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PubMed1991

HIV reverse transcriptase structure-function relationships.

HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented.

Amino Acid Sequence↗
PubMed2004

Detection and characterization of cytoplasmic hepatitis B virus reverse transcriptase.

It was recently found that the Duck hepatitis B virus (DHBV) reverse transcriptase is primarily a non-encapsidated cytoplasmic molecule that is rapidly translated and has a very short half-life. Here, a non-encapsidated reverse transcriptase from the human Hepatitis B virus (HBV) was characterized. HBV polymerase accumulated in the cytoplasm in a manner similar to non-encapsidated DHBV polymerase. However, the HBV polymerase accumulated at an apparently lower concentration and had a longer half-life than the DHBV enzyme, and it displayed no evidence of the post-translational modifications observed for DHBV. Unlike the DHBV polymerase, immunofluorescence detection of the HBV polymerase in cells was suppressed by the core protein, and this suppression occurred independently of encapsidation. This implies an interaction between the polymerase and core in addition to encapsidation, but the polymerase and core did not co-immunoprecipitate, so the interaction might not be direct. These data indicate that production of cytoplasmic, non-encapsidated polymerase is conserved among the hepadnaviral genera. Furthermore, conservation of the cytoplasmic form of the polymerase suggests that it might have function(s) in virus replication or pathology beyond copying the viral genome.

Cell Line, Tumor↗
PubMed2003

A conserved telomerase motif within the catalytic domain of telomerase reverse transcriptase is specifically required for repeat addition processivity.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The catalytic protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT) domain that mediates nucleotide addition. The RT domain of TERT is distinguishable from retroviral and retrotransposon RTs in having a sizable insertion between conserved motifs A and B', within the so-called fingers domain. Sequence analysis revealed the existence of conserved residues in this region, named IFD (insertion in fingers domain). Mutations of some of the conserved residues in Saccharomyces cerevisiae TERT (Est2p) abolished telomerase function in vivo, testifying to their importance. Significant effects of the mutations on telomerase activity in vitro were observed, with most of the mutants exhibiting a uniform reduction in activity regardless of primer sequence. Remarkably, one mutant manifested a primer-specific defect, being selectively impaired in extending primers that form short hybrids with telomerase RNA. This mutant also accumulated products that correspond to one complete round of repeat synthesis, implying an inability to effect the repositioning of the DNA product relative to the RNA template that is necessary for multiple repeat addition. Our results suggest that the ability to stabilize short RNA-DNA hybrids is crucial for telomerase function in vivo and that this ability is mediated in part by a more elaborate fingers domain structure.

Amino Acid Motifs↗
PubMed1997

The thiocarboxanilide nonnucleoside UC781 is a tight-binding inhibitor of HIV-1 reverse transcriptase.

The thiocarboxanilide nonnucleoside inhibitor (NNI) UC781 inhibited HIV-1 reverse transcriptase (RT) DNA polymerase activity at a 1:1 molar ratio of inhibitor to enzyme. Inhibition was linear uncompetitive with respect to template/primer (T/P) and mixed noncompetitive with respect to deoxynucleoside triphosphate (dNTP), typical of NNI. When the RT-T/P binary complex was incubated with UC781 and then separated from unbound inhibitor, recovery of enzyme activity was slow, with only about 60% activity recovered after 25 min. The inactivation of the RT-T/P complex was prevented by the presence of a large excess of UC84, another carboxanilide NNI that interacts with this RT mechanistic form. UC781 protected the RT-T/P-dNTP ternary complex from irreversible inactivation by a photoactivatable azido analog of nevirapine, implying that UC781 binds to the NNI pocket of this RT mechanistic form. UC781 did not photoprotect either the free enzyme or the RT-T/P binary complex; however, protein fluorescence quenching studies indicated that UC781 interacted with all RT mechanistic forms, with the order of affinity being RT-T/P-dNTP ternary complex > RT-T/P binary complex > free RT. Reaction progress curve analysis showed that the binding of UC781 to RT is rapid (k(on) approximately 1.7 x 10(6) M(-1) s(-1)), but that dissociation is slow (k(off) approximately 1.6 x 10(-3) s(-1)). UC781 is therefore a rapid tight-binding inhibitor of HIV-1 RT, the first NNI to demonstrate this property.

Anilides↗
PubMed2004

Studies of nonnucleoside HIV-1 reverse transcriptase inhibitors. Part 1: Design and synthesis of thiazolidenebenzenesulfonamides.

A random high-throughput screening (HTS) program to discover novel nonnucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out with MT-4 cells against a nevirapine-resistant virus, HIV-1(IIIB-R). The primary hit, a thiazolidenebenzenesulfonamide derivative, possessed good activity. A systematic modification program examining various substituents at the 3-, 4-, and 5-positions on the thiazole ring afforded compounds with enhanced anti-HIV-1 and reverse transcriptase (RT) inhibitory activities. These results confirm the important role of the substituents at these positions and the thiazolidenebenzenesulfonamide motif as a valuable lead series for the next generation NNRTIs.

Benzenesulfonates↗
PubMed1993

The inophyllums, novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, Calophyllum inophyllum Linn.

As part of a search for novel inhibitors of HIV-1 reverse transcriptase, the acetone extract of the giant African snail, Achatina fulica, was shown to be active. Fractionation of the extract yielded inophyllums A, B, C, and E and calophyllolide (1a, 2a, 3a, 3b, and 6), previously isolated from Calophyllum inophyllum Linn., a known source of nutrition for A. fulica. From a methanol/methylene chloride extract of C. inophyllum, the same natural products in considerably greater yield were isolated in addition to a novel enantiomer of soulattrolide (4), inophyllum P (2b), and two other novel compounds, inophyllums G-1 (7) and G-2 (8). The absolute stereochemistry of inophyllum A (1a) was determined to be 10(R), 11(S), 12(S) from a single-crystal X-ray analysis of its 4-bromobenzoate derivative, and the relative stereochemistries of the other inophyllums isolated from C. inophyllum were established by a comparison of their 1H NMR NOE values and coupling constants to those of inophyllum A (1a). Inophyllums B and P (2a and 2b) inhibited HIV reverse transcriptase with IC50 values of 38 and 130 nM, respectively, and both were active against HIV-1 in cell culture (IC50 of 1.4 and 1.6 microM). Closely related inophyllums A, C, D, and E, including calophyllic acids, were significantly less active or totally inactive, indicating certain structural requirements in the chromanol ring. Altogether, 11 compounds of the inophyllum class were isolated from C. inophyllum and are described together with the SAR of these novel anti-HIV compounds.

Acetylation↗
PubMed1995

Rationale and experience with reverse transcriptase inhibitors and protease inhibitors.

The majority of antiretroviral agents used in the treatment of HIV are targeted at the HIV reverse transcriptase (RT) enzyme. In a continuing effort to develop more effective antiviral regimens, drugs that target other specific enzymes in the virus replication cycle are under development. A promising new class of drugs is the protease inhibitors. These antiviral agents are potent and highly specific, giving excellent inhibition of viral replication. Unfortunately, the high level of inhibition may not be maintained in vivo because of the rapid emergence of resistance to these agents when they are used as monotherapy. However, the combination of protease inhibitors with reverse transcriptase inhibitors may represent a major advance in the treatment of HIV infection.

Drug Therapy, Combination↗
PubMed1991

Kinetic interaction of human immunodeficiency virus type 1 reverse transcriptase with the antiviral tetrahydroimidazo[4,5,1-jk]-[1,4]-benzodiazepine-2-(1H)-thione compound, R82150.

We examined the kinetic interaction of purified recombinant DNA-derived human immunodeficiency virus type 1 (HIV-1) reverse transcriptase with R82150, a member of the tetrahydroimidazo[4,5,1-jk]-[1,4]-benzodiazepin-2(1H)-thione family of compounds (Pauwels, R., Andries, K., Desmyter, J., Schols, D., Kukla, M.J., Breslin, H.J., Raeymaeckers, A., Van Gelder, J., Woestenborghs, R., Heykants, J., Schellekens, K., Janssen, M.A.C., De Clercq, E., and Janssen, P.A.J. (1990) Nature 343, 470-474). R82150 inhibited noncompetitively the utilization of homopolymeric and heteropolymeric template-primers (KI range 280-300 nM). Inhibition of dNTP substrate incorporation was also noncompetitive (KI range 100-890 nM). In contrast, 100 microM R82150 did not inhibit human DNA polymerases alpha, beta, or gamma. Gel electrophoresis was used to analyze the effect of inhibitors on extension of heteropolymeric template-primers by HIV-1 reverse transcriptase. ddCTP induced accumulation of partially extended primers which had been terminated at sites requiring incorporation of deoxycytidylate. Competing template-primers reduced accumulation of both fully and partially extended primers. In contrast, R82150 induced accumulation of shortened primers that were terminated at various sites that did not correspond to any one particular deoxynucleotide species. Our results suggest that R82150 does not interact with HIV-1 reverse transcriptase as an analog of either template-primer or deoxynucleoside triphosphate substrate, but may bind allosterically at a site unique to this replicase.

Antiviral Agents↗
PubMedDate not supplied

Detection of tumor cells in peritoneal lavages from patients with gastrointestinal cancer by multiplex reverse transcriptase PCR.

BACKGROUND/AIMS: Cytological examination of peritoneal lavages is a useful predictor of peritoneal recurrence in gastrointestinal carcinoma patients. Nevertheless, it may be inadequate for those patients with lavages containing only few cancer cells. In the present study, sensitive detection of free cancer cells could be achieved through amplification of cytokeratin 19, carcinoembryonic antigen, alpha-fetoprotein mRNAs by means of multiplex reverse transcriptase polymerase chain reaction and nested polymerase chain reaction. METHODOLOGY: The multiplex reverse transcriptase polymerase chain reaction assay was used to examine lavage samples from 64 patients with various gastrointestinal malignant lesions (colorectal n = 27; duodenal carcinoma n = 1; gastric n = 7; pancreatic n = 4; hepatocellular carcinoma n = 2; gallbladder n = 1; cholangiocellular carcinoma n = 2 and 20 colorectal liver metastases. Specificity was assessed by examination of 15 donors without malignancies. In addition, nested polymerase chain reaction was used to improve the sensitivity of the assay for the detection of alpha-fetoprotein transcripts. RESULTS: Peritoneal lavages from 12 of 64 gastrointestinal carcinoma patients were positive for carcinoembryonic antigen mRNA. Carcinoembryonic antigen proved a specific marker, as no false-positives were detected in any patients without gastrointestinal cancer. alpha-fetoprotein mRNA was detected exclusively in peritoneal lavages from tumor patients, i.e., in 16 of 27 colon cancer patients, 14 of 20 patients with colorectal liver metastasis, 2 of 7 patients with gastric cancer, two patients with hepatocellular carcinoma and 2 of 4 patients with pancreatic cancer. Cytokeratin 19 mRNA was not found a useful marker, since control patients without malignancies were also positive. CONCLUSIONS: Our data suggest that carcinoembryonic antigen- and alpha-fetoprotein mRNA in peritoneal lavage are potentially useful specific markers for early diagnosis of metastasis of gastrointestinal cancer. It has been shown that alpha-fetoprotein-specific nested reverse transcriptase polymerase chain reaction can detect not only hepatocellular carcinoma cells, but also malignant cells from other gastrointestinal carcinomas. In contrast, cytokeratin 19 mRNA lacks specificity for gastrointestinal cancer.

Biomarkers, Tumor↗
PubMed1991

Secondary structure of the ribonuclease H domain of the human immunodeficiency virus reverse transcriptase in solution using three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy.

The solution structure of the ribonuclease H domain of HIV-1 reverse transcriptase has been investigated by three-dimensional double and triple resonance heteronuclear magnetic resonance spectroscopy. The domain studied has 138 residues and comprises residues 427 to 560 of the 66 kDa reverse transcriptase with an additional four residues at the N terminus. Initial studies on the wild-type protein were hindered by severe differential line broadening, presumably due to conformational averaging. Mutation of the single tryptophan residue located in a loop at position 113 (position 535 in the reverse transcriptase sequence) to an alanine resulted in much improved spectral properties with no apparent change in structure. 1H, 15N and 13C backbone resonances were assigned sequentially using a range of three-dimensional double and triple resonance heteronuclear experiments on samples of uniformly (greater than 95%) 15N and 15N/13C-labeled protein, and the secondary structure was elucidated from a qualitative analysis of data derived from three-dimensional 15N- and 13C-edited nuclear Overhauser enhancement spectra. The secondary structure comprises three alpha-helices and five strands arranged in a mixed parallel/antiparallel beta-sheet with a +1, +1, -3x, -1x topology. The C-terminal region from residue 114 onwards appears to be conformationally disordered in solution as evidenced by an almost complete absence of sequential and medium range nuclear Overhauser effects.

Amino Acid Sequence↗
PubMed1978

Inhibition of viral reverse transcriptase and human sperm DNA polymerase by anti-sperm antibodies.

The IgG fraction of serum from a rabbit immunized with detergent-prepared human sperm nuclei inhibited the DNA polymerase activities in human sperm and seminal fluid as well as the partially purified reverse transcriptase of the baboon endogenous type-C retrovirus (BEV). The analogous enzymes from lysates of oncogenic type-C viruses was unaffected. IgG from the serum of individual partners from infertile marriages similarly inhibited both purified BEV reverse transcriptase and human sperm DNA polymerase, but not a DNA polymerase isolated from human prostatic fluid. The data suggest that BEV reverse transcriptase and the human sperm DNA polymerase are antigenically related. Furthermore, the sperm appears to be auto-antigenic and the antibodies thus formed may be capable of interfering with reproductive success.

Antigens↗
PubMed2006

Selective excision of chain-terminating nucleotides by HIV-1 reverse transcriptase with phosphonoformate as substrate.

A major mechanism for human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analogs involves the phosphorolytical removal of the chain-terminating nucleotide from the 3'-end of the primer. In this work, we analyzed the effect of phosphonoformate (PFA) and other pyrophosphate (PP(i)) analogs on PP(i)- and ATP-dependent phosphorolysis catalyzed by HIV-1 RT. Our experimental data demonstrated that PFA did not behave as a linear inhibitor but as an alternative substrate, allowing RT to remove AZT from a terminated primer through a PFA-dependent mechanism. Interestingly, in non-terminated primers, PFA was not a substrate for this reaction and competitively inhibited PP(i)- and ATP-dependent phosphorolysis. In fact, binding of PFA to the RT.template/primer complex was hindered by the presence of a chain terminator at the 3'-end of the primer. Other pyrophosphate analogs, such as phosphonoacetate, were substrates for the excision reaction with both terminated and nonterminated primers, whereas pamidronate, a bisphosphonate that prevents bone resorption, was not a substrate for these reactions and competitively inhibited the phosphorolytic activity of RT. As expected from their mechanisms of action, pamidronate (but not PFA) synergistically inhibits HIV-1 RT in combination with AZT-triphosphate in the presence of PP(i) or ATP. These results provide new clues about the mechanism of action of PFA and demonstrate that only certain pyrophosphate analogs can enhance the effect of nucleosidic inhibitors by blocking the excision of chain-terminating nucleotides catalyzed by HIV-1 RT. The relevance of these findings in combined chemotherapy is discussed.

Antiviral Agents↗
PubMed1997

A novel retron that produces RNA-less msDNA in Escherichia coli using reverse transcriptase.

Bacterial retroelements, or retrons, use reverse transcriptase (RT) to produce a multicopy single-stranded DNA (msDNA) molecule that is covalently linked to RNA. In these studies we show that a retron from Escherichia coli 110, a clinical isolate, produces a novel RNA-less msDNA with a 5' phosphate residue. The msDNA is a 74-nucleotide single-stranded DNA molecule with a stable stem-loop structure without a mismatched base pair. Only the genes encoding msDNA (msd), msdRNA (msr), and RT (ret) are required to produce the msDNA molecule. The organization of these genes on the retron was similar to that of other elements producing branched msDNA-RNA. The conserved guanine, which is the branched residue in msDNA-RNA complexes and is essential for branch formation, is also present. Site-directed mutagenesis showed that this guanine is essential for the production of RNA-less msDNA. We postulate that the RNA-less msDNA in strain 110 is produced by nucleolytic cleavage of the branched msDNA-RNA compound.

Amino Acid Sequence↗
PubMed1990

Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast.

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is comprised of two subunits of approximately 66kD and 51kD. We have defined the carboxyl terminus of the 51kD molecule using the 66kD RT and HIV-1 protease (PR) expressed in yeast. Precise constructs encoding the 66kD and 51kD molecules were expressed individually, in yeast, at high levels. The purified recombinant subunits were shown to associate into heterodimers that retained both RT and RNase H activities. Only the 66kD molecule could associate into homodimers. Such homodimers retained approximately 80% of the RT activity of the heterodimers. Our data demonstrates that the 51/66kD heterodimer, analogous to that found in vivo, can be reconstituted in vitro and is more efficient in both RT and RNase H activity than the homodimer.

Amino Acid Sequence↗
PubMed1990

Identification of a human immunodeficiency virus-1 protease cleavage site within the 66,000 Dalton subunit of reverse transcriptase.

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.

Amino Acid Sequence↗
PubMed1990

Presence of reverse transcriptase in human leukemias and lymphomas.

Reverse transcriptase (RT) transcribes viral RNA into DNA to be integrated into the host genome. To study epidemiological aspects of human leukemias and lymphomas which are known to express retroviruses, clinical specimens in this report were assayed for divalent cation-dependent viral-specific RT. The assay was carried out with cells solubilized with a detergent to release RT enzyme. RT was purified with poly(U)-Sepharose which fixed all DNA polymerases and assayed with 4 synthetic homopolymers, oligonucleotide primed-templates, poly(rA)-oligo(dT)12-18 or poly(dA)-oligo(dT)12-18 with Mg2+, poly(rC)-oligo(dG)12-18 or poly(rCm)-oligo(dG)12-18 with Mn2+ as divalent cation and [methyl-3H]thymidine 5'-triphosphate or deoxy[8-3H]guanosine 5-triphosphate respectively. Radioactivity incorporation of the precipitate allows quantitation of RT activity. One Hodgkin's disease, one out of 2 B lymphomas, one out of 2 T lymphomas, eight out of 12 leukemias were found to be positive for RT activity as well as acquired immunodeficiency syndrome (AIDS) patients, known to express RT. The obtained RT activity in hematological malignancies was found to be comparable to positive controls such as RT enzymes purified from avian myeloblastosis and Moloney murine leukemia viruses.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement.

Reverse transcriptase (RT) is an indispensable component of infectious retroviruses. We have developed an ultrasensitive RT test in which RNA of bacteriophage MS2 serves as the template for RT-mediated cDNA synthesis. A fragment of the cDNA is selectively amplified by polymerase chain reaction and the amplification product is analyzed by Southern blot hybridization or enzyme immunoassay. The procedure was 10(6) to 10(7) times more sensitive than a conventional RT test and detected as little as 10(-9) unit of murine leukemia virus RT, which corresponded to 2.1 x 10(2) molecules, a number present in 3-11 virions. As a screening assay for filterable particle-associated RT, it was positive with supernatants from cell cultures producing human immunodeficiency virus (HIV) type 1 or human T-cell leukemia virus (HTLV) type 1 or 2, but was negative with nonproducer cultures. It was positive with plasma samples from all tested individuals infected with HIV-1, HIV-2, or HTLV-1 and sera from cats infected with feline leukemia virus or feline immunodeficiency virus. Control samples from blood donors or uninfected cats were negative. Density banding experiments with culture supernatants showed that the RT activity was associated with virus particles. The assay should detect all replication-competent retroviruses or similar agents. It may be used as a screening assay for such agents, for quantitation of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.

Animals↗
PubMed1998

Characterization of RNA strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase.

The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H- mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H- RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.

Base Sequence↗