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PubMed2005

Inhibition of human telomerase by oligonucleotide chimeras, composed of an antisense moiety and a chemically modified homo-oligonucleotide.

Most tumor cells attain their immortality by reactivating telomerase. We report here the telomerase inhibitory potential of chimeric oligonucleotides composed of a 13mer antisense sequence targeting the telomerase RNA template region and a (s4dU)n moiety at its 3' or 5'-end. The increase of the thiolated chain length enhances the telomerase inhibitory potential, but decreases specificity, indicated by HIV reverse transcriptase inhibition. Chimeras with 5' (s4dU)(n)s were more potent inhibitors than the antisense alone or the 3' modified ones. Cy5-labeled (s4dU)4AS and (s4dU)8AS proved the internalization of the oligonucleotides, raising the possibility to be tested as cellular anti-telomerase agents.

Base Sequence↗
PubMed1998

Pausing of reverse transcriptase on retroviral RNA templates is influenced by secondary structures both 5' and 3' of the catalytic site.

In the most extensive examination to date of the relationship between the pausing of reverse transcrip-tase (RT) and RNA secondary structures, pause events were found to be correlated to inverted repeats both ahead of, and behind the catalytic site in vitro. In addition pausing events were strongly associated with polyadenosine sequences and to a lesser degree diadenosines and monoadenosine residues. Pausing was also inversely proportional to the potential bond strength between the nascent strand and the template at the point of termination, for both mono and dinucleotides. A run of five adenosine and four uridine residues caused most pausing on the HIV-1 template, a region which is the site of much sequence heterogeneity in HIV-1. We propose that homopolyadenosine tracts can act as termination signals for RT in the context of inverted repeats as they do for certain RNA polymerases.

Avian Myeloblastosis Virus↗
PubMed1999

Genotypic analysis methods for detection of drug resistance mutations in the HIV-1 proteinase and reverse transcriptase genes.

Understanding the basis of human immunodeficiency virus (HIV) drug resistance represents a key requirement for individualized HIV patient care. The genotypic data generated to date have already provided significant insight. However, it is clear that the relationship between genotype, phenotype and clinical outcome is complex and still poorly defined. In this review, we describe methods currently available to obtain genotypic data for the HIV-1 proteinase and reverse transcriptase genes. Different sample preparation strategies and DNA sequencing methods are discussed dividing the latter into two categories, those that give sequence information at specific positions and those that provide continuous sequence data for a particular region. In addition, we also address some of the broad biological and technical issues, which must be considered when interpreting the results of these tests and describe the advantages and disadvantages of individual methods.

Drug Resistance, Viral↗
PubMed1997

A pilot study of a combination of three reverse transcriptase inhibitors in HIV-1 infection.

The objective of this study was to evaluate the efficacy and tolerability of a combination of three reverse transcriptase inhibitors in patients with human immunodeficiency virus type 1 (HIV-1) infection. The investigation was an open pilot study of 48 weeks duration. Forty-five patients with CD4 cell counts between 50 and 500 cells/mm3 received a combination of oral zidovudine (200 mg three times daily) plus didanosine (200 mg twice daily) and lamivudine (150 mg twice daily). Plasma HIV-1 RNA and CD4 cell levels were measured weekly during the first month, at weeks 6, 8 and monthly thereafter. HIV-1 RNA levels were also measured sequentially in the lymph nodes of five patients after the initiation of therapy, and after several months of undetectable plasma RNA in 10 additional cases. Sequencing was performed on virus from the peripheral blood mononuclear cells of a subset of 14 patients after a mean period of 11+/-1 months on therapy. The mean (+/-SE) plasma viral load was 5.04+/-0.09 log10 copies/ml and the mean CD4 cell count was 339+/-14 cells/mm3 at baseline. Plasma HIV-1 RNA levels decreased exponentially in each case and became undetectable in 36 out of 42 cases who continued therapy for 24 weeks. HIV-1 RNA levels were < 20 copies/ml in 73% of these cases with undetectable HIV RNA. HIV-1 RNA decreased exponentially in lymph nodes after the initiation of therapy. The mean residual lymph node HIV-1 RNA level was 3.06+/-0.58 log10 copies/10(6) cells in 10 patients evaluated after several months of having undetectable plasma HIV RNA levels. A mean gain of 212 and 237 CD4 cells/mm3 was observed at 24 and 48 weeks, respectively. Proviral DNA sequencing showed that the main resistance codon mutations were absent in each case. Only one patient presented with a mutation resulting in the K219Q substitution, and one other with a T200I substitution. We conclude that this combination can achieve a significant decrease in HIV-1 replication in both plasma and lymph nodes in most cases. It is safe, able to delay the selection of resistant mutants, and keeps open the option for the use of protease inhibitors in case of therapeutic failure.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Enzymatic assay for measurement of intracellular DXG triphosphate concentrations in peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected patients.

DXG ([2R-cis]-2-amino-1,9-dihydro-9-[2-[hydroxymethyl]-1,3-dioxolan-4-yl]-6H-purin-6-one) and its prodrug DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir) are novel 2',3'-dideoxynucleosides (ddNs) displaying activity against human immunodeficiency virus type 1 (HIV-1). In this paper, we describe the development of an enzymatic assay for determining the intracellular active metabolite of DXG and DAPD, DXG triphosphate (DXGTP), in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients. The assay involves inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates (dNTPs) into a synthetic template primer. DXGTP (0.6 pmol) inhibited control product formation with or without a preincubation step. Inhibition was greatest when the template primer was most diluted. DAPDTP inhibited control product formation only at very high levels (50 pmol) and when a preincubation procedure was used. However, reduced template primer stability in assays using preincubation steps, coupled with potential interference by DAPDTP, led to the current assay method for DXGTP being performed without preincubation. Standard DXGTP inhibition curves were constructed. The presence of PBMC extracts or endogenous dGTP did not interfere with the DXGTP assay. Intracellular DXGTP and dGTP concentrations were determined in PBMCs from HIV-infected patients receiving oral DAPD (500 mg b.i.d.). Peak concentrations of DXGTP were obtained 8 h after dosing and were measurable through 48 h postdose. Levels of endogenous dGTP were also determined over 48 h. No direct relationship was observed between concentrations of DXGTP and dGTP. Quantification of DXGTP concentrations in PBMCs from patients receiving a clinically relevant dose of DAPD is possible with this enzymatic assay.

Dioxolanes↗
PubMed1993

The site of antiviral action of 3-nitrosobenzamide on the infectivity process of human immunodeficiency virus in human lymphocytes.

The C-nitroso compound 3-nitrosobenzamide, which has been shown to remove zinc from the retroviral-type zinc finger of p7NC nucleocapsid proteins, inhibits acute infection of human immunodeficiency virus type 1 in cultured human lymphocytes. The attachment of the virus to lymphocytes and the activities of critical viral enzymes, such as reverse transcriptase, protease, and integrase, are not affected by 3-nitrosobenzamide. However, the process of reverse transcription to form proviral DNA is effectively abolished by the drug, identifying the mode of action of 3-nitrosobenzamide as interrupting the role of p7NC in accurate proviral DNA synthesis during the infectious phase of the virus life cycle.

Antiviral Agents↗
PubMed1993

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

Capsid↗
PubMed1994

Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations.

The finding of G-->A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids. Using a RNA/reverse transcriptase ratio of approximately 1:30, comparable to that within the retroviral replication complex, G-->A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations--i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP. As G-->A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U-->C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G-->A and U-->C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.

Adenine↗
PubMed2001

Trifluoromethyl-containing 3-alkoxymethyl- and 3-aryloxymethyl-2-pyridinones are potent inhibitors of HIV-1 non-nucleoside reverse transcriptase.

3-Alkoxymethyl- and 3-aryloxymethyl-2-pyridinones were synthesized and evaluated for activity as non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1. It was found that several compounds were potent inhibitors of HIV-1 with the most potent compound 24 exhibiting an IC90 = 32 nM. Compound 24 also possessed a potent resistance profile as demonstrated by submicromolar IC90s against several clinically meaningful mutant virus strains.

Anti-HIV Agents↗
PubMed1995

Human immunodeficiency virus type 1 Nef increases the efficiency of reverse transcription in the infected cell.

We have analyzed the replication of Nef+ and Nef- isogenic human immunodeficiency virus in CEM, HUT78, MT4 lymphoid, and U937 monocytic cell lines. At each passage of infected cells, we have assessed the relative infectivity of the virus particles released in culture media by measuring the number of infections units per nanogram of p24 protein. Values appeared to be 3- to 10-fold higher for the Nef+ virus than for the Nef- number The positive effect of Nef was observed regardless of the cell line, the multiplicity of infection, and the number of virus replication cycles achieved. We showed, by using cells expressing glycosylphosphatidylinositol-linked CD4, that the enhancement of virion infectivity could be dissociated from the down-regulation of cell surface CD4 also induced by Nef. The gp120-to-p24 ratio and the RNA content of virus particles produced in the presence or in the absence of Nef were equivalent. Virions bound to cell surface CD4 receptors with equal efficiencies. Equivalent reverse transcriptase activities were measured both on exogenous substrate and on particle genomic RNAs. In contrast, reverse transcription in infected cells generated 5- to 10-fold less DNA when the virions were produced in the absence of Nef, indicating that these particles performed reverse transcription in a suboptimal environment. These data suggest that the expression of Nef in virus-producing cells is required for efficient processing of the early stages of virus replication in target cells, including the internalization in an appropriate cell compartment and the uncoating of the particle.

Cell Line↗
PubMed2003

Challenges of the unmet needs in the treatment-experienced patient.

Despite significant therapeutic advances in HIV medicine, virologic failures remain frequent in clinical practice. Issues such as suboptimal antiretroviral activity, poor tolerability, inconvenient dosing schedules, long-term toxicity, and low thresholds for the development of resistance all limit the clinical utility of HIV reverse transcriptase and protease inhibitors. Maintaining optimal viral suppression often necessitates that patients change treatment, but drug resistance and cross-resistance can rapidly reduce the number of effective treatments available. On the other hand, the therapeutic success achieved since the introduction of HAART has led to an increased overall exposure to antiretroviral drugs, and new toxicities have emerged. True second-generation agents and agents active against new targets will be essential to maximize the therapeutic impact of current antiretroviral therapy and minimize the risk of resistance development.

Antiretroviral Therapy, Highly Active↗
PubMed1995

Diagnosis of HIV infection from bloodstains by PCR. A further marker for identification.

The forensic usefulness of the detection of HIV infection in bloodstains is linked to the increasing spread of HIV infection and the consequent rise in the number of forensic cases involving HIV-positive subjects. This study was designed to detect HIV infection in bloodstains of various ages obtained from HIV-positive patients treated with zidovudine (3'-azido-3'-deoxythymidine, AZT) using PCR methods. Of the 3 kinds of extraction and amplification methods checked, only a nested PCR method for the amplification of a sequence located in the HIV Reverse Transcriptase (RT) region was successful. This method, involving 2 amplification steps (1: fragment of 768 bp; 2: codons 41-67-70-215-219), encompasses the mutations commonly observed during AZT therapy and overcomes the limitations inherent in serological testing. The discriminatory power of the method can detect specific mutation patterns in the RT gene linked to drug resistance and compares the specific pattern of the blood-stain with fresh blood or other specimens from the subject in question.

Antiviral Agents↗
PubMed2004

Pharmacokinetic and metabolic studies of retrojusticidin B, a potential anti-viral lignan, in rats.

The pharmacokinetics and metabolism of retrojusticidin B, an anti-HIV reverse transcriptase agent isolated from Phyllanthus myritifolius, were studied in rats. The phase II conjugated metabolites were characterized after solvolysis and enzymatic hydrolysis. The oral bioavailabilities of retrojusticidin B, suspended in Tween 80 and in corn oil, were found to be 22.1% and 33.1%, respectively. The elimination half-lives (T1/2) were 22.9 and 36.2 minutes, respectively. The T1/2, clearance, and the volume of distribution (Vz) of retrojusticidin B estimated from i.v. measurements were 24.5 min, 2.6 +/- 0.4 L/min, and 90.6 +/- 6.4 L, respectively. 9,9'-Secoretrojusticidin B was shown to be phase I metabolite.

Administration, Oral↗
PubMed2002

Development of enzymatic assays for quantification of intracellular lamivudine and carbovir triphosphate levels in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients.

In this paper, we describe the development and use of enzymatic assays to determine intracellular lamivudine triphosphate (3TCTP) and carbovir triphosphate (CBVTP) concentrations in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients. The assays involve inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates into a synthetic template primer. For the 3TCTP assay, a preincubation procedure was added whereby 3TCTP becomes incorporated before [(3)H]dCTP. At a 1:400 template primer dilution, control product formation was reduced by 88.0% with 0.8 pmol of 3TCTP. Standard 3TCTP inhibition curves were performed using this procedure. For the CBVTP assay, 0.1 pmol of CBVTP inhibited control product formation with and without the use of a preincubation step, so inhibition curves were constructed using both procedures. However, reduced template primer stability with assays using preincubation steps led to a single-incubation procedure being adopted for future studies. The presence of PBMC extracts interfered with the 3TCTP assay. However, this was overcome by the addition of CuSO(4). PBMC extracts did not interfere with the CBVTP assay. Intracellular 3TCTP and CBVTP concentrations were determined in PBMCs from HIV-infected patients over 24 h or greater. Peak concentrations were obtained 6 to 8 h after dosing, and the half-lives of the anabolites suggested the possibility of once-daily dosing. These assays are currently being used for determination of 3TCTP and CBVTP concentrations in clinical studies.

Chemistry Techniques, Analytical↗
PubMed2003

Impact of different HIV resistance interpretation by distinct systems on clinical utility of resistance testing.

PURPOSE OF REVIEW: Genotypic assays are widely used tools for determining HIV-1 drug resistance and for guiding treatment. Several systems have been developed to interpret the complex influence of amino acid substitutions in HIV reverse transcriptase or protease on the phenotypic susceptibility or clinical response to the 18 available antiretroviral agents. In this review we analyse both studies comparing interpretations by different systems and studies showing correlation of interpretations with clinical outcome, in order to identify discordance and how this may affect prediction of subsequent therapy outcomes. RECENT FINDINGS: During the last year, several studies analysing interpretation systems, individually or comparatively, have shown substantial variability of the predicted drug activities and therapeutic outcomes. Discrepant interpretation was detected mostly for nucleoside reverse transcriptase inhibitors and rarely for non-nucleoside reverse transcriptase inhibitors. Better correlation with treatment outcome was found with most recently updated systems, while a weaker prediction was found with systems interpreting activity of nucleoside reverse transcriptase inhibitors solely on the basis of phenotypic susceptibility. Virological, patient-related and treatment-related factors can all affect the results of systems' clinical validations. Refinement of resistance interpretation is possible by introducing rules derived from genotype-outcomes correlation or, at least for protease inhibitors, genotype-phenotype correlation. SUMMARY: Papers showing clinical validation of the available interpretation systems are presented with a critical view to help the readers' evaluation of their possible use. There is a need for developing a consensus towards common interpretations. Large clinical and virological databases with quality data will be useful for future improvements.

Acquired Immunodeficiency Syndrome↗
PubMed2005

G-->A hypermutation in protease and reverse transcriptase regions of human immunodeficiency virus type 1 residing in resting CD4+ T cells in vivo.

In vitro studies have shown that the host cytidine deaminase APOBEC3G causes lethal hypermutation in human immunodeficiency virus type 1 reverse transcripts unless its incorporation into virions is blocked by Vif. By examining stably archived sequences in resting CD4+ T cells, we show that hypermutation occurs in most if not all infected individuals. Hypermutated sequences comprised >9% of archived species in resting CD4+ T cells but were not found in plasma virus. Mutations occurred in predicted contexts, with notable hotspots. Thus, defects in Vif function in vivo give rise to hypermutated viral genomes that can be integrated but do not produce progeny viruses.

Antiretroviral Therapy, Highly Active↗
PubMed1989

1-(2,3-Dideoxy-beta-D-glycero-pent-2-enofuranosyl)thymine. A highly potent and selective anti-HIV agent.

The nucleoside analogue 1-(2,3-dideoxy-beta-D-glycero-pent-2-enofuranosyl)thymine (d4T, 1) was prepared by ring opening of the 3',5'-anhydro compound 5. This method has been refined such that it can be used to prepare d4T on a large scale. The triphosphate of d4T was also synthesized from 1 in order to examine the mode of action. The in vitro inhibitory activity of d4T was found to be comparable to that of AZT in HIV-infected CEM cells. The triphosphate of d4T (8) and that of AZT inhibited the HIV reverse transcriptase with poly(rA):oligo(dT) as the template:primer with Ki values of 0.032 and 0.007 microM, respectively. The in vitro toxicity of d4T against normal human hematopoietic progenitor cells (CFU-GM) was measured in comparison to AZT. While d4T reduces colony-forming units by 50% at a concentration of 100 microM, it takes only 1 microM AZT to have a similar toxic effect. With erythrocyte burst forming units (BFU-E) the in vitro toxicities for d4T and AZT have comparable ID50 values of 10 and 6.7 microM, respectively.

Antiviral Agents↗
PubMed2005

Marked depletion of glycosylation sites in HIV-1 gp120 under selection pressure by the mannose-specific plant lectins of Hippeastrum hybrid and Galanthus nivalis.

The plant lectins from Hippeastrum hybrid (HHA) and Galanthus nivalis (GNA) are 50,000-D tetramers showing specificity for alpha-(1,3) and/or alpha-(1,6)-mannose oligomers. They inhibit HIV-1 infection at a 50% effective concentration of 0.2 to 0.3 microg/ml. Escalating HHA or GNA concentrations (up to 500 microg/ml) led to the isolation of three HIV-1(III(B)) strains in CEM T cell cultures that were highly resistant to HHA and GNA, several other related mannose-specific plant lectins, and the monoclonal antibody 2G12, modestly resistant to the mannose-specific cyanovirin, which is derived from a blue-green alga, but fully susceptible to other HIV entry inhibitors as well as HIV reverse transcriptase inhibitors. These mutant virus strains were devoid of up to seven or eight of 22 glycosylation sites in the viral envelope glycoprotein gp120 because of mutations at the Asn or Thr/Ser sites of the N-glycosylation motifs. In one of the strains, a novel glycosylation site was created near a deleted glycosylation site. The affected glycosylation sites were predominantly clustered in regions of gp120 that are not involved in the direct interaction with either CD4, CCR5, CXCR4, or gp41. The mutant viruses containing the deleted glycosylation sites were markedly more infectious in CEM T-cell cultures than wild-type virus.

Anti-HIV Agents↗