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PubMed1991

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase.

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.

Amino Acid Sequence↗
PubMed2000

DNA-encoding enzymatically active HIV-1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV-1 challenge.

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.

AIDS Vaccines↗
PubMed1995

Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors.

The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 A resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded beta-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.

Antiviral Agents↗
PubMed1998

Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism.

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3' end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3'-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (Ki approximately 5 microM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3' nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

DNA, Viral↗
PubMed1995

Comprehensive chemical modification interference and nucleotide substitution analysis of an RNA pseudoknot inhibitor to HIV-1 reverse transcriptase.

We had previously used in vitro RNA selection techniques to describe a consensus RNA pseudoknot that binds and inhibits HIV-1 reverse transcriptase (HIV-RT). In this work we constructed variants of this consensus pseudoknot in order to evaluate the contributions of individual nucleotide identities and secondary structure to affinity for HIV-RT. We have also used chemical modification of ligand RNAs to corroborate the predicted structure of the pseudoknot, to discover which modifiable groups are protected from chemical attack when bound to HIV-RT, and to find which modifications interfere with binding to HIV-RT. A novel interference study is presented which involves selection of ligands from a pool created by mixed reagent oligonucleotide synthesis in order to rapidly determine allowed substitutions of 2'-OCH3 groups for the usual 2'-OH group in such RNA ligands.

Antiviral Agents↗
PubMed1992

HIV-1 and HIV-2 reverse transcriptases: a comparative study of sensitivity to inhibition by selected natural products.

One hundred and fifty six pure natural products, which had previously been tested against HIV-1 reverse transcriptase, were evaluated for HIV-2 reverse transcriptase inhibitory activity. Compounds that lacked effect in the HIV-1 reverse transcriptase system were found also to be inactive against HIV-2 reverse transcriptase. However, compounds belonging to the benzophenanthridine and protoberberine classes of alkaloids, certain flavonoids, the iridoid, fulvoplumierin, and the ansamycin antibiotic, daunomycin, exhibited similar potencies in both enzyme systems. In contrast, HIV-2 reverse transcriptase was observed to be four-fold more sensitive toward the inhibitory effects of the ipecac alkaloids, O-methylpsychotrine sulfate heptahydrate and psychotrine dihydrogen oxalate. Such differences in susceptibilities to inhibitors may indicate subtle dissimilarities in enzyme structure and function.

Alkaloids↗
PubMed1995

Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase.

Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). BHAP-resistant HIV-1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline-to-leucine substitution at amino acid 236 (P236L) of HIV-1 RT. To understand the basis for the in vitro sensitization-resistance phenomenon, single base pair mutations at amino acid P236 in HIV-1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV-1 RT mutants. Active HIV-1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV-2 RT, were also cloned, expressed, and purified. Three BHAPs (U-88204E, U-87201E, and U-90125S) and the pyridinone L-697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L-697,661 and resistance to the BHAPs. The HIV-1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV-2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV-1 RT mutants demonstrated a direct correlation between sensitization to L-697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L-697,661.

Amino Acid Sequence↗
PubMed2006

HIV-1 reverse transcriptase gene 103K/N and 184M/V combinations in tandem: detection and quantification of HIV-1 populations in the CD45RO+ T-cell compartment by a double-ARMS real-time PCR assay.

BACKGROUND: The proviral HIV-1 reverse transcriptase gene for the 103K/N and 184M/V combinations were studied in tandem. The CD45RO T (memory) cell compartment was investigated. METHODS: A new double-ARMS (amplification refractory mutation system) real-time polymerase chain reaction assay was developed to detect and quantify 4 populations (103K-184M, 103K-184V, 103N-184M, and 103N-184V) in the CD45RO T-cell compartment. Twenty-one patients, 18 lamivudine and efavirenz/nevirapine experienced, were enrolled in a cross-sectional study. RESULTS: None of the mutation combinations were detected in patients on highly active antiretroviral therapy (HAART) (naive at start) with viremia suppression below detection limits. Conversely, all patients exposed to mono- or dual therapy (prior to HAART) carried at least 1 mutation combination regardless of viral load. In 9 patients, 17 mutations were detected in a mosaic of combinations. This study provides definite evidence of the existence of 103N and 184V mutation quasi-populations in tandem, and separately in combination with the wild-type codons, 184M and 103K, in the CD45RO T-cell compartment. CONCLUSIONS: The initiation and continuation of potent antiretroviral therapy effectively hinders the appearance of 103N and 184V mutations alone or in tandem in memory cells. When switching therapies because of failure, caution should be exercised with drugs associated with single-mutation threshold; they can appear in tandem with contemporary resistant virus populations, leading to multidrug resistance.

Adult↗
PubMed2006

Identification of alternative amino acid substitutions in drug-resistant variants of the HIV-1 reverse transcriptase.

OBJECTIVE/DESIGN: To identify new drug-resistance-associated mutations in the HIV-1 reverse transcriptase (RT) protein, we screened the RT sequence database of our hospital for alternative amino acid substitutions at known RT drug-resistance positions. METHOD: The genotypic database used for this analysis contained 1322 RT sequences from 1015 patients. We analysed this RT database with a focus on alternative mutations at RT positions known to be involved in drug resistance. The patterns of drug resistance associated with these alternative mutations were investigated in a separate database containing genotype and drug-susceptibility results. RESULTS: We identified multiple alternative resistance-associated mutations at amino acid positions 44, 62, 67, 69, 70, 74, 75, 103, 181, 190, 210, and 219 in RT. Phenotypic analysis indicated that drug-resistance properties of the alternative Y181V and L74I mutants are similar, but not identical, to that of the well-known Y181C and L74V mutations. CONCLUSION: This initial survey indicates that many resistance-associated phenomena can be distilled from existing data. These findings endorse a more extensive analysis by computerized methods.

Anti-HIV Agents↗
PubMed1996

Impact of the stereochemistry of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide-deoxyadenosine adducts on resistance to digestion by phosphodiesterases I and II and translesion synthesis with HIV-1 reverse transcriptase.

Spatial orientations of bulky DNA adducts can influence the extent of resistance to digestion by exonucleases and translesion synthesis by HIV-1 reverse transcriptase (HIV-1 RT). In order to determine how different diastereomers of benzo[a]pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE)-adducted DNAs influence the activity of these enzymes, 11-mer and 33-mer oligodeoxyribonucleotides were synthesized bearing site-specific and stereospecific BPDE adducts at adenine N6 on position two of the human N-ras codon 61. Phosphodiesterase I, which hydrolyzes DNA in the 3'-->5' direction, exhibited greater resistance opposite the lesion with C10-R BPDE-adducted templates than the corresponding C10-S adducts. However, the opposite stereoselective resistance to digestion was observed with phosphodiesterase II, which hydrolyzes DNA in the 5'-->3' direction. These results are complemented by the in vitro replication pattern exhibited with HIV-1 RT. Primer extension reactions under conditions defining single encounters between polymerase and substrate revealed adduct-dependent termination one base 3' to each of the lesions. When experimental conditions were altered to permit multiple encounters, HIV-1 RT was able to replicate past the damaged site on four of the six adducted templates, exhibiting little pausing opposite the lesion. Analyses of the replication pattern past these lesions revealed two general categories of replication blockage, which, like the exonucleolytic digestion data, were also based on the C10-R and C10-S configuration of the stereoisomers. Thus, the chirality of BPDE-dA adducts modulates enzymatic functions. Furthermore, the (+)- and (-)-anti-trans-BPDE-dA modified templates exhibited the most facile bypass, while the (+)- and (-)-anti-cis-BPDE adducts were most blocking.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2003

Cloning, expression, purification, and crystallisation of HIV-2 reverse transcriptase.

A purification procedure is described for the isolation of recombinant HIV-2 reverse transcriptase expressed in Escherichia coli. The p68 subunit is expressed, in the absence of induction, and use of a heparin-Sepharose column produces substantially pure protein. Concentration of the homodimeric p68 reverse transcriptase pool, followed by incubation at room temperature for several days, results in full conversion by E. coli proteases to the heterodimer (p68/p55). This extended incubation simplifies the purification process and improves the yield of heterodimeric reverse transcriptase, which shows a truncation of the smaller subunit to 427 residues. The protein is then purified further by hydroxyapatite and gel-filtration chromatography to homogeneity. The HIV-2 RT is active and has been used to produce crystals that diffract to beyond 3.0 A.

Cloning, Molecular↗
PubMed1999

Inhibitors of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

The discovery and characterization of new inhibitors of HIV-1 reverse transcriptase (RT) is an important step toward understanding the mechanism of this multifunctional polymerase. We describe the identification of novel inhibitors of HIV-1 RT-catalyzed reactions utilizing a nucleic acid model system designed to mimic the essential features of DNA strand transfer reactions catalyzed by HIV-1 RT. This reaction requires the DNA polymerase and RNase H activities of RT, as well as the translocation of DNA from one template strand to another. In addition to the discovery of new inhibitors of DNA polymerase activity, two classes of inhibitors were identified that inhibit different steps of the DNA strand transfer reaction. One class of these, exemplified by actinomycin D, inhibits DNA strand transfer by interfering with the transfer of the DNA intermediate onto the acceptor template. The second class of strand transfer inhibitor, exemplified by the chlorophenylhydrazone of mesoxalic acid, was found to inhibit the ribonuclease H (RNase H) activity of HIV-1 RT under strand transfer conditions. This inhibitor is a potent and specific inhibitor of RNase H activity, which displays no inhibition of either DNA-dependent or RNA-dependent DNA polymerase activity. Together, these three inhibitors block different steps reverse transcription and will be valuable in studying the mechanism of multistep reactions such as DNA strand transfer. In addition, these new inhibitors of in vitro reverse transcription point to new strategies for the intervention of retroviral DNA replication and could be useful in the development of new HIV-1 therapeutic strategies.

Anti-HIV Agents↗
PubMed1994

The mycoplasma-related inhibitor of HIV-1 reverse transcriptase has a DNase activity and is present in the particle-free supernatants of contaminated cultures.

Drastic inhibition of the human immunodeficiency virus (HIV) reverse transcriptase (RT) by mycoplasma has been noted in many laboratories causing confusion in data interpretation. The mycoplasma-related inhibitor of HIV-1 RT was identified as a soluble protein in the particle-free supernatant of a contaminated culture. Gel filtration studies revealed the molecular mass of this protein to be about 70 kDa. This RT-inhibitor contained a DNase with strong activity on both linear and circular DNAs. Addition of this inhibitor after completion of reverse transcription still reduced the final outcome of the RT assay significantly, implying that the inhibitory mechanism occurred mainly by its DNase activity. Treatment of the culture with an antimycoplasma drug cured the mycoplasma contamination, removed the RT-inhibitor and abolished the DNase activity.

Cells, Cultured↗
PubMed2005

Relationship between enzyme activity and dimeric structure of recombinant HIV-1 reverse transcriptase.

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.

Binding Sites↗
PubMed1999

Mechanistic studies comparing the incorporation of (+) and (-) isomers of 3TCTP by HIV-1 reverse transcriptase.

Among the nucleoside inhibitors used clinically as anti-HIV drugs which target HIV-1 reverse transcriptase (RT), (-)-2', 3'-dideoxy-3'-thiacytidine [(-)SddC or 3TC] is the only analogue with the unnatural L(-) nucleoside configuration. 3TC has been shown to be more potent and less toxic than the D(+) isomer, (+)SddC, which has the natural nucleoside configuration. The mechanistic basis for the stereochemical selectivity and differential toxicity of the isomeric SddC compounds is not completely understood although a number of factors may clearly come into play including differences in uptake, metabolic activation, degradation, and transport. We used a pre-steady-state kinetic analysis to determine the maximum rate of incorporation, kpol, nucleotide-binding affinity, Kd, and efficiency of incorporation, kpol/Kd, for the (-) and (+) isomeric SddCTP compounds as well as the corresponding dideoxy and natural nucleoside triphosphates into a primer-template complex using HIV-1 reverse transcriptase. The affinity (Kd) of the dNTP was much tighter and the efficiency (kpol/Kd) of incorporation by enzyme into the primer-template complex was much higher for the DNA/RNA primer-template compared to DNA/DNA. The maximum rate of incorporation, kpol, followed the trend of dCTP > ddCTP > (+)SddCTP > (-)SddCTP while the Kd values determined for the DNA/RNA primer-template followed the order (-)SddCTP congruent with (+)SddCTP congruent with ddCTP > dCTP. The corresponding efficiency of incorporation followed the trend dCTP > ddCTP > (+)SddCTP > (-)SddCTP. These data suggest that perturbations on the ribose ring of cytidine analogues (C --> S) decrease the rate and efficiency of incorporation but enhance the binding affinity. These results are discussed in the context of a computer modeled structure of the ternary complexes of RT, DNA/RNA primer-template, and SddCTP analogues as well as implications for structure-activity relationships and further drug design. This information provides a mechanistic basis for understanding the inhibition of HIV-1 reverse transcriptase by 3TC.

Anti-HIV Agents↗
PubMed2006

Insertions and deletions in HIV-1 reverse transcriptase: consequences for drug resistance and viral fitness.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target of drugs fighting HIV infection. The introduction of potent antiretroviral therapies based on the use of RT inhibitors and/or protease inhibitors has been an important achievement towards the control of AIDS. However, the development of drug resistance constitutes a major hurdle towards long-term efficacy of those therapies. With the increasing complexity of the antiretroviral regimens, novel mutational patterns conferring high-level resistance to nucleoside and nonnucleoside RT inhibitors have been identified in viral isolates. Among them, insertions and deletions in the beta3-beta4 hairpin-loop-coding region of HIV-1 RT have been identified in heavily-treated patients. Insertions of one, two or several residues appear to have a significant impact on nucleoside analogue resistance. The frequently found combination of a dipeptide insertion and thymidine analogue resistance mutations (i.e. T215Y) in the viral RT confers an ATP-dependent phosphorolytic activity that facilitates the removal of the inhibitor from primers terminated with zidovudine or stavudine. Furthermore, this mechanism appears to be relevant for resistance mediated by one amino acid-deletions appearing in combination with thymidine analogue resistance mutations. However, in other sequence contexts (i.e. in the presence of Q151M), the effects of the deletion are not fully understood. Drugs targeting the excision repair mechanism could be an important aid in the fight against multinucleoside-resistant HIV isolates bearing complex mutational patterns in their RT-coding region.

Amino Acid Sequence↗
PubMed2003

Hybrid canonical-correlation neural-network approach applied to nonnucleoside HIV-1 reverse transcriptase inhibitors (HEPT derivatives).

Beneficial antiviral HIV-1 chemotherapy is associated with adverse reactions. To optimize the desired actions and to lower the side effects of nonnucleoside HIV-1 reverse transcriptase (RT) inhibitors (NNRTIs), quantitative structure-activity relationships (QSARs) were studied by using a series of HEPT derivatives of NNRTIs. Hypothesis testing requires that certain assumptions are approximately satisfied in statistically based QSARs, however. A complementary approach is based on artificial neural network analysis. Model building can be made without the manifold assumptions of statistically based QSAR approaches but the problem is that the number of neural weights increase exponentially (danger of overfitting) under certain circumstances. A way to get more reliable results is to reduce the dimensionality of the two subsets (biological and chemical variables). A suitable method is the canonical correlation analysis. The two subsets of canonical variates are used as outputs (biologically derived variates) and inputs (chemically derived variates) of an optimized backpropagation neural network approach. The contribution summarizes the most recent results of this canonical-correlation backpropagation-neural network QSAR approach. It is shown that noncovalent interactions (lipophilic, steric, hydrogen-bonding, and inductive forces of the substituents) are responsible for the antiviral and cytotoxic actions. The outcome of this analysis produces an internally highly self-consistent result (model robustness). The predictive performance is tested. The butterfly-like conformation of the predicted compound is consistent with the butterfly-like model of other NNRTIs. Molecular simulation shows that the complexed drug interacts with the Tyr181 and Tyr188 residues of the RT. The uracil ring of the drug binds directly with Lys101, and the acyclic side chain (with an intact free hydroxyl function) binds with Lys103. The suggested noncovalent interaction forces are equivalent with that found by the QSAR analysis.

Anti-HIV Agents↗
PubMed2006

Molecular docking studies on 4-thiazolidinones as HIV-1 RT inhibitors.

Flexible docking simulations were performed on two series of 4-thiazolidinones as HIV-1 reverse transcriptase (HIV-1 RT) inhibitors. This was done by analyzing the interaction of these compounds with the allosteric site of the HIV-1 reverse transcriptase enzyme. The binding scores for these compounds were also congruent with their anti-HIV activity. A good correlation between the predicted binding free energies and the experimentally observed inhibitory activities (EC(50)) suggest that the identified binding conformations of these inhibitors are reliable. The results of docking studies provide an insight into the pharmacophoric structural requirements for the HIV-1 RT inhibitory activity of this class of molecules.

Allosteric Site↗