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PubMed1972

Immunological relationships of reverse transcriptases from ribonucleic acid tumor viruses.

Antiserum to partially purified reverse transcriptase from the Schmidt-Ruppin strain of Rous sarcoma virus has been prepared and characterized. Antibody to the avian polymerase inhibited the reverse transcriptase activity of avian C-type viruses but had no effect on the polymerase activity from C-type viruses of other classes. The known mammalian C-type viral polymerases were significantly inhibited only by the antiserum to murine C-type viral polymerases; reverse transcriptases from four other mammalian viruses were immunologically distinct from both avian and mammalian C-type viral polymerases. Partially purified murine leukemia viral DNA polymerase activity was comparably reduced by specific antibody regardless of the template used for enzyme detection.

Animals↗
PubMed1988

Reverse transcriptases from human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIVMAC) are susceptible to inhibition by foscarnet and 3'-azido-3'-deoxythymidine triphosphate.

Reverse transcriptases from human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV) were investigated with respect to susceptibilities to the reverse transcriptase inhibitors foscarnet and 3'-azido-3'-deoxythymidine triphosphate (AZTTP). The different reverse transcriptases had the same sensitivity to foscarnet (50% inhibition at 0.10 to 0.16 microM). The Ki values for AZTTP were 0.01 to 0.02 microM for HIV-1 reverse transcriptase and 0.02 to 0.03 microM for HIV-2 and SIVMAC reverse transcriptases.

Animals↗
PubMed1988

Fidelity of HIV-1 reverse transcriptase.

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.

Avian Myeloblastosis Virus↗
PubMed1992

Regulation of the reverse transcriptase of human immunodeficiency virus type 1 by dNTPs.

Reverse transcriptase (RNA-directed DNA polymerase, EC 2.7.7.49) of human immunodeficiency virus type 1 has been examined with respect to the steady-state kinetics of polymerization of dNTPs into product DNA. With dNTPs as variable substrate, the kinetics of polymerization deviated from standard Michaelis-Menten kinetics. Substrate inhibition was observed at high substrate concentrations and negative cooperativity was seen at lower substrate concentrations. Examination of incorporation of substrate dNMPs in the presence of nucleotides not complementing the template demonstrated that dNTPs may act as noncompetitive inhibitors, as well as substrate. The Ki of the enzyme for dNTPs was 104 microM. A working model is presented that accounts for the substrate inhibition. In this model, the reverse transcriptase is a multisubunit holoenzyme, where noncompetitive inhibition is mediated by one subunit binding nucleotide and down-regulating the enzymatically active 64-kDa subunit. With additional assumptions, this model can accommodate the negative cooperativity observed.

Allosteric Regulation↗
PubMed1989

Ribonuclease H activities associated with viral reverse transcriptases are endonucleases.

A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy--that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H, including circular poly(A).linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNA.RNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes.

Avian Myeloblastosis Virus↗
PubMed1988

The alpha and beta chains of avian retrovirus reverse transcriptase independently expressed in Escherichia coli: characterization of enzymatic activities.

Reverse transcriptase of the avian sarcoma and leukosis retroviruses is a heterodimer composed of a 63-kDa alpha and a 95-kDa beta polypeptide chain, both of which are encoded in the pol gene and are produced by proteolytic processing of a larger precursor. We previously constructed a bacterial expression clone of the entire pol coding region that produces a protein 4 kDa larger than the mature viral beta subunit. By use of this clone and synthetic oligonucleotides to introduce stop codons, two derivatives have been constructed: one that directs synthesis of a protein equivalent to the mature beta subunit and the other that directs synthesis of a protein equivalent to alpha subunit. Predicted amino acid sequences of these proteins differ from their viral counterparts only by an initiator methionine that was added to the N termini for expression in Escherichia coli. Both bacterially expressed proteins exhibit reverse transcriptase activity and appear to function as homodimers. The properties of these proteins resemble those of the viral reverse transcriptase heterodimer; however, the bacterially produced alpha dimer protein could be distinguished from the other proteins by its increased sensitivity to heat inactivation, which also has been reported for the corresponding viral product. These results show that correct folding and expression of enzymatic function does not require formation of a precursor. The alpha and beta clones provide a convenient source of individual pol gene products for further evaluation of their roles in the synthesis and integration of retroviral DNA.

Amino Acid Sequence↗
PubMed1977

Interactions of murine leukemia virus core components: characterization of reverse transcriptase packaged in the absence of 70S genomic RNA.

Virions produced by cells in the presence of actinomycin D (Act D virions) contain reverse transcriptase but are deficient in 70S genomic RNA. To assess the role of genomic RNA in encapsidation of a functional reverse transcriptase and to study the interaction of the enzyme and its template in the cores of intact virions, the reverse transcriptase enzymes of normal and Act D virions were compared. The enzymes were indistinguishable by column chromatography, sedimentation velocity, or template/primer preferences. In addition, these enzymes showed equal sensitivity to inactivation by antibodies directed against Rauscher murine leukemia virus DNA polymerase. The enzymes from Act D and normal virions had similar thermal decay rates and were both protected against heat denaturation by natural and synthetic template/primers. By these criteria, the DNA polymerase molecules synthesized and assembled into virions in the absence of genomic RNA are identical to those packaged under normal conditions. Additional studies designed to measure protection of reverse transcriptase by genomic RNA were carried out by comparing the thermal lability of the enzyme in intact Act D and normal virions. The thermal decay rate of reverse transcriptase in Act D virions was identical to that in control virions. In contrast to the lability of the virion-associated enzyme, however, genomic RNA in control virions was stable to heat treatment.

AKR murine leukemia virus↗
PubMed1984

Increase of cellular murine leukemia virus reverse transcriptase in interferon-treated cells.

The addition of mouse interferon (IFN) to AKR murine leukemia virus (MuLV)-producing NIH3T3 cells inhibited the extracellular appearance of AKR MuLV when assayed for reverse transcriptase activity or infectious virus production. IFN treatment had no detectable effect on proviral DNA formation during infection nor on the level of viral RNA in virus-producing cells. However, addition of IFN did alter the level of cellular viral reverse transcriptase activity. Chromatography of extracts from virus-producing cells on poly(G)-Sepharose columns revealed two peaks of reverse transcriptase activity. Peaks I and II eluted at 0.45 M and 0.65 M NaCl, respectively, while the cellular DNA polymerase beta eluted earlier at 0.3 M NaCl. IFN treatment of these chronic virus producer cells resulted in a 5-fold increase in peak I whereas peak II and polymerase beta remained essentially unchanged. When reverse transcriptase from purified virions was similarly chromatographed on poly(G)-Sepharose, all of the enzymatic activity eluted as peak I. Thus, the reverse transcriptase in peak I from cell extracts appears to be the form which is present in mature virions. Contrary to the results with chronic virus-producing cells, IFN treatment prior to exogenous infection with MuLV did not alter levels of reverse transcriptase peaks I and II or polymerase beta. These results provide further evidence that the major effect of IFN occurs at the level of MuLV maturation and assembly.

AKR murine leukemia virus↗
PubMed1990

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity.

Amino Acid Sequence↗
PubMed1990

HIV-1 reverse transcriptase: structure predictions for the polymerase domain.

Reverse transcriptase (RT) plays an essential role in the life cycle of the human immunodeficiency viruses (HIV). A better understanding of this enzyme, and its two catalytic functions, the DNA polymerase and the RNase H, could lead to the development of new drugs that would specifically block HIV replication. The available genetic, sequence, biochemical, and immunological data on the reverse transcriptase of HIV-1 constrain the possible structure of the DNA polymerase domain. The purpose of this review is to correlate the data and to discuss, in light of that data, a model for the structure of the polymerase domain. In this model, the polymerase domain is approximately 50 to 60 A in diameter with a 20 A opening to accommodate the nucleic acid duplex. The most evolutionarily conserved region of RT (amino acids 20-190 of HIV-1 RT) is proposed to form the inner surface of the 20 A opening to which the nucleic acid hemiduplex is bound.

Amino Acid Sequence↗
PubMed1986

PPi analogs as inhibitors of human T-lymphotropic virus type III reverse transcriptase.

Twenty-six PPi analogs were tested for inhibitory effects on human T-lymphotropic virus type III reverse transcriptase. The structural requirements for inhibition and mechanism of action of the most active inhibitors have been investigated. Foscarnet (phosphonoformic acid) was the most potent inhibitor of human T-lymphotropic virus type III reverse transcriptase with 50% inhibition at 0.5 microM. The mechanism was a noncompetitive type of inhibition of a (riboadenylic acid)n . (deoxythymidylic acid)12-18 [(rA)n(dT)12-18]-directed transcription at varied dTTP concentration. At constant substrate (dTTP) concentration and varied amounts of template, (rA)n(dT)12-18, the inhibitory action of foscarnet was of an uncompetitive type. The same pattern of inhibition was seen when the less active inhibitor carbonyldiphosphonate was studied under identical conditions. The structural requirements for inhibition of human T-lymphotropic virus type III reverse transcriptase by PPi analogs were similar to those shown by other reverse transcriptases.

Antiviral Agents↗
PubMed1988

Low fidelity of cell-free DNA synthesis by reverse transcriptase of human immunodeficiency virus.

The fidelity of DNA synthesis by reverse transcriptases from human immunodeficiency virus and other retroviruses was compared by measuring the rates of misincorporation of dCMP in the place of TMP in cell-free DNA synthesis with polyadenylic acid as the template. The fidelity of human immunodeficiency virus reverse transcriptase was found to be about one-third of that of the reverse transcriptases of other retroviruses.

Autoradiography↗
PubMed1995

Detection of reverse transcriptase in culture medium for mammary tumour cell lines: a comparison of an established radio-labelling technique and a contemporary non-isotopic technique.

Classically, radio-label techniques have been employed to analyse biological samples for reverse transcriptase (RT) activity. More recently, however, non-isotopic kits have been developed for retroviral quantification. Nevertheless, until the present investigation it has not been known if these contemporary methods are more sensitive at detecting reverse transcriptase activity. In our study, a non-isotopic ELISA method was shown to be considerably more sensitive than the radio-label technique at detecting reverse transcriptase in growth medium used to culture the murine breast cancer cell line GR/A. Using the ELISA, less reverse transcriptase activity was demonstrated in growth medium from human mammary adenocarcinoma MCF-7 cells than the murine source. This ELISA did not detect reverse transcriptase activity from a pure source of Moloney murine leukaemia virus. In light of this, the broad applicability of this ELISA for reverse transcriptase from different viral sources must be investigated before it can be used to monitor biological supernatants for the presence of retroviruses.

Animals↗
PubMed1989

Co-expression of the subunits of the heterodimer of HIV-1 reverse transcriptase in Escherichia coli.

Expression of the 66-kDa form of human immunodeficiency virus, type 1 reverse transcriptase in Escherichia coli leads to isolation of small amounts of a 2 x 66-kDa homodimer and larger amounts of a heterodimer form of the enzyme in which the 66-kDa protein is complexed with its carboxyl-terminally truncated is complexed with its carboxyl-terminally truncated 51-kDa form. The latter arises via proteolysis by contaminating proteases. The heterodimer, which was characterized by gel filtration (apparent native molecular mass of 120-130 kDa), was the most active form of the enzyme (specific activity, 5000 units/mg, cf. less than 2000 for the 66-kDa fragment). The 66-kDa fragment alone was shown to be only partially dimerized, with the activity residing mainly in the dimer fraction. Proteolysis of the 66-kDa form resulting partially in the 51-kDa form led to an increase in reverse transcriptase activity. Expression of a truncated version of the protein containing the first 428 amino acids of the reverse transcriptase coding region led to a protein which had low but measurable reverse transcriptase activity (400-500 units/mg). Co-expression of the two proteins on a single plasmid led to expression in a 1:1 ratio. The 1:1 mixture behaved as a heterodimer, as shown by its chromatographic properties. It is likely that the mechanism for the production of heterodimers in vivo involves cleavage of 66-kDa monomers followed by rapid dimerization of the 51- and 66-kDa forms to give the heterodimeric form, which is stable toward further proteolysis.

Catalysis↗
PubMed1988

HIV-1 reverse transcriptase: crystallization and analysis of domain structure by limited proteolysis.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1977

A reverse transcriptase assay for detection of the bovine leukemia virus.

An RNA-dependent DNA polymerase or reverse transcriptase has been demonstrated in highly purified bovine leukemia virus (BLV) particles. The viral enzyme responded very effectively to the exogenous template primer polyneucleotide (poly) (rA)-oligonucleotide (oligo) (dT). Unlike the reverse transcriptases of most mammalian C type RNA viruses, and of the ubliquitous foamy-like bovine syncytial virus, the BLV enzyme prefers magnesium rather than manganese for optimal activity. The identification of several other conditions required for optimal activity of the viral reverse transcriptase led to the development of a rapid, sensitive, semiquantitative assay, which is comparable in sensitivity to the syncytia-infectivity assay for the detection of BLV in supernatant fluids of monolayer cell cultures. However, the reverse transcriptase assay is not sufficiently reproducible for obtaining routine detection of BLV in short-term cultures of bovine peripheral blood lymphocytes. Therefore, this assay does not seem to provide an accurate method for the diagnosis of BL virus infection in cattle.

Cells, Cultured↗
PubMed1991

Fidelity of reverse transcriptase of the simian immunodeficiency virus from African green monkey.

The in vitro fidelity of highly purified recombinant reverse transcriptase from simian immunodeficiency virus of African green monkeys (SIVagm) was determined. By using the phi X174am16 reversion assay an overall error rate of 1/19,000 was determined. This is 2.4-fold higher than the overall accuracy of purified recombinant HIV-1 reverse transcriptase, measured in parallel. The evaluation of error frequencies from nucleotide pool bias studies suggest an even higher accuracy for the SIVagm-derived reverse transcriptase. T:dGMP mismatches were formed most frequently with an error rate of 1/155,000, followed by G:dGMP (1/230,000), A:dGMP (1/315,000), G:dAMP (1/340,000), T:dCMP (1/540,000), T:dTMP (1/790,000), and A:dCMP (1/1,050,000) mispairs. Thus, according to pool bias effects and depending on the mismatch under consideration SIVagm reverse transcriptase appears to be 2 to 20-fold more accurate than the homologous enzyme from the human immunodeficiency virus type 1. This higher accuracy is not due to a co-purifying exonuclaease activity. Like the enzyme from HIV-1, the simian monkey-derived enzyme was found to be devoid of a proofreading 3' to 5' exonuclease.

Animals↗
PubMed1984

Detection of reverse transcriptase activity in association with the non-A, non-B hepatitis agent(s).

Particle-associated reverse transcriptase activity was detected in four human serum specimens and in two plasma-derived products, all of which had been shown to transmit non-A, non-B hepatitis (NANBH) to other human beings and/or chimpanzees. Reverse transcriptase activity was also detected in all twelve sera from patients with acute or chronic NANBH. In contrast, reverse transcriptase activity was found in only 2 of 49 serum specimens from healthy plasma donors and laboratory workers. Sucrose density gradient fractions of two of the infectious human sera (peak reverse transcriptase activity at 1.14 g/ml) transmitted NANBH to chimpanzees. Biochemical and enzymatic data indicate that the NANBH agent(s) is a retrovirus or is retrovirus-like.

Animals↗