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PubMed2000

High-level resistance to 3'-azido-3'-deoxythimidine due to a deletion in the reverse transcriptase gene of human immunodeficiency virus type 1.

A variant of human immunodeficiency virus type 1 (HIV-1) possessing a deletion in the reverse transcriptase (RT) gene at codon 67 was identified in a patient who had failed combination antiretroviral therapy. This deletion initially emerged under the selective pressure of combination therapy with 3'-azido-3'-deoxythymidine (AZT) plus 2',3'-dideoxyinosine. It has persisted for more than 3 years in association with the accumulation of a variety of other well-described drug resistance mutations and an uncharacterized mutation at RT codon 69 (T69G). Phenotypic studies demonstrated that the codon 67 deletion by itself had little effect on AZT sensitivity. However, in the context of the T69G mutation and three other mutations known to be associated with AZT resistance (K70R, T215F, and K219Q), this deletion led to a increase in AZT resistance from 8. 5-fold to 445-fold. A further increase in resistance (up to 1, 813-fold) was observed when two mutations associated with nonnucleoside RT inhibitor resistance (K103N and L74I) were added to the deletion T69G K70R T215F K219Q construct. Hence, these results establish that a deletion at RT codon 67 may be selected for in the presence of antiretroviral therapy and may lead to high-level resistance to AZT.

Anti-HIV Agents↗
PubMed1993

Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria.

Group II introns al1 and al2 of the yeast mtDNA cox1 gene encode reverse transcriptase-like proteins that function in RNA splicing and may play a role in intron mobility and excision. We find that ribonucleoprotein particles from yeast mitochondria contain a reverse transcriptase activity that is likely encoded by al1 and al2 and is highly specific for the introns and their flanking exons. Using a mutant strain with elevated activity, we show that the reverse transcriptase uses either excised intron RNA or cox1 pre-mRNA as template and initiates cDNA synthesis near the 3' end of al2 and immediately downstream in E3. Our results suggest that introns al1 and al2 are retroelements, which encode reverse transcriptases that have adapted to function in RNA splicing.

Base Sequence↗
PubMed2006

Regulation and effects of modulation of telomerase reverse transcriptase expression in primordial germ cells during development.

Telomere length maintenance in the germ line from generation to generation is essential for the perpetuation of eukaryotic organisms. This task is performed by a specialized reverse transcriptase called telomerase. While this critical function of telomerase has been well established, the mechanisms that regulate telomerase in the germ line are still poorly understood. We now show, using a Pou5f1-GFP transgenic mouse model, that telomerase suppression in quiescent male primordial germ cells (PGCs) is accompanied by a decrease in expression of murine telomerase reverse transcriptase (TERT). To further assess the role of TERT in quiescent PGCs, we developed a chicken Actb gene promoter/cytomegalovirus enhancer (CAG)-Tert transgenic mouse strain that constitutively expresses murine TERT. Telomerase activity was detected in quiescent PGCs from CAG-Tert transgenic embryos, demonstrating that re-activation of TERT expression is sufficient to restore telomerase activity in these cells and implying that TERT expression is an important mechanism of telomerase regulation in PGCs. Fluorescence-activated cell-sorting (FACS) analysis of PGC frequency and cell cycle status revealed no effect of either overexpression or deficiency of TERT in CAG-Tert transgenic mice or Tert knock-out mice respectively. These results demonstrate that TERT per se does not affect proliferation or development of PGCs, in contrast with recent studies that suggest that TERT has a telomere-independent effect in certain stem cells. It is possible that the direct effect of TERT on cell behavior may be dependent on cell type.

Animals↗
PubMed2004

Susceptibility of feline immunodeficiency virus/human immunodeficiency virus type 1 reverse transcriptase chimeras to non-nucleoside RT inhibitors.

To map the determinants of the lack of susceptibility of feline immunodeficiency virus (FIV) reverse transcriptase (RT) to anti human immunodeficiency virus type 1 (HIV-1) non-nucleoside RT inhibitors (NNRTIs), a variety of chimeric HIV-1/FIV RTs were constructed. The majority of chimeric RTs had an affinity (Km) for their natural substrates comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy was decreased. Whereas HIV-1 RT could be made entirely insensitive to NNRTIs by exchanging the amino acid sequence 97 through 205 of FIV RT, none of the reverse FIV/HIV-1 RT chimeras gained susceptibility to NNRTIs. The amino acids that are thought to be involved in NNRTI susceptibility and that are different from those in HIV-1 RT have also been introduced in FIV RT. These mutant RTs gained virtually no susceptibility to efavirenz or capravirine. Vice versa, when these HIV-1-specific amino acids were replaced by their FIV RT counterparts in HIV-1 RT, susceptibility to the NNRTIs was lost. Thus, replacing segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive toward NNRTIs and was often accompanied by a decrease or even total loss of polymerase activity. It is postulated that, in contrast to the results found for HIV-1/HIV-2 RT chimeras and supported by the crystal structure of HIV-2 RT, there exist significant differences in the structure and/or flexibility of FIV RTs that may prevent NNRTIs from interacting with the FIV RT.

Amino Acid Sequence↗
PubMed1995

Human immunodeficiency virus type 1 reverse transcriptase. 3'-Azidodeoxythymidine 5'-triphosphate inhibition indicates two-step binding for template-primer.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T.P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T.P binary complex (RTT.P). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T.P (for single-nucleotide incorporation). To explore cycling through these alternate rate-limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T.P system, poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Km should be equivalent to Ki, and since Km for AZTTP should be influenced by the dissociation rate constant for RTT.P, we examined the effect of altering RTT.P dissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different T.P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T.P binding. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16 was biphasic, with a burst followed by a slower steady-state phase representing kcat (0.42 min-1) which was similar to the rate constant for RTT.P dissociation. Additionally, Km for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (Ki,AZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT.P dissociation rate constant and Ki,AZTTP was not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT.P exist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T.P where the binary RTT.P complex undergoes an isomerization before binding of the deoxynucleotide substrate.

Antiviral Agents↗
PubMed1997

A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron.

The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

Bacterial Proteins↗
PubMed1982

Reverse transcriptase from simian foamy virus serotype 1: purification and characterization.

Chromatography on heparin-Sepharose, known for its affinity for nucleotide-binding polypeptides, was used to purify the viral RNA-dependent DNA polymerase (reverse transcriptase) from the core polypeptides of simian foamy virus type 1. This procedure allowed the recovery of highly purified enzyme with a high specific activity. The average molecular weight of this monomeric enzyme is 81,000 and is thus comparable to that found for other known primate retroviruses. Reverse transcriptase activity of simian foamy virus type 1 requires a ribonucleotide template as a primer or otherwise a DNA with 3'-OH ends. Other optimal conditions of activity are reviewed. Heat inactivation studies led to the concept of an enzyme with two loci, one specific for the substrate and the other for the template-primer.

Hot Temperature↗
PubMed2005

Cumulative exposure to nucleoside analogue reverse transcriptase inhibitors is associated with insulin resistance markers in the Multicenter AIDS Cohort Study.

OBJECTIVE: To estimate insulin resistance and its relationship to antiretroviral therapy (ART) in a cohort of HIV-infected persons with comparison to HIV-seronegative controls. DESIGN: Prospective cohort of 533 HIV-infected and 755 HIV-seronegative men in the Multicenter AIDS Cohort Study evaluated at 6-month intervals between 1999 and 2003. METHODS: Recent ART exposure was assessed by type of treatment in the preceding 6 months [i.e., no ART, monotherapy, combination ART, or highly active antiretroviral therapy (HAART) with and without a protease inhibitor (PI)]. Cumulative exposure was determined for the three major ART classes and for individual medications within each class. Two endpoints, a modified QUICKI index, 100 x 1/[log10(glucose) + log10(insulin)] and fasting hyperinsulinemia (insulin > 15 microU/ml), were assessed. All statistical models were adjusted for age, body mass index, race, nadir CD4 cell count, hepatitis C serostatus and family history of diabetes mellitus. RESULTS: Each of the HIV-infected groups had higher odds of hyperinsulinemia and lower mean QUICKI than the HIV-seronegative men. Each additional year of exposure to nucleoside analogue reverse transcriptase inhibitors (NRTI) was associated with increased odds of hyperinsulinemia [odds ratio (OR), 1.08; 95% confidence interval (CI), 1.02-1.13) and a lower QUICKI (-0.04; 95% CI, -0.07 to -0.01). Cumulative exposure to non-nucleoside analogue reverse transcriptase inhibitors or PI drugs was not associated with either insulin resistance marker. Of individual medications examined, stavudine was associated with the highest risk of hyperinsulinemia (OR, 1.2; 95% CI, 1.2-1.3). CONCLUSIONS: Fasting surrogate markers suggest increased insulin resistance in HIV-infected men, which is related to cumulative NRTI exposure.

Adult↗
PubMed1995

Inhibitory effects of Egyptian folk medicines on human immunodeficiency virus (HIV) reverse transcriptase.

Extracts of 41 medicinal plants used in Egyptian folk medicine were screened for their inhibitory effects on human immunodeficiency virus-1 reverse transcriptase. The extracts of fruits of Phyllanthus emblica, Quercus pedunculata, Rumex cyprius, Terminalia bellerica, Terminalia chebula and Terminalia horrida showed significant inhibitory activity with IC50 < or = 50 micrograms/ml. Through a bioassay guided-fractionation of the methanol extract of the fruit of P. emblica, putranjivain A (1) was isolated as a potent inhibitory substance with IC50 = 3.9 microM, together with 1,6-di-O-galloyl-beta-D-glucose (2), 1-O-galloyl-beta-D-glucose (3), kaempferol-3-O-beta-D-glucoside (4), quercetin-3-O-beta-D-glucoside (5) and digallic acid (6). The inhibitory mode of action by 1, 2 and 6 was non-competitive with respect to the substrate but competitive with respect to a template-primer. Furthermore, the stereochemistry of 1 was established in this paper by nuclear magnetic resonance spectroscopy.

Carbohydrate Conformation↗
PubMed1994

Kinetic studies with the non-nucleoside human immunodeficiency virus type-1 reverse transcriptase inhibitor U-90152E.

The bisheteroarylpiperazine U-90152E is a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses excellent anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound inhibits both the RNA- and DNA-directed DNA polymerase functions of HIV-1 RT. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-90152E. 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 Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of 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 essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The results obtained indicate that U-90152E acts exclusively as a mixed inhibitor with respect to the template: primer and dNTP binding sites for both the RNA- and DNA-directed DNA polymerase domains of the enzyme. The inhibitor shows a significantly higher binding affinity for the enzyme-substrate complexes than for the free enzyme and consequently does not directly impair the functions of the substrate binding sites. Therefore, U-90152E appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond.

Antiviral Agents↗
PubMed1993

Characterization of novel reverse transcriptase encoding human endogenous retroviral sequences similar to type A and type B retroviruses: differential transcription in normal human tissues.

The polymerase chain reaction was used to amplify genomic DNA and reverse-transcribed RNA from human lymphocytes, using primers derived from conserved regions within the retroviral reverse transcriptase. Sequencing of 33 cloned amplification products revealed that a variety of sequences with similarity to mouse mammary tumor virus, mouse intracisternal A particle, and human endogenous retrovirus K10 were detected with this primer pair. The sequences were divided into six subgroups, with a nucleotide sequence dissimilarity of about 25% between the subgroups. Members within five of the subgroups were most closely related to human endogenous retrovirus K10 and mouse mammary tumor virus, whereas sequences of the sixth subgroup also showed similarity to mouse intracisternal A particle. Ten of the sequences had open reading frames with preference for silent mutations at conserved sites. Southern blot analysis showed that some HML (human endogenous MMTV-like) subgroups (HML-4 and HML-5) were present in a few copies (about 5), whereas others (HML-1 to HML-3 and HML-6) were present in at least 10 to 20 copies per genome. Northern (RNA) blot analysis revealed that several of the subgroups are differentially expressed in human normal tissues. A complex pattern of transcripts from about 12 to 1.4 kb was found in several of the tissues tested. However, the most abundant expression was detected in lung (all subgroups), skeletal muscle (HML-4 and HML-5), placenta (HML-2 and HML-5), and kidney (HML-2, HML-3 and HML-5). Expression of reverse transcriptase sequences in human tissues may have biological consequences. The described sequences are similar to elements which cause carcinoma and are immunoregulatory in mice. It remains to be seen whether human sequences also have such functions.

Base Sequence↗
PubMed1983

Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid.

The synthesis of single-stranded globin cDNA by the RNA-directed DNA polymerase activity of reverse transcriptase in the presence of oligothymidylate primers was investigated in order to determine the limitations to higher yields. The results indicated that the associated ribonuclease H activity, an integral part of reverse transcriptase, plays a large role in the synthesis of the first strand of cDNA and that the interplay of the two enzyme activities for any specific set of conditions determines the yield of single-stranded products. In both the presence and the absence of polymerization, the associated ribonuclease H catalyzed the deadenylation of mRNA, producing molecules that were somewhat shorter, highly homogeneous in size, and fully translatable into globin protein. They were also entirely lacking in the ability to serve as templates for cDNA synthesis. The reaction was completely dependent on oligothymidylate and completely independent of deoxyribonucleoside triphosphates. The initial rate of deadenylation was one-fourth the initial rate of initiation of polymerization when saturating levels of deoxyribonucleoside triphosphates were used in the polymerase reaction. In the presence of ribonuclease H activity, the DNA polymerase catalyzed the synthesis of an array of cDNAs including some that were full length. The initiation of polymerization was rate limiting: once synthesis had begun, it required 1-1.5 min to transcribe globin mRNA. However, most primers that were elongated were aborted prematurely. Maximum synthesis of full-length cDNA required stoichiometric levels of enzyme and high triphosphate levels, but regardless of conditions, the sum of completed cDNA and deadenylated mRNA accounted for only 50% of the input mRNA. The data fit a model in which synthesis of full-length cDNA molecules depends on the arrangement of primers and transcription initiation complexes on the poly(A) "tail" of mRNA.

Animals↗
PubMed2003

Structure-activity relationship studies on a novel family of specific HIV-1 reverse transcriptase inhibitors.

We have previously reported the discovery and preliminary structure-activity relationships of a new class of specific HIV-1 reverse transcriptase (RT) inhibitors whose prototype compound is the 1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3-N-[(carboxy) methyl]-thymine. In an attempt to increase the inhibitory efficacy against HIV-1 RT of this new class of nucleosides, and to further explore the structural features required for anti-HIV-1 activity, different types of modifications have been carried out on the prototype compound. These include substitution of the tert-butyldimethylsilyl groups by other liphophilic groups, replacement of the carboxy group at the N-3 position of the nucleobase by other functional groups, change in the length of the spacer between the thymine and the carboxylic acid residue and substitution of the thymine moiety by other pyrimidine (uracil, 5-ethyluracil) or purine (hypoxanthine) nucleobases. In addition, the most salient structural features of this new class of HIV-1-specific nucleosides have been incorporated into classical HIV RT nucleoside inhibitors such as ddl, AZT, d4T. Our studies demonstrate that both the carboxymethyl moiety at the nucleobase and tert-butyldimethylsilyl groups at the sugar are important structural components since deletion of either of them is detrimental to the antiviral activity.

Anti-HIV Agents↗
PubMed1986

Putative reverse transcriptase intermediates of human hepatitis B virus in primary liver carcinomas.

Nucleocapsid-pol fusion proteins have been detected by serological screening hepatocellular carcinoma tissues that contain hepatitis B virus (HBV) DNA. The existence of these fusion proteins suggests that HBV may synthesize its reverse transcriptase in a fashion analogous to the way that retroviruses synthesize and process a precursor. The accumulation of HBV reverse transcriptase intermediates in tumorous tissues and not in other tissues may be related to the absence of viral core particles and possibly contributes to tumor development.

Animals↗
PubMed1994

Subunit specificity of mutations that confer resistance to nonnucleoside inhibitors in human immunodeficiency virus type 1 reverse transcriptase.

We constructed plasmid vectors that simultaneously express both the p66 and p51 subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in Escherichia coli. These vectors allow us to generate HIV-1 RT heterodimers in which either the p66 or the p51 subunit has the wild-type sequence and the other subunit has a specific amino acid substitution. We used these vectors to express HIV-1 RT heterodimers containing several different amino acid substitutions reported to confer resistance to nonnucleoside inhibitors. Most of the amino acid substitutions conferred resistance to nonnucleoside inhibitors R86183 (TIBO) and TSAO-m3T only when present in the p66 subunit of the p66-p51 heterodimer; heterodimers that contained a wild-type p66 subunit and a mutant p51 subunit remained sensitive to the inhibitors. However, there was one mutation, E138K, that conferred drug resistance when the mutation was present in the p51 subunit. The corresponding heterodimer with the E138K mutation in the p66 subunit and a wild-type p51 subunit remained sensitive to the inhibitors. Analysis of the three-dimensional structure of HIV-1 RT indicated that residue 138 of the p51 subunit is in the nonnucleoside inhibitor-binding pocket while residue 138 of the p66 subunit is not. The mutagenesis results, combined with structural data, support the idea that the nonnucleoside inhibitors exert their effects by binding to a hydrophobic pocket in the RT heterodimer and that mutations which give rise to drug resistance directly interfere with the interactions between the nonnucleoside inhibitors and HIV-1 RT.

Antiviral Agents↗
PubMed1998

A proline-to-histidine substitution at position 225 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) sensitizes HIV-1 RT to BHAP U-90152.

Two mutant virus strains in which the novel P225H mutation appeared in a V106A reverse transcriptase (RT)-mutated genetic background upon treatment of human immunodeficiency virus type 1 (HIV-1) with quinoxaline S-2720 were isolated. Surprisingly, the addition of the P225H mutation to the V106A RT mutant genetic background resensitized the V106A RT mutant virus to the non-nucleoside RT inhibitor (NNRTI) BHAP U-90152, but not to other NNRTIs. Construction of both recombinant viruses and recombinant RTs containing the V106A, P225H and V106A+P225H mutations revealed that P225H was indeed responsible for the marked potentiation of the antiviral activity of BHAP against the P225H single-mutant virus and the V106A+P225H double-mutant virus when compared to wild-type and V106A single-mutant viruses, respectively. An explanation for the markedly increased sensitivity of the P225H mutant HIV-1 RT to BHAP and not to the other NNRTIs was provided by the unique features of the X-ray structure of the RT-BHAP complex.

Amino Acid Substitution↗
PubMed2004

Nonnucleoside inhibitor binding affects the interactions of the fingers subdomain of human immunodeficiency virus type 1 reverse transcriptase with DNA.

Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the "open" configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.

Amino Acid Substitution↗