Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HIV Reverse Transcriptase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1995

Use of an oligoribonucleotide containing the polypurine tract sequence as a primer by HIV reverse transcriptase.

A primary site for initiation of plus strand DNA synthesis in human immunodeficiency virus (HIV) corresponds to a 19-nucleotide-long purine rich sequence located just upstream of the U3 region, designated the polypurine tract (PPT). The HIV reverse transcriptase (RT) uses its RNase H activity to cut the genomic RNA after minus strand DNA synthesis. A plus strand PPT primer is formed, extended, and then removed. In vitro, the HIV-RT recognizes this primer specifically, using it much more efficiently than other RNA primers. However, the PPT still primes significantly less efficiently than DNA primers. The 19-nucleotide PPT primer is partially resistant to degradation when compared with other oligoribonucleotides. Prior to initiation of DNA synthesis, several nucleotides are removed by the RT from the 3' ends of some of the PPT primers. Cleavage is enhanced in the absence of dNTPs. We suggest that DNA synthesis suppresses primer degradation, so that primer extension and cleavage occur in proper sequence. As a result of 3' end degradation, PPT elongation products contain 5'-RNA segments from 16 to 19 nucleotides in length. These shorter segments are also generated from a longer transcript containing the PPT sequence, indicating that they are not created as a result of binding of the RT to the 5' end of the PPT oligoribonucleotide. Full-length and shorter versions of the PPT primers are cleaved from the extended DNA by RT. These experiments show that HIV-RT has a specificity to generate a primer in the region of the PPT but that the ends of the primer are not well defined.

Base Sequence↗
PubMed1997

Anti-human immunodeficiency virus (anti-HIV) natural products with special emphasis on HIV reverse transcriptase inhibitors.

This review article aims at summarizing research findings concerning natural products which are endowed with the ability to inhibit human immunodeficiency virus (HIV). An emphasis is placed on HIV reverse transcriptase inhibitors because the bulk of the literature is focused on these compounds. It was found that a spectacular diversity of chemical structures encompassing proteins, terpenoids, coumarins, xanthones, alkaloids, flavonoids, polyphenols, and polysaccharides, which are elaborated by plant species as phylogenetically remote as the algae, gymnosperms and angiosperms, were capable of rendering the retroviral enzyme less active. The literature pertaining to natural products with HIV protease and integrase inhibitory activities is less voluminous.

Anti-HIV Agents↗
PubMed1999

The structural basis for the increased immunogenicity of two HIV-reverse transcriptase peptide variant/class I major histocompatibility complexes.

Designing altered peptide ligands to generate specific immunological reactivity when bound to class I major histocompatibility complexes is important for both therapeutic and prophylactic reasons. We have previously shown that two altered peptides, derived from human immunodeficiency virus (HIV)-reverse transcriptase (RT) residues 309-317, are more immunogenic in vitro than the wild-type peptide. One peptide variant, I1Y, was able to stimulate RT-specific cytotoxic T cells from the blood of three HIV-infected individuals better than the wild-type RT peptide. Both I1Y and I1F peptide variants increase the cell surface half-life of the peptide-class I complex approximately 3-fold over that of the RT peptide but have different immunological activities. These peptides are candidates for the design of vaccines for HIV due to their increased immunogenicity. To understand the basis for the increased cell surface stability compared with wild-type peptide and to understand the differences in T cell recognition between I1Y and I1F, we determined the x-ray crystal structures of the two class I MHC-peptide complexes. These structures indicate that the increased cell surface half-life is due to pi-pi stacking interactions between Trp-167 of HLA-A2.1 and the aromatic P1 residues of I1F and I1Y. Comparison of the structures and modeling potential T cell receptor (TCR) interactions suggests that T cell interactions and immunogenicity are different between I1Y and I1F for two reasons. First, subtle changes in the steric and polar properties of the I1Y peptide affect TCR engagement. Second, water-mediated hydrogen bond interactions between the P1-Tyr and the P4-Glu peptide residues increase peptide side chain rigidity of residues critical for TCR engagement.

Circular Dichroism↗
PubMed1990

Mechanism of HIV reverse transcriptase: enzyme-primer interaction as revealed through studies of a dNTP analogue, 3'-azido-dTTP.

Primer and dNTP recognition by purified HIV reverse transcriptase have been investigated. Earlier kinetic studies suggested that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence [Majumdar et al. (1988) J. Biol. Chem. 263, 15657-15665], and through use of a particularly high affinity template-primer analogue [r(I)n.Sd(C)28], rate values for formation of the first complex were calculated [Majumdar et al. (1989) Biochemistry 28, 1340-1346]. We now report rate values for first complex formation in the usual model replication system with poly[r(A)].oligo [d(T)] as template-primer. We find that 3'-azido-dTTP (AZTTP) is a linear competitive inhibitor of DNA synthesis against the substrate dNTP (dTTP) in the poly[r(A)].oligo[d(T)] replication system. This suggests that 3'-azido-dTTP and dTTP combine with the same form of the enzyme in the reaction scheme, i.e., the enzyme-primer complex. This is not trivial, since a second analogue, 3'-amino-dTTP, also is an inhibitor against dTTP, but the mechanism in this case is linear noncompetitive. Because the inhibition by 3'-azido-dTTP is linear competitive, the KD for physical binding to the enzyme is assumed to be the same as the Ki for inhibition (20 nM). Substrate kinetic studies of DNA synthesis using 3'-azido-dTTP as substrate revealed that the Michaelis constant is 3 microM. Therefore, the Km for this substrate analogue is 100-fold higher than the KD for binding of the analogue to the enzyme-primer complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗
PubMed1994

Steady-state kinetic studies with the polysulfonate U-9843, an HIV reverse transcriptase inhibitor.

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template: primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.

Binding Sites↗
PubMed1999

The connection domain is implicated in metalloporphyrin binding and inhibition of HIV reverse transcriptase.

We have shown that heme and zinc protoporphyrin inhibit both human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs) and, in combination with other nucleoside and non-nucleoside inhibitors, exert an additive effect on HIV-1 RT inhibition. Screening of a phage peptide library against heme resulted in the isolation of a peptide with sequence similarity to sequence 398-407 from the connection subdomain of both HIV-1 and HIV-2 RTs, suggesting that this highly conserved region of HIV RTs corresponds to the binding site for metalloporphyrins and a new site for inhibition of enzyme activity. Inclusion of a synthetic peptide corresponding to the exact sequence 398-407 of HIV-1 RT in RT inhibition assays had a protective effect on metalloporphyrin inhibition, as it was able to reverse the inhibitory effect of both metalloporphyrins on HIV-1 RT activity. Furthermore, intrinsic fluorescence assays indicated that these metalloporphyrins bind to synthetic peptide 398-407 as well as to intact dimeric HIV-1 RT. The identification of this novel inhibition site will help to expand our understanding of the mode of action of metalloporphyrins in RT inhibition and will assist in the design and development of more potent metalloporphyrin RT inhibitors for the management of HIV infection.

Amino Acid Sequence↗
PubMed1994

Site-directed mutagenesis of HIV reverse transcriptase to probe enzyme processivity and drug binding.

Site-directed mutagenesis has demonstrated that changes within the human immunodeficiency virus reverse transcriptase coding sequence alone can account for viral resistance to inhibitors. Inhibitor sensitivity of mutant enzymes in vitro correlates with the sensitivity of the virus to non-nucleoside inhibitors observed in vivo, but this is not the case with nucleoside analogs. Recent structural, kinetic, and site-directed mutagenesis studies demonstrate the importance of enzyme-nucleic acid contacts in determining enzyme sensitivity to inhibitors in vitro, as well as how accurately the reverse transcriptase synthesizes DNA.

Antiviral Agents↗
PubMed2000

The sequential mechanism of HIV reverse transcriptase RNase H.

Synthesis of the minus strand of viral DNA by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase is accompanied by RNase H degradation of the viral RNA genome. RNA fragments remain after synthesis and are degraded by the polymerase-independent mode of RNase H cleavage. Recently, we showed that this mode of cleavage occurs by a specific ordered mechanism in which primary cuts are first, secondary and 5-nucleotide cuts are next, and second primary cuts occur last (Wisniewski, M., Balakrishnan, M., Palaniappan, C., Fay, P., J., and Bambara, R., A. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 11978-11983). Ultimately the RNAs are cleaved into small fragments that can dissociate from the DNA template. Because the cleavage mechanism is an ordered series of events, we determined in this study whether any earlier cut is required for a later cut. By precisely inhibiting cleavage at each site, we examined the dependence of later cuts on cleavage at that site. We found that each cut is independent of the other cuts, demonstrating that the order of this stepwise mechanism is based on the rates of each cut. A mechanism for unlinked ordered cleavage consistent with these results is presented.

Base Sequence↗
PubMed1998

Enhanced impairment of chain elongation by inhibitors of HIV reverse transcriptase in cell-free reactions yielding longer DNA products.

We have studied the relationship between the length of HIV-1 reverse transcriptase (RT)-mediated nucleotide polymerization and inhibitors of these reactions in cell-free RT assays performed in the presence of either of two dideoxynucleoside triphosphates (ddNTPs), i.e. AZTTP or 3TCTP, or nevirapine, a non-nucleoside RT inhibitor. These reactions employed a heterologous RNA template and three DNA oligonucleotide primers, i.e. pAR, dPR and PA, that yielded distinct full-length products of 65, 192 and 376 nt, respectively, in the absence of inhibitor. We now show that the extent of inhibition of RT activity was greatest with use of the PA primer, which normally yielded the longest reaction product, and that lesser degrees of inhibition were noted in the reactions that generated shorter products. For example, at a concentration of 5 microM AZTTP, the extent of inhibition was 75% with the PA primer but only 40% and <10% when reactions were primed by the dPR and pAR primers, respectively. Similar results were obtained when either a mutated form of HIV RT (i.e. M184V), associated with resistance to 3TC, was tested in the presence of 3TCTP or when RT derived from Moloney murine leukemia virus (M-MuLV) was tested in the presence of AZTTP.

Cell-Free System↗
PubMedDate not supplied

Anti-AIDS agents, 1. Isolation and characterization of four new tetragalloylquinic acids as a new class of HIV reverse transcriptase inhibitors from tannic acid.

Four new tetragalloylquinic acids, 3,5-di-O-galloyl-4-O-digalloylquinic acid, 3,4-di-O-galloyl-5-O-digalloylquinic acid, 3-O-digalloyl-4,5-di-O-galloylquinic acid, and 1,3,4,5-tetra-O-galloylquinic acid, were isolated and characterized from a commercial tannic acid as a new class of human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitor. Compounds 2, 3, and 4 inhibit HIV RT activity 90, 89, and 84% at 100 microM and 73, 70, and 63% at 30 microM, respectively. Compounds 2-5 also inhibit the HIV growth in cells in the range of 61-70% with low cytotoxicity at 25 microM. The HIV cell growth inhibitory effects of these compounds at 25 microM and 6.25 microM (44-57%) are comparable to their effects against the HIV RT at 30 microM and 10 microM, respectively. The inhibitory effect of 3 against DNA polymerases indicates that the selective antiviral action of 3 is determined by more than its action with HIV RT.

Antiviral Agents↗
PubMed1993

Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase.

An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.

Biotin↗
PubMed1993

Three-dimensional structural resemblance between the ribonuclease H and connection domains of HIV reverse transcriptase and the ATPase fold revealed using graph theoretical techniques.

Using 3D searching techniques based on algorithms derived from graph theory, we have established two previously unreported structural similarities involving the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT). First, we report that there is a strong similarity between the 3D folds of the RNase H domain of RT and the 'ATPase folds' of hexokinase, the 70 kDa heat-shock cognate protein and actin. Like RNase H, these enzymes are involved in nucleotide binding and metal ion-catalysed cleavage of a phosphodiester bond. Similarities of the folding motif and the position of the metal-binding site in these enzymes suggest possible functional analogies and evolutionary relationships with RNase H. Second, we find there is a strong resemblance between the folds of the RNase H domain and of the p66 and p51 'connection' domains of RT. It is possible that this striking similarity within the RT structure indicates a possible ancestral gene doubling event. The similarity may also indicate that the connection domains possess functional roles in addition to those previously suggested, and they may therefore represent a further target for the design of therapeutic agents.

Adenosine Triphosphatases↗
PubMed1993

Comparison of interactions of 5'-derivatives of deoxyoctathymidylate with human DNA polymerize alpha and HIV reverse transcriptase.

Km and Vmax values for d(pT8) and its derivatives containing various 5'-end groups were estimated in the reaction of DNA polymerization alpha catalyzed by DNA polymerase alpha and HIV-RT. The effect of 5'-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT8 is 200-fold higher than that of d(pT8). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase alpha the influence of modifiers on primer affinity is much weaker. The effect of 5'-end residues on the Vmax values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.

DNA↗
PubMed1995

Photoaffinity labeling of HIV reverse transcriptase: utilization of 2',3'-dideoxyuridylate analog bearing aryl(trifluoromethyl)diazirine moiety as a chain terminator.

In order to develop a photoaffinity labeling reagent for HIV-1 reverse transcriptase, we utilized 2',3'-dideoxy-E-5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl] styryl]UTP (TDS-ddUTP) for formation of a photolabile primer. This compound was incorporated into the 3'-terminus of the primer by reverse transcriptase activity, and thus the generated photo-reactive primer was able to bind to the enzyme molecule by photo-irradiation.

Affinity Labels↗
PubMedDate not supplied

HIV-reverse transcriptase and human DNA polymerase alpha share amino acid sequence homologies to bacterial penicillin-binding proteins.

Penicillin-binding proteins are the specific targets for the beta-lactam antibiotics. Recently it was observed that beta-lactam antibiotics also have targets in proliferating eukaryotic cells (1), one of which most likely is the replicative DNA polymerase alpha. Here we show that HIV-reverse transcriptase and human DNA polymerase alpha share amino acid sequence homologies to five bacterial penicillin-binding proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Pharmacodynamic studies (PD) of didanosine (ddI) alone and in combination with azidothymidine (AZT) in human T-cells; a stochastic biochemical approach to antiretroviral nucleoside drug combination in inhibiting HIV-reverse transcriptase (RT).

Didanosine (ddI) is used in the treatment of HIV-1 infection alone and in combination with azidothymidine (AZT). When combined with AZT, patients exhibit improved patterns of surrogate markers after sequential combination regimens of ddI and AZT compared to either drug monotherapy. We have investigated the biochemical mechanism(s) of this synergistic drug combination in human PBMC cells and in human T-cell lines sensitive and resistant to AZT due to lack of thymidine kinase (TK). DdI is preferentially activated to its triphosphate anabolite, ddATP, at 3:1 ratio in human T-lymphocytes compared to monocytes from the same individual. There are no apparent differences in the intracellular concentrations of ddATP in Jurkat/0 and Jurkat/AZT-10, an AZT resistant human T-cell line, when ddI is administered alone or in combination with AZT, hence there appears to be a case of collateral sensitivity. Intracellular increases of AZTTP concentrations in patient's PBMC cells have been determined clinically after AZT alone and in a combination regimen with ddI. A stochastic biochemical model has been developed that estimates the velocity of HIV-RT under uninhibited and inhibited conditions by the active anabolites, AZTTP and ddATP. This model provides a rational explanation for the greater inhibition of HIV-RT in the presence of both inhibitors, AZTTP and ddATP, as compared to the presence of either anabolite triphosphate alone. Expanding this model to describe the inhibition of HIV-RT in the presence of three competitive inhibitors, AZTTP, ddATP and 3TCTP demonstrated that the presence of these HIV-RT inhibitors resulted in an even greater inhibition of this viral enzyme necessary for HIV integration and replication. Hence, a more effective inhibition of HIV-RT enzyme is achieved by the combination of the three drugs, AZT, ddl and 3TC. In an effort to verify this model with experimental data the kinetics of HIV-RT were studied in the absence and after inhibition by AZTTP or ddATP alone, both AZTTP + ddATP or AZTTP + ddATP + 3TCTP. Treatment of HIV-RT with high concentrations of these triphosphate inhibitors, as high as 3Kis, inhibited this enzyme to greater than 90% of untreated control. However, a small percentage of residual HIV-RT, 6%, was uninhibited even after exposure to 3Ki concentrations of each inhibitor. These studies strongly suggested that: 1) AZT plus ddI or AZT plus ddI plus 3TC are synergistic at the active anabolite level against HIV-RT; 2) the combination of the three nucleoside analog drugs (AZT, ddI 3TC) is needed for more effective inhibition of HIV-RT; 3) that the combination of the triphosphates at concentrations much greater than those pharnacologically achieved in T-Cells or PBMC under treatment conditions did not inhibit completely HIV-RT. Hence, the three nucleoside HIV-RT inhibitors must be combined with other classes of antiviral drugs or T-cell specific inhibitor drugs.

Anti-HIV Agents↗