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PubMed2006

Design, synthesis, and biological evaluations of novel quinolones as HIV-1 non-nucleoside reverse transcriptase inhibitors.

A novel series of quinolones was discovered as HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) using a structure-based approach. The lead quinolones exhibited single digit nanomolar potency in the HIV-1 replication assays. The preliminary SAR of these quinolones was also established via systematic structural modifications. These novel and potent quinolones could serve as advanced leads for further optimization.

Binding Sites↗

PubMed1997

Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases.

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases alpha and beta. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

DNA Polymerase II↗

PubMed1983

Rose Bengal mediated inhibition of DNA polymerases: mechanism of inhibition of avian myeloblastosis virus reverse transcriptase under photooxidative conditions.

DNA polymerases from prokaryotic, eukaryotic, and oncornaviral sources are irreversibly inactivated upon exposure to Rose Bengal in the presence of light (photooxidation conditions). Inactivation of these enzymes under dark (nonoxidative) conditions is totally reversible [Srivastava, S. K., & Modak, M. J. (1982) Biochemistry 21, 4633-4639]. The primary effect of photooxidation on the enzyme-Rose Bengal complex was found to be the loss of template-primer binding ability within 5 min of exposure to light. The presence of template-primer but not the substrate deoxynucleotides consistently provided partial protection against Rose Bengal mediated photooxidative inactivation. Preformed enzyme-template-primer complexes were not dissociated by Rose Bengal under these conditions. However, prolonged exposure (25-30 min) of this complex to light in the presence of Rose Bengal led to complete inactivation of catalytic activity without affecting the ability of enzyme to bind to template-primer. These results clearly indicate that oxidative inactivation of avian myeloblastosis virus reverse transcriptase in the presence of Rose Bengal involves a domain within the enzyme that contains the template-primer binding site as well as an additional site which is required for the expression of both the polymerization and nuclease activities of the reverse transcriptase.

Avian Leukosis Virus↗

PubMed2006

A rapid and sensitive real-time PCR assay for the K65R drug resistance mutation in SIV reverse transcriptase.

Macaques infected with simian immunodeficiency virus (SIV) provide a suitable model for assessing the efficacy of antiretroviral (ARV) drug interventions and drug resistance selection associated with treatment. Resistance to the HIV reverse transcriptase inhibitor tenofovir continues to be examined in different treatment strategies in the macaque model. Evaluations of treatment interventions and drug resistance are hampered by the limited sensitivity of conventional population sequencing and the substantial effort involved in testing various tissue compartments in which viruses may reside. Therefore, a sensitive assay that permits simple and rapid testing for drug-resistant viruses would benefit appraisals of ARV treatments using in vivo models. To have this capability, we developed a real-time PCR-based assay for the detection of the SIV K65R reverse transcriptase mutation, a key marker for reduced susceptibility to tenofovir. Evaluations of SIV sequences yielded an assay detection limit mean of 0.4% mutant virus (range = 0.1-2%) in a wild-type background. In testing longitudinal plasma specimens from four SIV-infected macaques that received an active daily regimen of 30 mg/kg of tenofovir subcutaneously, the assay was able to detect K65R-positive viruses in all animals within 1-7 weeks after treatment began. The emerging mutants were initially present at frequencies estimated between 0.4% and 3%, below the detection capability of population sequencing. We propose the SIV K65R real-time PCR assay provides improved sensitivity and simplicity in studying tenofovir resistance in macaque models.

Adenine↗

PubMed1975

Studies on reverse transcriptase of RNA tumor viruses. I. Localization of thermolabile DNA polymerase and RNase H activities on one polypeptide.

Purified reverse transcriptase from avian myeloblastosis virus or Rous sarcoma virus consists of two subunits of average mol wt of 100,000 and 60,000. The lower-molecular-weight subunit, alpha, has been isolated from avian myeloblastosis virus, Rous sarcoma virus and a temperature-sensitive mutant of Rous sarcoma virus, LA337. Subunit alpha manifests both the DNA polymerase and RNase H activities associated with purified reverse transcriptase of avian RNA tumor viruses. The thermal inactivation of these enzymatic activities of alpha subunit from the wild-type virus. The results show that both DNA polymerase and RNase H activities associated with the alpha subunit of LA337 are five to seven times more thermolabile then the corresponding alpha subunit from the wild-type virus. It is concluded that (i) both the polymerase and nuclease activities reside on the same polypeptide chain, and (ii) at least the lower-molecular-weight subunit alpha is coded for by the viral RNA.

Avian Leukosis Virus↗

PubMed1996

Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis.

Tyr115 is located in the vicinity of the polymerase catalytic site of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Site-directed mutagenesis was used to generate variant enzymes having Phe, Trp, Ala, Ser, Asp or Lys instead of Tyr115. The substitution of Tyr115 by Phe renders a fully active polymerase, displaying similar kinetic parameters, processivity and misinsertion fidelity of DNA synthesis as the wild-type enzyme. In contrast, the replacement of Tyr by Asp or Lys produced enzymes with a very low polymerase activity. The activity of the variant enzymes having Trp, Ala or Ser instead of Tyr115 was reduced significantly, particularly when poly(rA)484 was used as template. This effect was caused by a dramatic increase in the Km value for dTTP, and was detected using a DNA template mimicking a proviral HIV-1 gag sequence. Misinsertion fidelity assays revealed that mutants Y115W, Y115A and Y115S had a higher misinsertion efficiency than the wild-type reverse transcriptase. The low fidelity of these mutants appears to be related to nucleotide recognition rather than altered DNA-DNA template-primer interactions. The effects observed on the steady state kinetic constants, processivity and fidelity were mediated by the 66 kDa subunit, as demonstrated using chimeric heterodimers with the Y115A substitution in either p66 or p51.

DNA Replication↗

PubMed1996

Targeting HIV reverse transcriptase for anti-AIDS drug design: structural and biological considerations for chemotherapeutic strategies.

The reverse transcriptase of HIV is a key target for the antiviral treatment of AIDS. Numerous potent inhibitors of RT have been described including all of the drugs that have been currently licensed for the treatment of AIDS, but their efficacy has been limited by the emergence of drug-resistant HIV variants. Extensive biochemical, genetic, and clinical data about HIV RT enzymatic mechanisms, inhibition, and drug resistance have been reported. This information, taken together with structural data from crystallographic studies of HIV-1 RT, has set the stage for structure-based design of improved inhibitors of this essential viral enzyme. Comparisons of the different crystal structures of HIV-1 RT shows that the enzyme has great conformational flexibility, providing additional possibilities for drug targeting. Recent clinical and virological data suggest that HIV-1 RT enzymes that carry drug-resistance mutations can be substantially impaired and that combinations of RT inhibitors can produce significant clinical benefit in the treatment of AIDS. An immediate goal is to use the available information to design specific inhibitors or combination therapies that will select for relatively less fit HIV variants.

Binding Sites↗

PubMed1979

In vitro synthesis of infectious transforming DNA by the avian sarcoma virus reverse transcriptase.

Infectious DNA molecules, capable of transforming chicken embryo fibroblasts, can be synthesized by the Rous sarcoma virus-associated reverse transcriptase in vitro. The optimal enzymatic conditions employed for infectious DNA synthesis also facilitate maximum synthesis of genome length DNA. Analysis of the DNA product synthesized by detergent-disrupted Rous sarcoma virus under these conditions indicates that DNA complementary to viral RNA (minus-strand DNA) is genome length in size, whereas DNA complementary to genome length minus-strand DNA (plus-strand DNA) appears as subgenomic-length molecules ranging between 300 and 3,500 nucleotides in length. These features of the DNA product synthesized by the Rous sarcoma virus reverse transcriptase in vitro are similar to those identified in the cytoplasm of cells shortly after infection and lend credence to studies of the mechanism of reverse transcription in vitro and their significance to proviral DNA synthesis in vivo.

Avian Sarcoma Viruses↗

PubMed2002

Ectopic expression of a COOH-terminal fragment of the human telomerase reverse transcriptase leads to telomere dysfunction and reduction of growth and tumorigenicity in HeLa cells.

The COOH-terminus of telomerase reverse transcriptase (hTERT) has been shown to participatein the nuclear translocation of TERT. Here, we constructed plasmids expressing the COOH-terminal M(r) 27,000 polypeptide of hTERT (hTERTC27) withthe telomerase RNA-binding domains and the reverse transcriptase domains deleted. We showed that ectopic overexpression of this polypeptide caused a defect in telomere maintenance in hTERT-positive HeLa cells, which led to senescence-like growth arrest and apoptosis. The hTERTC27 appears to work by inducing telomere dysfunction, exemplified by significantly increased anaphase chromosome end-to-end fusion events in transfected cells. Significantly, it had no effect on the cellular telomerase enzymatic activity or telomere length. The in vivo effect was further demonstrated as HeLa cells stably expressing hTERTC27 have significantly lower growth rate and reduced tumorigenicity in nude mice xenografts. Results from this study revealed an important function for the COOH terminus of hTERT in maintaining the integrity of telomere structure and chromosome ends, as well as in cell senescence and apoptosis. Furthermore, hTERTC27 provides a new strategy for cancer therapy by inducing telomere dysfunction in cancer cells without affecting the telomerase enzymatic activity.

Animals↗

PubMed2001

Telomerase activity and expression of telomerase reverse transcriptase correlated with cell proliferation in meningiomas and malignant brain tumors in vivo.

Telomerase activity was examined in intracranial tumors and compared with gene expression of the two core components of telomerase--the reverse transcriptase subunit (hTERT) and the RNA subunit (hTR)--and the proliferative index. We investigated 32 tumors across three to five sampled regions (20 meningiomas, 1 acoustic schwannoma, 1 pituitary adenoma, 8 gliomas, and 2 medulloblastomas). Telomerase activity was demonstrated by the telomeric repeat amplification protocol (TRAP) assay in seven (22%) intracranial tumors (four malignant brain tumors, two atypical meningiomas and one ependymoma) but could not be detected in the 18 (100%) benign meningiomas. hTERT and hTR mRNA were detected using reverse-transcription polymerase chain reaction (RT-PCR). hTERT mRNA was present in 20 (63%) intracranial tumors. Whereas hTERT mRNA transcripts were consistently low or absent in meningiomas, malignant brain tumors exhibited elevated hTERT mRNAs. Multiple regions of glioblastomas showed differences in telomerase activity and in the presence of hTERT mRNA. RT-PCR analysis revealed, for the first time in intracranial tumors, the presence of hTERT mRNA spliced products, corresponding to full-length mRNA as well as spliced mRNAs with critical reverse transcriptase motifs deleted. Only tumors with marked telomerase activity showed all hTERT spliced messages simultaneously. The absence of a positive correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT spliced variants. We found a significant correlation between telomerase activity scores and Ki-67 proliferation index. A positive association is also seen between Ki-67 staining and the degree of hTERT mRNA expression. This shows that there seems to be a relationship between telomerase activity or the degree of hTERT expression and proliferation rate in intracranial tumors.

5' Untranslated Regions↗

PubMed1996

The nucleoside analog-resistant E89G mutant of human immunodeficiency virus type 1 reverse transcriptase displays a broader cross-resistance that extends to nonnucleoside inhibitors.

The alteration of a glutamic acid (E) to a glycine (G) amino acid residue at position 89 (E89G alteration) in the human immunodeficiency virus type 1 reverse transcriptase confers decreased susceptibility to several nucleoside analog inhibitors. Because the nonnucleoside inhibitor-binding pocket is adjacent to the deoxynucleoside triphosphate substrate-binding site, the impact of the E89G reverse transcriptase has decreased susceptibility to TIBO R82150, nevirapine, and to a lesser extent, delavirdine. Human immunodeficiency viruses bearing the same mutation displayed decreased susceptibility to inhibition by these compounds in a cell culture virus replication assay.

Anti-HIV Agents↗

PubMed2005

Suppression of virus load by highly active antiretroviral therapy in rhesus macaques infected with a recombinant simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type 1.

We have modeled highly active antiretroviral therapy (HAART) for AIDS in rhesus macaques infected with a chimera (RT-SHIV) of simian immunodeficiency virus containing reverse transcriptase from human immunodeficiency virus type-1 (HIV-1). Seven RT-SHIV-infected macaques were treated with a combination of efavirenz (200 mg orally once daily), lamivudine (8 mg/kg subcutaneously once daily), and tenofovir (30 mg/kg subcutaneously once daily). Plasma viral RNA levels in all animals were reduced by more than 1,000-fold after 4 weeks and, in six of the seven animals, were reduced to undetectable levels after 10 weeks. Virus loads increased slightly between 12 and 16 weeks of treatment, associated with problems with the administration of efavirenz. After a change in the method of efavirenz administration, virus loads declined again and remained undetectable in the majority of animals for the duration of therapy. Treatment was stopped for three animals after 36 weeks of therapy, and virus loads increased rapidly. Posttreatment RT-SHIV isolates had no mutations associated with resistance to any of the three drugs. Efavirenz treatment was stopped, but lamivudine and tenofovir treatment for two other macaques was continued. The virus load in one of these two animals rebounded; virus from this animal was initially free of drug-resistance mutations but acquired the K65R mutation in reverse transcriptase at 11 weeks after efavirenz treatment was withdrawn. These results mimic HAART of HIV-1-infected humans. The RT-SHIV/rhesus macaque model should be useful for studies of tissue reservoirs and sites of residual replication that are not possible or practical with humans.

Animals↗

PubMed2006

[Construction and selection of siRNA expression cassettes targeting human telomerase reverse transcriptase gene in vitro].

OBJECTIVE: To determine whether the human telomerase reverse transcriptase (hTERT) gene silencing could be effectively induced by PCR-derived siRNA expression cassettes (SEC) transfected by the fifth generation polyamidoamine dendrimer (G5 PAMAM-D) in Tca8113 cells. METHODS: Four SEC were rationally designed and constructed based on a two-step PCR reaction. The SEC were then transferred into Tca8113 cells using G5 PAMAM-D, and hTERT expression was investigated by real-time fluorescence-quantitative reverse transcriptase-PCR and western blot analysis. RESULTS: The RNA interference effects of the SEC targeted for varying hTERT mRNA positions showed a significant disparity. Among them, SEC-A revealed the most potent inhibitory effects (above 95% of reduction), followed by SEC-D and SEC-C, and SEC-B had no effect on hTERT expression (P > 0.05). That the endogenous hTERT gene silencing induced by G5 PAMAM dendrimer-mediated SEC-A was highly sequence-specific, and multiple transfection as well as properties of the vectors were routinely attributable to the specific suppression. CONCLUSIONS: Specific inhibition of endogenous hTERT expression by use of a PCR-based short hairpin siRNA technique and dendrimer transfer system may serve as a novel strategy for treatment of tongue cancers expressing hTERT in vitro.

Carcinoma, Squamous Cell↗

PubMed2006

Effects of nucleotides and nucleotide analogue inhibitors of HIV-1 reverse transcriptase in a ratchet model of polymerase translocation.

A single cycle of nucleotide incorporation by the reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) involves the initial binding of an incoming nucleotide, a conformational change that traps the substrate, the formation of a new phosphodiester bond, the release of pyrophosphate (PPi), and ultimately polymerase translocation, which clears the nucleotide binding site. This article reviews different mechanistic models for polymerase translocation with emphasis placed on HIV-1 RT. Structure-function analyses of stalled complexes of HIV-1 RT provide strong evidence to suggest that the enzyme can oscillate between pre- and post-translocational states. Nucleotide hydrolysis is not required for the movement of the polymerase in a stalled configuration; thermal energy is sufficient to allow random bidirectional sliding. The next complementary nucleotide, following the incorporated chain-terminator, acts like a pawl of a ratchet that traps the enzyme in the post-translocation state and prevents the reverse movement. Quantitative footprinting experiments have shown that the concentration of the templated nucleotide required to shift the translocational equilibrium forward depends crucially on the structure of the 3'end of the primer. Changes in the relative population of pre- and post-translocation complexes can influence rates of excision of incorporated NRTIs, which, in turn, affects drug susceptibility. The concept of a ratchet model of HIV-1 RT translocation and its implications for drug action and resistance, and the discovery and development of novel antiviral compounds is discussed.

Catalysis↗

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↗

PubMed1998

Vertical-scanning mutagenesis of a critical tryptophan in the minor groove binding track of HIV-1 reverse transcriptase. Molecular nature of polymerase-nucleic acid interactions.

While sequence-specific DNA-binding proteins interact predominantly in the DNA major groove, DNA polymerases bind DNA through interactions in the minor groove that are sequence nonspecific. Through functional analyses of alanine-substituted mutant enzymes that were guided by molecular dynamics modeling of the human immunodeficiency virus type 1-reverse transcriptase and DNA complex, we previously identified a structural element in reverse transcriptase, the minor groove binding track (MGBT). The MGBT is comprised of five residues (Ile94, Gln258, Gly262, Trp266, and Gln269) which interact 2-6 base pairs upstream from the polymerase active site in the DNA minor groove and are important in DNA binding, processivity, and frameshift fidelity. These residues do not contribute equally; functional analysis of alanine mutants suggests that Trp266 contributes the most to binding. To define the molecular interactions between Trp266 and the DNA minor groove, we have analyzed the properties of eight mutants, each with an alternate side chain at this position. A refined molecular dynamics model was used to calculate relative binding free energies based on apolar surface area buried upon complex formation. In general, there was a strong correlation between the relative calculated binding free energies for the alternate residue 266 side chains and the magnitude of the change in the properties which reflect template-primer interactions (template-primer dissociation rate constant, Ki,AZTTP, processivity, and frameshift fidelity). This correlation suggests that hydrophobic interactions make a major contribution to the stability of the polymerase-DNA complex. Additionally, tyrosine and arginine substitutions resulted in mutant enzymes with DNA binding properties better than predicted by buried surface area alone, suggesting that hydrogen bonding could also play a role in DNA binding at this position.

Antiviral Agents↗

PubMed1990

Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5'-triphosphate and 3'-azido-3'-deoxythymidine 5'-triphosphate.

3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was an efficient substrate for the human immunodeficiency virus 1 reverse transcriptase. It was incorporated into both homopolymer and defined sequence DNA-primed RNA templates and DNA-primed DNA templates. The substrate and inhibitor kinetics of both AZTTP and dTTP were dependent on the template-primer and reaction conditions used. dTMP was incorporated into poly(rA).oligo(dT) and into a defined sequence DNA-primed RNA template (when the other three 2'-deoxynucleoside 5'-triphosphates were present) as a conventional substrate, with steady-state Km values of 5-10 microM. The results suggest that the reverse transcriptase was capable of processive DNA polymerization on these DNA-primed RNA templates. In contrast, in the absence of the other three 2'-deoxynucleoside 5'-triphosphates, the time course for incorporation of dTMP into the same defined sequence DNA-primed RNA template was biphasic. A burst of product formation was observed followed by a slow steady-state rate with a Km value of 0.082 microM. AZTMP incorporation into poly(rA).oligo(dT) and into the defined sequence DNA-primed RNA template produced similar biphasic time courses and steady-state Km values. These results were consistent with rate-limiting dissociation of the polymerase.template-primer complex after "forced" termination of polymerization. AZTMP and dTMP were both incorporated into the homopolymer DNA-primed DNA template, poly(dA).oligo(dT), and a defined sequence DNA-primed DNA template as conventional substrates. Their Km values were similar (2-10 microM). The absence of biphasic time courses suggested that dissociation of the DNA-primed DNA templates from the enzyme, after forced termination, was not rate-limiting. This was consistent with a more distributive mode of DNA polymerization. With the defined sequence template-primers and poly(dA).oligo(dT), Ki values for both dTTP and AZTTP were comparable to their Km values. Thus, AZTTP appeared to be a simple competitive substrate-inhibitor with respect to dTTP. AZTTP inhibition of dTMP incorporation into poly(rA).oligo(dT) was linear competitive at low concentrations (0-100 nM) of AZTTP (Ki = 35 nM) but became hyperbolic (decreasing potency) at concentrations of AZTTP above this range. A mechanism for this nonlinear inhibition is discussed.

Antiviral Agents↗

PubMed1995

Isolation from human brain of six previously unreported cDNAs related to the reverse transcriptase of human endogenous retroviruses.

cDNAs prepared from total RNA extracted from plaques of multiple sclerosis were amplified by the polymerase chain reaction. The 11-bp degenerate primers used were derived from conserved sequences of reverse transcriptase. Amplified cDNAs were fractionated according to size by electrophoresis in polyacrylamide gels under denaturing conditions. cDNAs of the proper size were cloned, grouped according to the sequence of their insert by differential hybridization, and sequenced. Six cDNAs were isolated and found to belong to new members of two groups of human endogenous retroviruses: the group related to ERV9 and that related to HERVK10 and HUMMTV. These sequences were expressed in all human organs tested, including normal white matter of brain. The approach described in this article is a powerful tool with which to isolate new members of the reverse transcriptase gene family.

Amino Acid Sequence↗