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PubMed1986

Serological relationship between reverse transcriptases from human T-cell lymphotropic viruses defined by monoclonal antibodies. Evidence for two forms of reverse transcriptases in the AIDS-associated virus, HTLV-III/LAV.

The immunological relationship between reverse transcriptases purified from human T-cell lymphotropic viruses (HTLV-I, HTLV-II, HTLV-III) was defined using monoclonal antibodies specific for HTLV-III reverse transcriptase, secreted by a mouse/mouse hybridoma clone (4F8) developed in our laboratory. The viral proteins from HTLV-I and HTLV-II do not bear any cross-reactive epitope to antibodies secreted by this clone. These antibodies specifically cross-react with HTLV-III reverse transcriptase. The antibodies failed to neutralize the catalytic activity of reverse transcriptase; however, after immunoprecipitation with a magnetic conjugate of goat anti-mouse IgG, the residual activity was completely inhibited. This shows that the antibodies are not directed towards the catalytic active center of the enzyme. Using an immunoblotting technique (Western blotting), we have found two cross-reactive proteins with HTLV-III lysate with molecular masses of 53 and 66 kDa. This suggests that HTLV-III possesses two reverse transcriptase activities with a common determinant recognized by the same epitope.

Acquired Immunodeficiency Syndrome↗
PubMed1987

Immunogenicity of human immunodeficiency virus (HIV) reverse transcriptase: detection of high levels of antibodies to HIV reverse transcriptase in sera of homosexual men.

Immunoglobulin isolated from sera of homosexual men infected with human immunodeficiency virus (HIV) inhibited the reverse transcriptase (RT) activity of HIV. The inhibitory activity was specifically directed against HIV RT, and not against other mammalian retrovirus RT, including human T-lymphotropic virus type I. The relative titer of anti-RT antibody was significantly higher in asymptomatic men than in patients with lymphadenopathy or acquired immune deficiency syndrome (AIDS)-related complex. There was no correlation between the relative titer of anti-RT antibody and the relative titers of antibodies to major virion structural protein as determined by the enzyme-linked immunosorbent assay (ELISA) technique. These data suggest that antibodies to HIV RT may be related to the clinical status and possibly to the different degree of HIV replication in HIV-infected homosexual men.

AIDS-Related Complex↗
PubMed1989

A novel in situ colony screening method to detect human immunodeficiency virus reverse transcriptase activity expressed in bacteria. Isolation of pseudorevertants of reverse transcriptase mutants.

An in situ screening procedure is described that permits the detection of RNA-dependent DNA polymerase activity in bacterial colonies expressing the reverse transcriptase of the human immunodeficiency virus. The procedure has been applied to the isolation of rare pseudorevertants of an inactive parental mutant.

Amino Acid Sequence↗
PubMed1993

Kinetic analysis of template.primer interactions with recombinant forms of HIV-1 reverse transcriptase.

The reverse transcriptase (RT) from the human immunodeficiency virus (HIV) exists predominantly as a heterodimer (p66/p51), but can also form a homodimer of p66 subunits (p66/p66). RT binds to template-primer (T/P) tightly to form the first complex in the reaction sequence poised to conduct DNA synthesis upon the addition of dNTP and Mg2+. We have made use of this property to kinetically analyze poly(rA)-(dT)n interactions with recombinant homo- and heterodimeric HIV-1 RT derived from HXB2R proviral DNA. A T/P challenge assay was used to quantitatively follow RT-T/P complex formation. The homo- and heterodimeric forms of RT bound to poly(rA)-(dT)16 in a kinetically similar fashion. There was no more than a 2-fold difference in kcat or for any T/P parameter examined: Km, Kd, kon, koff determined from a binary complex or from a complex incorporating dTMP, processivity, and stoichiometry of binding. In contrast, it was found that the T/P Km with heterodimeric RT derived from the NY5 strain was significantly greater than that determined for HXB2R enzyme, indicating that a kinetic diversity exists between RT derived from different viral strains. Since HXB2R RT binds to poly(rA)-(dT)16 tightly, Kd < 1 nM, active-site titrations are facilitated. At saturation, one T/P binds per two polypeptides, suggesting that RT binds substrate productively as a dimer and that if monomers are present they must rapidly form dimers in the presence of T/P.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1978

Reverse transcriptase of RNA tumor viruses. V. In vitro proteolysis of reverse transcriptase from avian myeloblastosis virus and isolation of a polypeptide manifesting only RNase H activity.

Purified avian myeloblastosis virus reverse transcriptase contains two subunits that are structurally related. The large subunit, beta (molecular weight, 95,000), was converted in vitro by chymotrypsin into a polypeptide of molecular weight 63,000. This polypeptide was indistinguishable from the small subunit, alpha (molecular weight, 65,000), in its chromatographic behavior on the phosphocellulose column and its tryptic peptide composition. During this proteolytic conversion, a polypeptide of molecular weight 32,000 (fragment B) was obtained. It was composed of tryptic peptides unique to beta and appeared to be derived from the portion of the beta subunit that was cleaved off during the conversion of beta into alpha. Upon continued proteolysis, a smaller polypeptide of molecular weight 24,000 (fragment A) was generated. This polypeptide manifested only RNase H activity and shared common amino acid sequences with beta and alpha subunits. Fragment A did not share any amino acid sequence homology with fragment B.

Avian Leukosis Virus↗
PubMed1993

The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor.

We show that the reverse transcriptase (RT) encoded by the Mauriceville mitochondrial plasmid of Neurospora closely resembles viral RNA-dependent RNA polymerases in initiating cDNA synthesis opposite the penultimate C residue of a 3' tRNA-like structure and has the unprecedented ability for a DNA polymerase to initiate DNA synthesis at a specific site in a natural template without a primer. The Mauriceville plasmid enzyme can also use DNA or RNA primers in a manner suggesting how a primitive RT could have evolved from an RNA-dependent RNA polymerase into retroviral and other types of RTs. The characteristics of the Mauriceville plasmid RT suggest that it may be related to the progenitor of present-day RTs and DNA polymerases.

Base Sequence↗
PubMed1993

HIV-1-specific reverse transcriptase inhibitors show differential activity against HIV-1 mutant strains containing different amino acid substitutions in the reverse transcriptase.

Serial passage of HIV-1 in CEM or MT-4 cell cultures in the presence of different HIV-1-specific reverse transcriptase (RT) inhibitors yielded mutant viruses which were resistant (i.e., 200- to 1000-fold less sensitive) to the homologous compounds. The RT of these mutant HIV-1 strains showed different amino acid substitutions depending on the class of the HIV-1-specific RT inhibitors. The following amino acid substitutions were found: 138 Glu-->Lys (TSAO-T), 181 Tyr-->Cys (nevirapine), 181 Tyr-->Cys (pyridinone), and 100 Leu-->Ile (TIBO R82150). Four TIBO (R82913)-resistant HIV-1 strains contained different amino acid substitutions: 103 Lys-->Asn (strain 2), 100 Leu-->Ile and 138 Glu-->Lys (strain B02), 100 Leu-->Ile and 181 Tyr-->Cys (strain 1), 100 Leu-->Ile and 188 Tyr-->His (strain B22). The level of cross-resistance (or sensitivity) highly depends on the nature of the amino acid substitutions. As a rule, the TSAO-resistant HIV-1 strains (138 Glu-->Lys) and TIBO (R82150 or R82913)-resistant HIV-1 strains (Leu 100-->Ile or 103 Lys-->Asn) are sensitive to the other HIV-1-specific RT inhibitors, whereas the amino acid change 181 Tyr-->Cys results in a significant reduction of sensitivity to all classes of the HIV-1-specific RT inhibitors.

Amino Acid Sequence↗
PubMed1995

Sensitivity/resistance profile of a simian immunodeficiency virus containing the reverse transcriptase gene of human immunodeficiency virus type 1 (HIV-1) toward the HIV-1-specific non-nucleoside reverse transcriptase inhibitors.

To develop an animal model for the therapy of AIDS with human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitors, we recently constructed a hybrid simian immunodeficiency virus (SIV)/HIV-1 in which the RT gene of SIV was replaced by the RT gene of HIV-1. This chimaeric virus, designated RT-SHIV, was found to be markedly sensitive to the inhibitory effects of both nucleoside (ddN) and non-nucleoside RT inhibitors (NNRTIs). In contrast, SIV was inhibited only by ddNs (i.e., 3TC and AZT), but not NNRTIs. When RT-SHIV was grown in the presence of 3TC, nevirapine, TSAO-m3T or the thiocarboxanilide UC-42 drug-resistant mutant virus strains emerged in cell culture as rapid as for HIV-1(IIIB). The antiviral sensitivity/resistance spectrum of the mutant RT-SHIV strains against NNRTIs and ddNs, and the nature of the mutations that appeared in their RT were similar to those of the mutant HIV-1 strains that were selected under identical experimental conditions. Infection of macaques with RT-SHIV may be a useful tool for studying the mechanism of NNRTI-resistance development and the therapy of NNRTI-resistant viruses in an animal model.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Engineering of the human-immunodeficiency-virus-type-1 (HIV-1) reverse transcriptase gene to prevent dimerization of the expressed chimaeric protein: purification and characterization of a monomeric HIV-1 reverse transcriptase.

We report here a human-immunodeficiency-virus-type-1 (HIV-1) recombinant reverse transcriptase (RT) engineered to contain a 26-amino-acid linker insertion from the tether domain of feline leukaemia virus (FLV) RT. The chimaeric protein was expressed in Escherichia coli and migrated on SDS/PAGE as a 68 kDa band. A monomeric form of the chimaeric HIV-1 RT has been prepared by the coordinated applications of immobilized-metal-affinity chromatography and gel filtration on Superose 12 columns. The monomeric nature of this chimaeric HIV-I RT was further characterized by cross-linking studies using disuccinimidyl suberate. The RNA-dependent DNA polymerase activity of the monomeric chimaeric HIV-1 RT was 35% that of the heterodimeric (p66/p51) HIV-1 RT. These results support our recent studies on the monomeric polymerase domain (p51 RT) which exhibited an RNA-dependent DNA polymerase activity equal to 33% of that of the p66/p51 heterodimeric HIV-1 RT (Evans, Kezdy, Tarpley and Sharma [1993] Biotechnol. Appl. Biochem. 17, 91-102). The inability of the monomeric chimaeric HIV-1 RT to display polymerase activity like that of the heterodimeric HIV-1 RT is attributed to a decrease in the processive rate of DNA synthesis (75%) and DNA binding (65%). However, the monomeric chimaeric HIV-1 RT (p68) exhibited RNAase H activity like that of the heterodimeric form (p66/p51) of HIV-1 RT. These results suggest that the linker insertion from FLV RT does not interfere with the RNAase H activity associated with the monomeric HIV-1 RT.

Amino Acid Sequence↗
PubMed1995

Novel non-nucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. 4. 2-Substituted dipyridodiazepinones as potent inhibitors of both wild-type and cysteine-181 HIV-1 reverse transcriptase enzymes.

The major cause of viral resistance to the potent human immunodeficiency virus type 1 reverse transcriptase (RT) inhibitor nevirapine is the mutation substituting cysteine for tyrosine-181 in RT (Y181C RT). An evaluation, against Y181C RT, of previously described analogs of nevirapine revealed that the 2-chlorodipyridodiazepinone 16 is an effective inhibitor of this mutant enzyme. The detailed examination of the structure-activity relationship of 2-substituted dipyridodiazepinones presented below shows that combined activity against the wild-type and Y181C enzymes is achieved with aryl substituents at the 2-position of the tricyclic ring system. In addition, the substitution pattern at C-4, N-5, and N-11 of the dipyridodiazepinone ring system optimum for inhibition of both wild-type and Y181C RT is no longer the 4-methyl-11-cyclopropyl substitution preferred against the wild-type enzyme but rather the 5-methyl-11-ethyl (or 11-cyclopropyl) pattern. The more potent 2-substituted dipyridodiazepinones were evaluated against mutant RT enzymes (L100I RT, K103N RT, P236L RT, and E138K RT) that confer resistance to other non-nucleoside RT inhibitors, and compounds 42, 62, and 67, with pyrrolyl, aminophenyl, and aminopyridyl substituents, respectively, at the 2-position, were found to be effective inhibitors of these mutant enzymes also.

Cell Line↗
PubMed2025

A bacterial PrimPol-reverse transcriptase hybrid protein has a proofreading exonuclease activity that can be transferred to other reverse transcriptases.

Gene disruption analysis revealed that an E. coli PPRT protein, which has an N-terminal Primase-Polymerase (PrimPol) domain fused to a group II intron-like reverse transcriptase (RT) domain followed by a long C-terminal domain (CTD), contributes to a cellular oxidative DNA damage response in addition to its previously described function in phage defense. Biochemical analysis showed that the PrimPol domain has an error-prone DNA polymerase activity that enables read through of oxidation-induced DNA damage. Surprisingly, we found that the RT-like domain, in addition to synthesizing protein-primed DNAs for phage defense, has a 3' to 5' DNA exonuclease activity that functions in proofreading DNAs synthesized by the PrimPol domain. Extending these findings, we identified structural features that contribute to this proofreading activity, enabling us to associate it with both a group II intron-encoded and retroviral RT and suggesting general methods for incorporating proofreading activity into RTs.

DNA sequencing↗
PubMed1989

Reverse transcriptase in bacteria.

Reverse transcriptase, discovered in 1970 in retroviruses, has until recently been found only in eukaryotic organisms. Recently it was shown to occur in two groups of bacteria: myxobacteria and Escherichia coli. The gene for reverse transcriptase is part of a chromosomal genetic element that codes for the production of a branched DNA-RNA compound. In this compound a single-stranded DNA is connected to RNA at a specific G residue by a 2'-5' phosphodiester linkage. The precursor for the DNA-RNA compound is a folded messenger RNA, in which the specific G residue is the initiation point for reverse transcription. In the final DNA-RNA compound, the portion of the RNA transcribed by reverse transcriptase is eliminated by RNase H. The DNA-RNA compound is present in several hundred copies per cell. Its biological function is unknown at present.

Chromosomes, Bacterial↗
PubMed1992

3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors.

Nonnucleoside reverse transcriptase (NNRT) inhibitors (R82913; (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione; Cl-TIBO; and BI-RG-587, nevirapine) were used to select resistant human immunodeficiency virus type 1 (HIV-1) variants by passage in cell cultures of wild-type or 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant strains. Similar to other NNRT inhibitors, Cl-TIBO induced a single mutation (Y181 to C) in reverse transcriptase (RT) that accounted for the resistance. BI-RG-587 induced a different mutation (V106-->A) in AZT resistance backgrounds. A series of viable HIV-1 variants was constructed by site-directed mutagenesis of the RT, which harbored multiple drug resistance mutations, including Y181 to C. HIV-1 that was co-resistant to NNRT inhibitors and 2',3'-dideoxyinosine resulted when a 2',3'-dideoxyinosine resistance mutation (L74 to V) was also present in RT. By contrast, however, the Y181 to C mutation in an AZT resistance background significantly suppressed resistance to AZT, while it conferred resistance to NNRT inhibitors. However, the V106-->A substitution did not cause suppression of preexisting AZT resistance. Since certain combinations of nucleoside analogs and NNRT inhibitors might result in the development of co-resistance, careful analysis of clinical isolates obtained during combination therapy will be needed to determine the potential significance of these observations.

Antiviral Agents↗
PubMed1994

Structural basis of asymmetry in the human immunodeficiency virus type 1 reverse transcriptase heterodimer.

The reverse transcriptase from human immunodeficiency virus type 1 is a heterodimer consisting of one 66-kDa and one 51-kDa subunit. The p66 subunit contains both a polymerase and an RNase H domain; proteolytic cleavage of p66 removes the RNase H domain to yield the p51 subunit. Although the polymerase domain of p66 folds into an open, extended structure containing a large active-site cleft, that of p51 is closed and compact. The connection subdomain, which lies between the polymerase and RNase H active sites in p66, plays a central role in the formation of the reverse transcriptase heterodimer. Extensive and very different intra- and intersubunit contacts are made by the connection subdomains of each of the subunits. Together, contacts between the two connection domains constitute approximately one-third of the total contacts between subunits of the heterodimer. Conversion of an open p66 polymerase domain structure to a closed p51-like structure results in a reduction in solvent-accessible surface area by 1600 A2 and the burying of an extensive hydrophobic surface. Thus, the monomeric forms of both p66 and p51 are proposed to have the same closed structure as seen in the p51 subunit of the heterodimer. The free energy required to convert p66 from a closed p51-like structure to the observed open p66 polymerase domain structure is generated by the burying of a large, predominantly hydrophobic surface area upon formation of the heterodimer. It is likely that the only kind of dimer that can form is an asymmetric one like that seen in the heterodimer structure, since one dimer interaction surface exists only in p51 and the other only in p66. We suggest that both p51 and p66 form asymmetric homodimers that are assembled from one subunit that has assumed the open conformation and one that has the closed structure.

Antibodies, Viral↗
PubMed1995

Laboratory-scale production and purification of recombinant HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase from the HIV-1 strain WMF 1.13 was expressed in Escherichia coli JM 105 using a pKK233-2 vector. The bacteria were cultivated in a 20-l fermentor with 14-l net volume using M9ZB medium containing bactotryptone and yeast extract. After induction of reverse transcriptase (RT) expression by addition of isopropyl-beta-D-thiogalactopyranoside the enzyme concentration was monitored. Both soluble and inclusion-body deposited RT were detected by Western blots. Inclusion-body formation was confirmed by transmission electron microscopy. Further purification of soluble and insoluble RT was investigated. After cell desintegration by enzymatic treatment combined with osmotic shock and centrifugation, the supernatant was desalted by size-exclusion chromatography and further purified by DEAE-Sepharose FF, AF-Heparin Toyopearl 650 M and Fractogel EMD TMAE 650 (S). The results of the purification steps were monitored by SDS-PAGE with silver staining, non-radioactive RT assay and protein determination with Coomassie Blue. The sediment was extracted with 6 M GuHCl and after clarification and conventional refolding, treated in the same manner as soluble RT. This method is well suited for studying fermentation conditions as well as purification conditions. The RT is expressed in approximately equal amounts as soluble and insoluble enzyme.

Chromatography↗
PubMed1995

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1984

Mechanism of the action of the inhibitor of reverse transcriptase.

An inhibitor of reverse transcriptase of Moloney leukemia virus was reported previously in the cytoplasm of the cultured cells (1). In this report, the mechanism of the inhibition was examined. The inhibitory activity was completely abrogated by treatment at 55 degrees C for 20 min. Destruction of the reaction products of reverse transcriptase was not observed. Strong inhibition was observed by preincubation of poly rA oligo dT used as a template-primer, together with the inhibitor, but was not by incubation of poly rA or oligo dT with the inhibitor separately. It is suggested that the inhibitor reacts with poly rA oligo dT complex and interferes with attachment of the reverse transcriptase to the template-primer complex.

Cell Line↗