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At least 1,207 records · Page 67Linked to original sources
PubMed1995

Anti-human immunodeficiency virus (HIV) activities of halogenated gomisin J derivatives, new nonnucleoside inhibitors of HIV type 1 reverse transcriptase.

Halogenated gomisin J (a derivative of lignan compound), represented by the bromine derivative 1506 [(6R, 7S, S-biar)-4,9-dibromo-3,10-dihydroxy-1,2,11,12-tetramethoxy-6, 7-dimethyl-5,6,7,8- tetrahydrodibenzo[a,c]cyclo-octene], was found to be a potent inhibitor of the cytopathic effects of human immunodeficiency virus type 1 (HIV-1) on MT-4 human T cells (50% effective dose, 0.1 to 0.5 microM). Gomisin J derivatives were active in preventing p24 production from acutely HIV-1-infected H9 cells. The selective indices (toxic dose/effective dose) of these compounds were as high as > 300 in some systems. 1506 was active against 3'-azido-3'-deoxythymidine-resistant HIV-1 and acted synergistically with AZT and 2',3'-ddC. 1506 inhibited HIV-1 reverse transcriptase (RT) in vitro but not HIV-1 protease. From the time-of-addition experiment, 1506 was found to inhibit the early phase of the HIV life cycle. A 1506-resistant HIV mutant was selected and shown to possess a mutation within the RT-coding region (at position 188 [Tyr to Leu]). The mutant RT expressed in Escherichia coli was resistant to 1506 in the in vitro RT assay. Some of the HIV strains resistant to other nonnucleoside HIV-1 RT inhibitors were also resistant to 1506. Comparison of various gomisin J derivatives with gomisin J showed that iodine, bromine, and chlorine in the fourth and ninth positions increased RT inhibitory activity as well as cytoprotective activity.

Antiviral Agents↗
PubMed1994

Inhibition of HIV type 1 reverse transcriptase assay by nucleases produced by contaminating mycoplasmas.

Mycoplasmal contamination of HIV-1-infected cells has been found to induce reduction of reverse transcriptase (RT) activity; however, the exact mechanism of this phenomenon was not clearly elucidated. Our results indicate that the apparent reduction in RT activity is due to a calcium-dependent nuclease(s) that is (are) produced by contaminating mycoplasmas. The interference with the RT assay was found to be due to the degradation of products of the RT activity. Addition of EGTA at a 1 mM concentration was sufficient to remove the inhibitory effect. The particular HIV-1-producing cell line that was under study was found to be contaminated with Mycoplasma fermentans and Mycoplasma pirum and the latter was isolated in pure culture. Nuclease activity was also observed with pure cultures of mycoplasmas from different species. The activity was found to be of the endonuclease type because it was active with both supercoiled and linear DNAs.

Cell Line↗
PubMed1998

Lamivudine resistance of HIV type 1 does not delay development of resistance to nonnucleoside HIV type 1-specific reverse transcriptase inhibitors as compared with wild-type HIV type 1.

We compared the development of resistance toward BI-RG-587 (nevirapine) and alpha-APA R89439 (loviride) starting from the wild-type HIV-1 strain IIIB and the 3TC-resistant HIV-1 strain containing the M184V mutation. The reverse transcriptase of the M184V mutant has been reported to have a higher fidelity. Our experiments showed that there was no significant delay in virus breakthrough of the M184V mutant as compared with the wild-type virus. We therefore conclude that the reported higher fidelity of the M184V mutant does not lead to a delay in the development of resistance to the nonnucleoside reverse transcriptase inhibitors nevirapine and loviride.

Acetamides↗
PubMed2001

Evaluation of viral resistance to reverse transcriptase inhibitors (RTI) in HIV-1-infected patients before and after 6 months of single or double antiretroviral therapy.

We evaluated samples of peripheral blood mononuclear (PBMC) cells from 46 AIDS patients, before starting therapy with HIV-1 reverse transcriptase inhibitors (RTI), and after 6 months of drug use. PBMC were stored and tested by a Line Probe Assay (LiPA), in order to assess the frequency of RT mutations in this population. Six patients were taking AZT before initial blood collection (1 to 16 weeks of drug use) and 40 patients had no prior therapy. After baseline evaluation, 19 patients received AZT, 23 AZT plus DDI, 3 started AZT only with DDI added after 3 months, and 3 received a combination of AZT plus 3TC. Detection of at least one mutation was found in 33% (15/46) of patients at baseline, and 83% (38/46) had at least 1 mutation after 6 months of therapy. In the majority of cases, samples presented with the wild type and variants of HIV, simultaneously. Patients receiving monotherapy had a higher frequency of mutations (L41 and F214, Y215) than did patients receiving double-drug therapy (19 vs. 10). No specific mutation associated with DDI was identified in 26 patients so treated. Despite the finding of a mean increase in CD4 count and a mild decrease in viral load, patients tended to have an inverse correlation between the CD4 variation and number of mutations detected after 6 months, suggesting potential loss of drug efficacy in the presence of these genotypic changes.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Specific inhibition of in vitro reverse transcription using antisense oligonucleotides targeted to the TAR regions of HIV-1 and HIV-2.

Antisense oligonucleotides (ODNs) overlapping the stem-loop structure of the trans-activating responsive (TAR) element at the 5' end of HIV-1 and HIV-2 viral RNAs were tested for their inhibitory effect on cDNA synthesis by HIV-1 and HIV-2 reverse transcriptases (RT). Inhibition of reverse transcription is sequence-specific and enhanced by the presence of the RT-associated RNase H activity. The degree of inhibition obtained with the anti-TAR antisense is significantly higher than with other HIV-1 targeted antisense ODNs used before [1]. Gel retardation showed a stable specific complex between the 16- and 25-mer anti-TAR HIV-1 selected ODNs and the target region. No complex was observed with a non-inhibitor 22-mer anti-TAR ODN and with the corresponding control sequences. Targeting of the first stem-loop in the 5' region of HIV-2 RNA by anti-TAR ODNs inhibited very strongly reverse transcription by HIV-2 RT. The structure of the antisense and the target sequence affect annealing efficiency and hence the degree of inhibition of reverse transcription.

Base Sequence↗
PubMed1989

Further characterization of sulfated homopolysaccharides as anti-HIV agents.

Fucoidan and dextran sulfate showed anti-HIV activities against mononuclear cells from AIDS patients, and they abrogated HIV reverse transcriptase (RT) activity by interacting with the HIV envelope in the membranes of target cells. Furthermore, they showed a synergistic effect with azidothymidine (AZT).

Acquired Immunodeficiency Syndrome↗
PubMed2004

Evolution of human immunodeficiency virus type 1 (HIV-1) resistance mutations in nonnucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1-infected patients switched to antiretroviral therapy without NNRTIs.

We studied the evolution of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations among 29 human immunodeficiency virus type 1 (HIV-1)-infected patients who experienced virologic failure when receiving an NNRTI-containing regimen (nevirapine, delavirdine, or efavirenz) and subsequently switched to antiretroviral therapy without NNRTIs. Genotypic resistance was determined from plasma samples collected at the time of NNRTI withdrawal (baseline) and during follow-up. At baseline, 83% of patients had more than two thymidine analog resistance mutations (TAMs), and all had NNRTI resistance mutations. Mutations at codons 103, 181, and 190 were found in 62, 62, and 34% of the patients, respectively. Follow-up samples were available after a median time of 6 months in all patients and at 12 months in 22 patients. The mean number of resistance mutations to NNRTIs was significantly lower at months 6 (1.34 +/- 1.04) and 12 (1.18 +/- 1.05) than at month 0 (2.03 +/- 1.02) (P < 0.009). The percentages of patients with at least one NNRTI resistance mutation were 100, 76, and 73% at baseline, month 6, and month 12, respectively (P < 0.0044). Overall, 70% of the patients had a mutation at codon 103 or 181 at month 12. The mean number of TAMs did not vary significantly during follow-up. Our data show that, in the context of maintained antiretroviral therapy, NNRTI resistance mutations persist in two-thirds of the patients in spite of NNRTI withdrawal. These results argue for the low impact of NNRTI resistance mutations on viral fitness and suggest that resistance mutations to different classes of drugs are associated on the same genome, at least in some of the resistant strains.

Antiretroviral Therapy, Highly Active↗
PubMed1989

Human immunodeficiency virus reverse transcriptase-associated RNase H activity.

Biochemical characteristics of the RNase H activity associated with immunoaffinity purified human immunodeficiency virus (HIV) reverse transcriptase (RT) were examined. Glycerol gradient centrifugation of HIV RT resulted in a single peak of RNase H, associated with RT activity, with an apparent molecular weight of 110,000. HIV RNase H exhibited a marked substrate preference for poly(dC).[3H]poly(rG) compared to poly(dT).[3H]poly(rA). It did not hydrolyze single-stranded RNA or the DNA component of DNA.RNA hybrids. Products of the HIV RT-associated RNase H reaction consisted primarily of monomers, dimers, and trimers with 3' OH groups. This reaction was Mg2+ dependent, with greater than 90% of maximum activity at MgCl2 concentrations between 4 and 12 mM. The optimum KCl concentration for HIV RT catalyzed polymerization with a poly(rA).(dT)10 template. The optimum pH for HIV RNase H activity was between 8.0 and 8.5, in contrast to an optimum pH of 7.5 to 8.0 for HIV RT activity. The association of RNase H activity with the p66 component of HIV RT was demonstrated by activity gel analysis. These results indicate that HIV RT has an integral RNase H activity; however, some of its properties are different from those of RNase H associated with other retroviral RT's, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization.

Centrifugation, Density Gradient↗
PubMed1998

Contacts between reverse transcriptase and the primer strand govern the transition from initiation to elongation of HIV-1 reverse transcription.

HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA3Lys, and RT. We have previously shown that extension of an oligodeoxyribonucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primer-template complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2'-hydroxyl group at the 3' end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400-fold between addition of the sixth and seventh nucleotide to tRNA3Lys. The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA3Lys. The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix alphaH in the p66 thumb subdomain and the RNase H domain of RT, respectively.

Animals↗
PubMed1995

Comparative anti-HIV evaluation of diverse HIV-1-specific reverse transcriptase inhibitor-resistant virus isolates demonstrates the existence of distinct phenotypic subgroups.

We have biologically and biochemically evaluated a structurally diverse group of HIV-1-specific reverse transcriptase (RT) inhibitors and determined that the members of this class share many common properties. These include reproducible and selective antiviral activity against a panel of biologically distinct laboratory and clinical strains of HIV-1, activity against HIV-1 in a wide variety of cultured and fresh human cells, and potent inhibition of HIV-1 RT when evaluated using a heteropolymeric ribosomal RNA template assay. Each of the HIV-1-specific compounds was capable of inhibiting HIV replication when challenged at high m.o.i., further distinguishing them from the nucleoside analogs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC). When tested in combination with AZT, each of the HIV-1-specific compounds synergistically inhibited the replication of HIV-1. HIV-1 isolates resistant to different HIV-1-specific inhibitors exhibited heterogeneous patterns of cross-resistance to other members of this pharmacologic class. Four distinct phenotypic classes have been defined through the use of drug-resistant virus isolates which derive from distinct mutations in the RT. These results indicate that the various subgroups of HIV-1-specific inhibitors interact differently with HIV-1 RT, suggesting important potential implications for drug combination therapeutic strategies.

Antiviral Agents↗
PubMed1996

Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens.

We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.

Amino Acid Sequence↗
PubMed1995

Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cell-free assays and in HIV-infected cells.

We have investigated two regions of the viral RNA of human immunodeficiency virus type 1 (HIV-1) as potential targets for antisense oligonucleotides. An oligodeoxynucleotide targeted to the U5 region of the viral genome was shown to block the elongation of cDNA synthesized by HIV-1 reverse transcriptase in vitro. This arrest of reverse transcription was independent of the presence of RNase H activity associated with the reverse transcriptase enzyme. A second oligodeoxynucleotide targeted to a site adjacent to the primer binding site inhibited reverse transcription in an RNase H-dependent manner. These two oligonucleotides were covalently linked to a poly(L-lysine) carrier and tested for their ability to inhibit HIV-1 infection in cell cultures. Both oligonucleotides inhibited virus production in a sequence- and dose-dependent manner. PCR analysis showed that they inhibited proviral DNA synthesis in infected cells. In contrast, an antisense oligonucleotide targeted to the tat sequence did not inhibit proviral DNA synthesis but inhibited viral production at a later step of virus development. These experiments show that antisense oligonucleotides targeted to two regions of HIV-1 viral RNA can inhibit the first step of viral infection--i.e., reverse transcription--and prevent the synthesis of proviral DNA in cell cultures.

Base Sequence↗
PubMed2005

Evaluation of a low cost reverse transcriptase assay for plasma HIV-1 viral load monitoring.

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.

Alkynes↗
PubMed2006

In vitro synthesis of long DNA products in reactions with HIV-RT and nucleocapsid protein.

In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.

DNA Primers↗
PubMed1996

Risks and synergies from drug interactions.

AIM: To review what is known from in vitro and in vivo studies about the interactions, both potentially beneficial and potentially harmful, of antiretroviral agents with each other and with other classes of drugs. INTERACTIONS WITH NUCLEOSIDE ANALOGUES: Some interactions between nucleoside HIV reverse transcriptase inhibitors and between nucleoside analogues and HIV protease inhibitors result in greater antiretroviral activity (e.g. zalcitabine with saquinavir). Others may increase the risks of toxicity and there are a number of combinations of nucleoside agents with other drugs that should be used with caution or avoided completely. INTERACTIONS WITH PROTEASE INHIBITORS: These drugs are metabolized by cytochrome P450 CYP3A4 in the liver; because they have the potential to inhibit this enzyme they may interact with many other drugs that are metabolized by this pathway. Ritonavir also inhibits other cytochrome P450 enzymes and so interacts with numerous drugs from a range of classes. Potentially beneficial interactions between protease inhibitors include the increase in saquinavir levels brought about by ritonavir. CONCLUSIONS: Knowledge of additive and synergistic interactions between antiretroviral agents should facilitate development of therapeutic regimens with prolonged antiretroviral activity. Thorough investigation of possibly harmful interactions with co-administered drugs and education of clinicians and patients about the risks of these interactions is required.

Anti-HIV Agents↗
PubMed2006

Overcoming HIV drug resistance through rational drug design based on molecular, biochemical, and structural profiles of HIV resistance.

There are 20 available drugs for the treatment of human immunodeficiency virus (HIV) infection. With a single exception, all of these drugs inhibit either HIV reverse transcriptase or protease. Reverse transcriptase inhibitors can be further categorized as nucleoside/nucleotide analogs or non-nucleoside reverse transcriptase inhibitors. Resistance that has emerged against all available antiretroviral drugs represents a major challenge in the therapy of HIV infection. Nevertheless, extensive analysis of the molecular and structural mechanisms by which such mutations confer resistance has accumulated over the years. This understanding has driven the development and refinement of novel compounds capable of maintaining antiviral activity against both wild-type and drug-resistant HIV strains. The molecular, biochemical, and structural profiles of reverse transcriptase inhibitor and protease inhibitor resistance are discussed. In addition, how this knowledge has been utilized to generate a new generation of antiviral drugs with activity against drug-resistant HIV is reviewed.

Anti-HIV Agents↗