(s)
Anti-HIV Agents/pharmacology , Foscarnet/pharmacology , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , Mutation , Zidovudine/pharmacology , Amino Acid Sequence , Drug Resistance , HIV Reverse Transcriptase/chemistry , Humans , Molecular Sequence Data , Polymerase Chain Reaction
10.
AIDS Res Hum Retroviruses ; 14(12): 1059-64, 1998 Aug 10.
Article in English | MEDLINE | ID: mdl-9718121

ABSTRACT

Foscarnet (PFA) is a pyrophosphate analogue antiviral active against human immunodeficiency virus (HIV-1) and herpesviruses. Strains of HIV-1 resistant to PFA have mutations in the HIV-1 reverse transcriptase (RT). We examined the influence of PFA resistance mutations, in different genetic backgrounds, on HIV-1 replication competency in both replication kinetics and growth competition assays. In replication kinetics assays, the recombinant strains HX89K, HX92I, and HX156A (encoding RT mutations E89K, L92I, and S156A, respectively, in the HXB2-D genetic background) replicated to lower titers than the wild-type parent in the absence of drug, and the degree of replication impairment increased as PFA resistance increased. PFA-resistant strains LAI 92I and LAI 156A (encoding RT mutations L92I and S156A, respectively) were replication impaired in comparison to the wild-type parent LAI to a similar degree as observed for strains in the HXB2D background. In growth competition assays with wild-type LAI, strains LAI 92I and LAI 156A had relative fitness values of 0.5 and 0.8, respectively. These results show that the RT mutations E89K, L92I and S156A, observed in PFA-resistant strains selected in cell culture, reduce replication competence. Furthermore, these data show a correlation of increasing PFA resistance and decreasing replication competence mediated by single amino acid substitutions in the RT.


Subject(s)
Antiviral Agents/pharmacology , Foscarnet/pharmacology , HIV-1/drug effects , Cell Line , Drug Resistance, Microbial/genetics , HIV Reverse Transcriptase/genetics , HIV-1/genetics , HIV-1/physiology , Virus Replication
11.
J Virol ; 70(11): 8010-8, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8892925

ABSTRACT

Sequencing of the reverse transcriptase (RT) region of 26 human immunodeficiency virus type 1 (HIV-1) isolates from eight patients treated with 3'-azido-3'-deoxythymidine (AZT) revealed a mutation at codon 210 from TTG (leucine) to TGG (tryptophan) exclusively in association with resistance to AZT. The mutation Trp-210 was observed in 15 of the 20 isolates phenotypically resistant to AZT, being more commonly observed than resistance-associated mutations at codons 67, 70, and 219. Trp-210 was never observed before the emergence of resistance-associated mutations Leu-41 and Tyr-215, and in a sequential series of five isolates from one patient the order of emergence of mutations was found to be Tyr-215, Leu-41, and then Trp-210. Trp-210 was also found in association with the Leu-41, Asn-67, Arg-70, and Tyr-215 resistance genotype. To define the role of Trp-210 in AZT resistance, molecular HIV-1 clones were constructed with various combinations of RT mutations at codons 41, 67, 70, 210, and 215 and tested for susceptibility to AZT. In clones with polymerase genes derived either from HXB2-D or clinical isolates, Trp-210 alone did not increase AZT resistance, whereas in conjunction with Leu-41 and Tyr-215, Trp-210 contributed to high-level resistance (50% inhibitory concentration of >1 microM). In HXB2-D, Trp-210 with Tyr-215 generated a virus with resistance comparable to one with Leu-41, Tyr-215, and Trp-210. Inserting Trp-210 into the genetic context of mutations at codons 41, 67, 70, and 215 further enhanced resistance from a 50% inhibitory concentration of 1.44 microM to 8.41 microM. Molecular modeling of the tertiary structure of HIV-1 RT revealed that the distance between the side chains of Trp-210 (in helix alphaF) and Tyr-215 (in strand beta11a) approximated 4 A (1 A = 0.1 nm), sufficiently close to result in significant energetic interaction between these two aromatic side chains. In conclusion, Trp-210 contributes significantly to phenotypic AZT resistance of HIV-1 by augmenting resistance at least three- to sixfold in the context of two resistant genotypes, and its effect may require an interaction with an aromatic amino acid at position 215.


Subject(s)
HIV Infections/virology , HIV Reverse Transcriptase/chemistry , HIV-1/enzymology , Leucine , Reverse Transcriptase Inhibitors/pharmacology , Tryptophan , Zidovudine/pharmacology , Base Sequence , Cell Line , Cell Line, Transformed , DNA, Viral , Drug Resistance, Microbial , HIV Infections/drug therapy , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/drug effects , HIV-1/genetics , HIV-1/isolation & purification , Humans , Models, Molecular , Molecular Sequence Data , Point Mutation
12.
J Virol ; 70(10): 7171-81, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8794364

ABSTRACT

Both foscarnet (PFA) and zidovudine (AZT) select for drug-resistant variants of human immunodeficiency virus type 1 (HIV-1), but the interactions between the mutations causing such resistance are unknown. The introduction of the previously identified PFA resistance mutation W to G at codon 88 (W88G), E89K, L92I, or Q161L into an HIV-1 strain having the four known AZT resistance mutations completely reversed high-level AZT resistance. Two additional PFA resistance mutations, W88S and S156A, partially suppressed AZT resistance. Phenotypic reversion of AZT resistance by W88S, W88G, E89K, L921, and S156A was associated with a concomitant suppression of PFA resistance. The degree to which PFA resistance mutations reversed AZT resistance was directly correlated with each mutation's ability to confer high-level PFA resistance (> or = 5.0-fold) and AZT hypersusceptibility in a wild-type genetic background. Highly PFA-resistant HIV- 1 strains were hypersusceptible to AZT; conversely, AZT-resistant strains with M41L and T215Y; M41L, L210W, and T215Y; or M41L, D67N, K70R, and T215Y mutations were 2.2- to 2.5-fold hypersusceptible to PFA. Prolonged in vitro selection of wild-type or AZT-resistant HIV-1 strains with the combination AZT and PFA failed to generate coresistant virus, indicating that dual resistance was relatively difficult to achieve. Strains selected by passage in PFA plus AZT were phenotypically PFA resistant and AZT susceptible despite multiple reverse transcriptase mutations known to confer AZT resistance. These data show that PFA resistance mutations can phenotypically reverse AZT resistance and that AZT and PFA resistance might be mutually exclusive. The reciprocal interactions between AZT and PFA resistance-conferring mutations have implications for structure-function studies of the HIV-1 reverse transcriptase.


Subject(s)
Antiviral Agents/pharmacology , Drug Resistance/genetics , Foscarnet/pharmacology , HIV-1/drug effects , Mutation , Zidovudine/pharmacology , HIV-1/genetics , Humans , Molecular Sequence Data
13.
Virology ; 212(1): 58-68, 1995 Sep 10.
Article in English | MEDLINE | ID: mdl-7545854

ABSTRACT

Foscarnet is a broad-spectrum viral DNA polymerase inhibitor active in vitro and in vivo against human immunodeficiency virus type 1 (HIV-1). Strains of HIV-1 resistant to foscarnet were selected by in vitro passage in increasing concentrations of drug. Reduced susceptibility to foscarnet was evident at the levels of both HIV-1 replication and reverse transcriptase. Biologically cloned, foscarnet-resistant strains with distinct genotypes were hypersensitive to zidovudine, azidodeoxyuridine, nevirapine, and R82913 but had unchanged susceptibility to zalcitibine and didanosine. The reverse transcriptase of foscarnet-resistant strains had unique substitutions Glu89-Lys, Leu92-Ile, or Ser156-Ala, the third being associated with six polymorphic changes. Introduction of these mutations into wild-type HIV-1 by site-directed mutagenesis confirmed their role in foscarnet resistance. In the three-dimensional structure of the reverse transcriptase enzyme these amino acids are located close to the template strand of the template primer and far away from the putative pyrophosphate binding site, suggesting that the mechanism by which HIV-1 becomes resistant to foscarnet is indirect. Foscarnet resistance is thus likely to be mediated through an altered interaction of the mutant enzyme with the template strand of the template primer which distorts the geometry of the polymerase active site and thereby decreases foscarnet binding.


Subject(s)
Foscarnet/pharmacology , HIV-1/drug effects , Amino Acid Sequence , Base Sequence , DNA Primers/chemistry , Drug Resistance, Microbial , Genes, pol , HIV Reverse Transcriptase , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA-Directed DNA Polymerase/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship
14.
J Med Virol ; 46(3): 238-43, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7561796

ABSTRACT

Sequential human immunodeficiency virus type 1 (HIV-1) isolates were obtained over a 29-month period from a person before, during, and after AZT therapy. DNA sequence analysis of polymerase chain-amplified reverse-transcriptase gene showed a gradual accumulation of mutations to peak resistance (IC50 2.13 microM AZT) in association with mutations at codons 44, 210, and 369, as well as at 41, 67, 70, and 215. Eight months after cessation of AZT therapy, when an HIV-1 isolate from the patient was again sensitive to AZT, these mutations had all returned to the pretherapy sequence.


Subject(s)
Acquired Immunodeficiency Syndrome/virology , HIV-1/enzymology , Mutation , RNA-Directed DNA Polymerase/genetics , Zidovudine/therapeutic use , Acquired Immunodeficiency Syndrome/drug therapy , Adult , Amino Acid Sequence , Base Sequence , Cells, Cultured , DNA, Viral , Drug Resistance, Microbial , Female , Follow-Up Studies , Genes, Viral , HIV Reverse Transcriptase , HIV-1/drug effects , HIV-1/genetics , HIV-1/isolation & purification , Humans , Microbial Sensitivity Tests , Molecular Sequence Data
15.
J Med Virol ; 42(2): 207-11, 1994 Feb.
Article in English | MEDLINE | ID: mdl-7512615

ABSTRACT

Sequential human-immunodeficiency virus type 1 (HIV-1) isolates from an acquired immunodeficiency syndrome (AIDS) patient on long-term foscarnet and zidovudine therapy were examined for the emergence of foscarnet resistance. One isolate obtained before therapy, and three post-foscarnet therapy HIV isolates had similar foscarnet sensitivity profiles, despite the emergence of foscarnet-resistant herpes simplex virus type 2 (HSV-2) during the period of therapy. Virion reverse transcriptases from these isolates were also equally inhibited by foscarnet. In contrast, sequential HIV isolates taken pre- and postzidovudine therapy showed a gradual increase in IC50 to this drug. In this patient, long-term foscarnet and zidovudine therapy selected foscarnet-resistant HSV and zidovudine-resistant HIV; in contrast, HIV remained susceptible to foscarnet. Concurrent administration of other anti-HIV drugs (zidovudine and interferon-alpha) may have hindered the development of foscarnet resistant HIV-1 in vivo.


Subject(s)
Acquired Immunodeficiency Syndrome/microbiology , Foscarnet/therapeutic use , HIV-1/genetics , Herpes Genitalis/drug therapy , Acquired Immunodeficiency Syndrome/complications , Dose-Response Relationship, Drug , Drug Resistance, Microbial , Foscarnet/pharmacology , HIV Reverse Transcriptase , Herpes Genitalis/complications , Herpesvirus 2, Human/genetics , Humans , RNA-Directed DNA Polymerase/drug effects , Time Factors , Zidovudine/therapeutic use
16.
AIDS Res Hum Retroviruses ; 9(12): 1245-50, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8142141

ABSTRACT

Topoisomerase I activity was detected in detergent-disrupted human immunodeficiency virus type 1 (HIV-1) particles. The enzyme did not require ATP for its conversion of SC DNA to an RC form, had divalent cation requirements similar to those of eukaryotic topoisomerase I, and was significantly inhibited by the specific topoisomerase I inhibitor camptothecin. However, camptothecin failed to inhibit replication of HIV in infected cells at nontoxic concentrations, and an active site motif for topoisomerase I could not be detected on the HIV genome. These results suggests that HIV does not encode a novel topoisomerase I, but rather packages the cellular enzyme.


Subject(s)
DNA Topoisomerases, Type I/metabolism , HIV-1/enzymology , Amino Acid Sequence , Blotting, Western , Camptothecin/pharmacology , Cell Line , Consensus Sequence , DNA Topoisomerases, Type I/genetics , HIV Infections/enzymology , HIV Infections/microbiology , HIV-1/genetics , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Topoisomerase I Inhibitors , Virus Replication/drug effects
17.
J Infect Dis ; 166(1): 108-12, 1992 Jul.
Article in English | MEDLINE | ID: mdl-1318909

ABSTRACT

Three AIDS patients with severe cutaneous herpes simplex virus (HSV) infection refractory to therapy with acyclovir and foscarnet (2 patients) were treated with a topical preparation of trifluorothymidine (TFT) and interferon-alpha. Complete healing of lesions occurred in 1 patient; a second had significant regression of the infected area. In the third, the lesion was stabilized twice after application of the preparation and reduced in size after a subsequent treatment. In vitro studies confirmed that isolates from these patients were acyclovir- or acyclovir/foscarnet-resistant. In addition, they revealed strong synergy between TFT and interferon-alpha for these isolates and for strains with wild-type drug sensitivity profiles. Topical TFT/interferon-alpha may be of benefit in the therapy of mucocutaneous HSV infections, especially when they are resistant to treatment with systemic antiviral agents.


Subject(s)
Acquired Immunodeficiency Syndrome/complications , Herpes Simplex/therapy , Interferon Type I/therapeutic use , Trifluridine/therapeutic use , Acyclovir/pharmacology , Acyclovir/therapeutic use , Adult , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Combined Modality Therapy , Cytopathogenic Effect, Viral/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Microbial , Female , Foscarnet , Herpes Simplex/complications , Herpes Simplex/drug therapy , Humans , Interferon Type I/pharmacology , Male , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/pharmacology , Phosphonoacetic Acid/therapeutic use , Recombinant Proteins , Recurrence , Simplexvirus/drug effects , Trifluridine/pharmacology , Virus Replication/drug effects
18.
J Infect Dis ; 162(3): 731-4, 1990 Sep.
Article in English | MEDLINE | ID: mdl-2167340

ABSTRACT

Acyclovir (ACV)-resistant herpes simplex virus type 2 (HSV-2) was isolated from a patient with acquired immunodeficiency syndrome after long-term but intermittent ACV therapy. These thymidine kinase-defective isolates were sensitive in vitro to foscarnet. While combined therapy with ACV and interferon produced only partial clinical improvement, the in vitro effect of this combination against an ACV-resistant isolate from the patient was strongly synergistic. A short course (10-12 days) of intravenous foscarnet controlled severe ulceration, and clinical improvement lasted 6 months. After recurrence and further courses of foscarnet, however, the patient responded poorly, and subsequent HSV isolates were resistant to both ACV and foscarnet and hypersensitive to aphidicolin.


Subject(s)
Acquired Immunodeficiency Syndrome/complications , Acyclovir/pharmacology , Antiviral Agents/pharmacology , Herpes Simplex/complications , Simplexvirus/drug effects , Acquired Immunodeficiency Syndrome/drug therapy , Acyclovir/therapeutic use , Adult , Aphidicolin , Deoxyguanosine/pharmacology , Diterpenes/pharmacology , Drug Resistance, Microbial , Female , Foscarnet , Herpes Simplex/microbiology , Humans , Phosphonoacetic Acid/analogs & derivatives , Phosphonoacetic Acid/pharmacology
19.
J Med Virol ; 31(2): 90-7, 1990 Jun.
Article in English | MEDLINE | ID: mdl-1696959

ABSTRACT

Primary duck hepatocyte (PDH) cultures, congenitally infected with the duck hepatitis B virus (DHBV), were grown on feeder cell layers of irradiated human embryonic lung fibroblasts and then exposed to a number of compounds with recognized or potential antiviral activity. These compounds included conventional antiviral agents, reverse transcriptase inhibitors, compounds with activity to supercoiled-DNA, and DNA-binding agents. Twenty-three compounds were evaluated, and 13 were found to inhibit significantly viral DNA replication. Seven of these compounds (ellipticine, amsacrine, coumermycin A1, Adriamycin, mitozantrone, chloroquine, and neocarzinostatin) acted at the level of viral SC DNA and significantly inhibited production of duck hepatitis B surface antigen (DHBsAg). Conventional agents that inhibited DHBV DNA replication included ganciclovir, acyclovir, bromovinyldeoxyuridine, ribavirin, phosphonoformate, and dideoxyadenosine. Except for dideoxyadenosine, these inhibitors of viral DNA synthesis did not significantly inhibit DHBsAg production. Two additional compounds, novobiocin and nalidixic acid, altered the pattern of viral DNA replication, especially the generation and processing of viral SC DNA, and also inhibited the production of DHBsAg. Several compounds acting at the level of viral SC DNA have now been identified and may offer potential for the management of chronic hepatitis B virus infection.


Subject(s)
Antiviral Agents/pharmacology , Disease Models, Animal , Ducks , Hepatitis B Virus, Duck/drug effects , Hepatitis, Viral, Animal/pathology , Animals , Antiviral Agents/classification , Cells, Cultured , DNA Damage , DNA Replication/drug effects , DNA, Superhelical/drug effects , DNA, Viral/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Hepatitis B Virus, Duck/genetics , Hepatitis B Virus, Duck/physiology , Humans , Intercalating Agents/pharmacology , Liver/pathology , Reverse Transcriptase Inhibitors , Virus Replication/drug effects
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