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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↗
PubMed1995

The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design.

BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

Benzodiazepines↗
PubMed1997

Reverse transcriptase motifs in the catalytic subunit of telomerase.

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

Amino Acid Sequence↗
PubMed1993

Characterization of HIV-1 reverse transcriptase with antibodies indicates conformational differences between the RNAse H domains of p 66 and p 15.

Antibody binding to the p 66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511-527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p 66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p 66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p 66 subunit and the RNase H p 15 polypeptide.

Amino Acid Sequence↗
PubMedDate not supplied

[Study of reverse transcriptase selectivity. Substrate properties of new 2',3'-unsaturated acyclic nucleotide analogs].

A new series of nucleotide analogs, (Z)-pyrophosphoryl (phosphonyloxymethyl) but-2-enyl derivatives of pyrimidines and purines, was synthesized. Their substrate and inhibitory properties toward some DNA polymerases and reverse transcriptases were evaluated. They were shown to be selective inhibitors of HIV reverse transcriptase. The structure-substrate properties relationships for nucleotide analogs were discussed.

Base Sequence↗
PubMed2000

Quantitation of human immunodeficiency virus type 1 group O load in plasma by measuring reverse transcriptase activity.

We have evaluated the use of an ultrasensitive reverse transcriptase (RT) activity assay to monitor plasma viremia in two human immunodeficiency virus type 1 (HIV-1) group O-infected patients treated with stavudine, lamivudine, and indinavir. After a initial decline in RT levels observed at 4 weeks of therapy, RT-based plasma viremia returned to baseline values at 28 or 44 weeks of treatment. The rebound in levels of RT activity was associated with the detection of phenotypic resistance to lamivudine and with the Met184Val mutation. Analysis of RT activity in plasma provides a sequence-independent means of monitoring virus loads in HIV-1 group O-infected patients.

Adult↗
PubMed1992

Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation.

Steady-state and pre-steady-state kinetic constants were determined for reverse transcriptase catalyzed incorporation of nucleotides and nucleotide analogues into defined-sequence DNA primed-RNA templates. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was almost as efficient a substrate (kcat/Km) as dTTP for the enzyme. In contrast, the four 2',3'-dideoxynucleoside 5'-triphosphates and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (d4TTP) were 6-30-fold less efficient substrates of the enzyme. The kcat values for all nucleotide analogues were similar, consistent with a kinetic model in which the steady-state rate-limiting step was dissociation of the template-primer from the enzyme [Reardon, J. E., & Miller, W. H. (1990) J. Biol. Chem. 265, 20302-20307]. The pre-steady-state kinetics of single-nucleotide incorporation were consistent with the kinetic model: [formula: see text] where E, TP, and dNTP represent reverse transcriptase, a defined-sequence DNA primed-RNA template, and 2'-deoxynucleoside 5'-triphosphate (or analogue), respectively. The dissociation constant (Kd1) for template-primer binding was 10 nM, and the estimated rate constants for association and dissociation of the enzyme.template-primer complex were 4 x 10(6) M-1 s-1 and 0.04 s-1, respectively. The dissociation constants (Kd2) for dTTP, AZTTP, and 3'-deoxythymidine 5'-triphosphate (ddTTP) were 9, 11, and 4.6 microM, respectively. Thus, the differences in steady-state Km values were not due to differences in binding of the nucleotide analogues to the enzyme. In contrast, the rate-limiting step during single-nucleotide incorporation (kp) was sensitive to the structure of the nucleotide substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗
PubMed1998

Bioluminometric method for real-time detection of reverse transcriptase activity.

A simple and sensitive technique for detection of reverse transcriptase (RT) activity in real time has been developed. The technique is based on continuous detection of the inorganic pyrophosphate formed in the RT-catalyzed reaction by a luminometric method. The technique has been used for continuous monitoring of RT-catalyzed DNA synthesis on both homo- and heteropolymeric templates. The assay is sensitive and yields linear responses between 1.5-960 mU of avian myeloblastosis virus RT (AMV-RT). The assay was used for detection of the inhibitory effect of dideoxythymidine (ddTTP) on the AMV-RT activity and also for real-time detection of single-base incorporation events catalyzed by AMV-RT. The possibility of using the new technique for other applications is discussed.

Avian Myeloblastosis Virus↗
PubMed2001

HIV-1 reverse transcriptase interaction with model RNA-DNA duplexes.

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer-templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66(E478Q)/p51) (analytes) were estimated by surface plasmon resonance at 25 degrees C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA-DNA hybrids with 5'-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5'-end of RNA or DNA strand does not change the equilibrium constant (K(D)) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66(E478Q)/p51 enzyme, orientation effects are notable even altering the K(D) value. Binding of the p66(E478Q)/p51 to any RNA-DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

Algorithms↗
PubMed1982

5-Mercaptopolyuridylic acid (MPU), a potent inhibitor of the reverse transcriptase from avian myeloblastosis virus.

The effects of partially thiolated polyuridylic acid (MPU), partially thiolated polycytidylic acid (MPC), and their unmodified counterparts, poly(U) and poly(C), respectively, on the reverse transcriptase of avian myeloblastosis is virus (AMV) have been determined, using different template-primers. Poly(C) stimulated and MPC inhibited the polymerization reaction catalyzed by the AMV reverse transcriptase in the presence of the natural viral template (endogenous RNA or purified 70S RNA), or with poly(C) . oligo(dG)12-18 but not with poly(A) . oligo(dT)12-18 as the only template-primer present in the purified enzyme system. Both poly(U) and MPU inhibited the reaction in the presence of either of the synthetic or natural templates. Using the purified enzyme with the 70S RNA template, MPU was by far the most potent inhibitor, with I50 = 0.8 microM based on the epsilon (P) value, or I50 less than 3 x 10(-9) M based on the actual, macromolecular weight of the polynucleotide.

Avian Leukosis Virus↗
PubMed1991

Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate.

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.

Antiviral Agents↗
PubMed2004

Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase.

Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein's back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.

Amino Acid Sequence↗
PubMed2003

Mitochondrial dysfunction and nucleoside reverse transcriptase inhibitor therapy: experimental clarifications and persistent clinical questions.

Nucleoside reverse transcriptase inhibitors (NRTIs) in combination with other antiretrovirals (HAART) are critical in current AIDS therapy, but mitochondrial side effects have come to light with the increased use of these compounds. Clinical experience, pharmacological, cell and molecular biological evidence links altered mitochondrial (mt-) DNA replication to the toxicity of NRTIs in many tissues, and conversely, mtDNA replication defects and mtDNA depletion in specific target tissues are observed. The shared features of mtDNA depletion and energy depletion became key observations and related the clinical and in vivo experimental findings to inhibition of mtDNA replication by NRTI triphosphates in vitro. Subsequent to those findings, other observations suggested that mitochondrial energy deprivation is concomitant with or the result of mitochondrial oxidative stress in AIDS (from HIV, for example) or from NRTI therapy itself. With increased use of NRTIs, mtDNA mutations may become increasingly important pathophysiologically. One important future goal is to prevent or attenuate the side effects so that improved efficacy is achieved.

Anti-HIV Agents↗
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↗
PubMed2002

Assembly, purification and crystallization of an active HIV-1 reverse transcriptase initiation complex.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates DNA synthesis from the 3' end of human tRNA(Lys3). We have used cis-acting hammerhead ribozymes to produce homogeneous-length transcribed tRNA(Lys3) and have developed conditions for purifying highly structured RNAs on a modified tube-gel apparatus. Titration experiments show that this RNA can assemble into an initiation complex that contains equimolar amounts of HIV-1 RT, transcribed tRNA(Lys3), and chemically synthesized template RNA. We have purified this complex using gel-filtration chromatography and have found that it is homogeneous with respect to molecular weight, demonstrating that the initiation complex forms a single discrete species at micromolar concentrations. When this initiation complex is supplied with deoxynucleotides, essentially all of the tRNA is used as a primer by HIV-1 RT and is fully extended to the 5' end of the template. Thus, in vitro transcribed tRNA can be used efficiently as a primer by HIV-1 RT. We have also obtained crystals of the HIV-1 initiation complex that require the precisely defined ends of this in vitro transcribed tRNA(Lys3) to grow.

Base Sequence↗
PubMed1994

Mutational analysis of the fingers and palm subdomains of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase.

We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 and between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting RNase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the RNase H functions.

Amino Acids↗
PubMed1998

Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration.

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Crystallization↗
PubMed2001

Human monocytes possess a serine protease activity capable of degrading HIV-1 reverse transcriptase in vitro.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) plays a central role in the virus replication cycle. We found that HIV-1 RT was rapidly degraded when incubated with cell extracts obtained from human peripheral blood cells. The proteolytic activity responsible for the in vitro degradation of RT was present in monocytes and their precursors. Interestingly, this activity was downregulated upon cell activation or differentiation along the macrophage pathway. The proteolytic process appears specific for HIV-1 RT since other HIV-1 proteins were not degraded upon incubation in the same extracts. Although the degradation of RT was unaffected by specific proteasome inhibitors, it could be inhibited by PMSF and aprotinin, suggesting the involvement of a serine protease. Upon cell fractionation, this serine protease was found to be associated with the microsomal fraction and displayed an apparent molecular weight of approximately 2000 kDa, as determined by gel filtration. Our results suggest that a giant serine protease, different from tripeptidyl peptidase II, is involved in the in vitro degradation of HIV-1 RT. The possibility of an in vivo interaction between HIV-1 RT and a cell-type-specific serine protease is discussed.

Capsid↗