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PubMed1977

Radioimmunoassay for mammalian type C viral reverse transcriptase.

Radioimmunological techniques were applied to the analysis of reverse transcriptase of mammalian type C RNA viruses. The polymerase of Rauscher mouse leukemia virus was purified by ion exchange and sequential affinity chromatography. Radioimmunoassays that utilized the viral enzyme as a probe detected as little as 1 ng of purified polymerase. No cross-reactivity could be demonstrated between the reverse transcriptase and other known virus-coded proteins. By comparing the immunological reactivity of the purified enzyme with the reactivity of detergent-disrupted virions, Rauscher mouse leukemia virus was shown to contain the antigenic equivalent of 40 molecules of reverse transcriptase. In a homologous competition immunoassay, the Rauscher viral enzyme demonstrated type-specific antigenic determinants, which distinguish it from other mouse type C viral polymerases. In a broadly reactive interspecies immunoassay, the reverse transcriptases of a number of mammalian type C viruses were cross-reactive, indicating their shared antigenic determinants. Various treatments that inhibit or inactivated DNA polymerase activity had little or no effect on the immunological properties of the enzyme. Thus, radioimmunoassays should be useful in the search for type C viral reverse transcriptase as a marker of subviral expression.

Cross Reactions↗

PubMed1994

Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors.

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.

Antiviral Agents↗

PubMed1993

A short-term clinical evaluation of L-697,661, a non-nucleoside inhibitor of HIV-1 reverse transcriptase. L-697,661 Working Group.

BACKGROUND: The non-nucleoside reverse transcriptase inhibitors are novel antiretroviral agents with selective activity in vitro against human immunodeficiency virus type 1 (HIV-1). They act through direct inhibition of reverse transcriptase and are not incorporated into DNA. METHODS: We evaluated a pyridinone non-nucleoside reverse transcriptase inhibitor, L-697,661, in separate six-week double-blind trials in patients with HIV-1 infection whose CD4 counts ranged from 200 to 500 cells per cubic millimeter (68 patients) or less than 200 cells per cubic millimeter (67 patients). Eligible patients were randomly assigned to receive L-697,661 orally in one of three doses (25 mg twice a day, 100 mg three times a day, or 500 mg twice a day) or zidovudine (100 mg five times a day). Clinical and laboratory assessments were performed weekly. Viral isolates were obtained from a subgroup of patients before and after treatment and were evaluated for in vitro sensitivity to L-697,661. RESULTS: Both L-697,661 and zidovudine were well tolerated. Transient increases in CD4 counts were noted in the patients with fewer than 200 CD4 cells per cubic millimeter who received the two higher doses of L-697,661, but not in those who received the lowest dose or zidovudine. Patients who received L-697,661 had rapid, dose-related decreases in plasma p24 antigen levels. However, this response virtually disappeared after six weeks in some patients receiving L-697,661, coincidently with the emergence of resistant viruses. This change in susceptibility was more frequent among patients receiving the higher doses of L-697,661 and was associated with amino acid substitutions at positions 103 and 181 in the HIV-1 reverse transcriptase gene. CONCLUSIONS: L-697,661 is safe and well tolerated and has significant dose-related activity against HIV-1. However, resistant strains of the virus emerge rapidly and may limit the effectiveness of non-nucleoside reverse transcriptase inhibitors as monotherapy for HIV-1 infection.

Adult↗

PubMed1978

Binding of tryptophanyl-tRNA to the reverse transcriptase of replication-defective avian sarcoma viruses.

The ability of reverse transcriptase to bind to [3H]tryptophanyl-tRNA and to function as DNA polymerase was compared for five temperature-sensitive mutants of avian sarcoma virus. Both activities of the reverse transcriptase were found to be heat labile in LA 335 and LA 336 as compared with the wild-type parents. For the other mutant viruses, LA 338, LA 343, and LA 672, grown at the permissive temperature, the reverse transcriptase was nearly as heat stable as for the wild-type parents in terms of tRNA binding and DNA polymerase. LA 338, LA 343, and LA 672 showed characteristic defects in their reverse transcriptase when propagated at the nonpermissive temperature; namely, tryptophanyl-tRNA binding and DNA polymerase activities were coordinately decreased in these virions. The reduced enzymatic activities were not entirely due to an inactive reverse transcriptase present in the virions, however, but rather lower amounts of enzyme protein incorporated into the virions contributed to the effect, according to assays of reverse transcriptase antigen by radioimmune competition.

Animals↗

PubMed1996

Inhibitory RNA ligand to reverse transcriptase from feline immunodeficiency virus.

High-affinity, high-specificity RNA ligands for reverse transcriptase from feline immunodeficiency virus (FIV) were isolated from an RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. The selected RNA ligands bound to FIV reverse transcriptase with dissociation constants in the nanomolar range. One of the ligands was a potent inhibitor of the RNA-dependent DNA polymerase activity of both the recombinant and the virion-derived FIV reverse transcriptase. It also inhibited the reverse transcriptase from an FIV mutant that is resistant to 3'-azido-3'-deoxythymidine (AZT). The inhibition of FIV reverse transcriptase was competitive with respect to template-primer and noncompetitive with respect to deoxyribonucleoside 5'-triphosphates. This ligand was specific for the FIV enzyme and did not inhibit other reverse transcriptases tested (avian myeloblastosis virus, Moloney murine leukemia virus, and human immunodeficiency virus type 1).

Animals↗

PubMed1992

In vitro enzymatic activity of human immunodeficiency virus type 1 reverse transcriptase mutants in the highly conserved YMDD amino acid motif correlates with the infectious potential of the proviral genome.

Reverse transcriptases contain a highly conserved YXDD amino acid motif believed to be important in enzyme function. The second amino acid is not strictly conserved, with a methionine, valine or alanine occupying the second position in reverse transcriptases from various retroviruses and retroelements. Recently, a 3.5-A (0.35-nm) resolution electron density map of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase positioned the YMDD motif within an antiparallel beta-hairpin structure which forms a portion of its catalytic site. To further explore the role of methionine of the conserved YMDD motif in HIV-1 reverse transcriptase function, we have substituted methionine with a valine, alanine, serine, glycine, or proline, reflecting in some cases sequence motifs of other related reverse transcriptases. Wild-type and mutant enzymes were expressed in Escherichia coli, partially purified by phosphocellulose chromatography, and assayed for the capacity to polymerize TTP by using a homopolymeric template [poly(rA)] with either a DNA [oligo(dT)] or an RNA [oligo(U)] primer. With a poly(rA).oligo(dT) template-primer, reverse transcriptases with the methionine replaced by valine (YVDD), serine (YSDD), or alanine (YADD) were 70 to 100% as active as the wild type, while those with the glycine substitution (YGDD) were approximately 5 to 10% as active. A proline substitution (YPDD) completely inactivated the enzyme. With a poly(rA).oligo(U) template-primer, only the activity of mutants with YVDD was similar to that of the wild type, while mutants with YADD and YSDD were approximately 5 to 10% as active as the wild-type enzyme. The reverse transcriptases with the YGDD and YPDD mutations demonstrated no activity above background. Proviruses containing the reverse transcriptase with the valine mutation (YVDD) produced viruses with infectivities similar to that of the wild type, as determined by measurement of p24 antigen in culture supernatants and visual inspection of syncytium formation. In contrast, proviruses with reverse transcriptases containing the YADD and YSDD mutations were less infectious than wild-type virus. These results point to the critical role of methionine of the YMDD motif in the activity of HIV-1 reverse transcriptase and subsequent replication potential of the virus.

Amino Acid Sequence↗

PubMed1989

Identification of a doubly spliced viral transcript joining the separated domains for putative protease and reverse transcriptase of hepatitis B virus.

Hepatitis B virus (HBV), like retroviruses, replicates through reverse transcription. However, the identity and mechanism for the synthesis of HBV reverse transcriptase remain unknown. The open reading frame (ORF) for HBV putative reverse transcriptase (pol), as a consequence of overlapping with the whole ORF of envelope proteins (hepatitis B surface antigens), includes a hypervariable region at the N terminus. Thus, compared with retroviruses, it is unlikely that HBV reverse transcriptase is translated from complete pol ORF in the full-length pregenomic RNA. We have now detected in infected human livers a novel doubly spliced RNA in which one splicing event removed the hypervariable region of the pol gene but retained the conserved region homologous to retroviral reverse transcriptase. The other splicing event deleted the central region of hepatitis B core antigen and thus brought the protease domain which is important for maturation of reverse transcriptase close to that of pol. For this sequence organization, the spliced RNA as the possible template for the synthesis of HBV reverse transcriptase is discussed.

Base Sequence↗

PubMed1994

Sensitivity of HIV-1 reverse transcriptase and its mutants to inhibition by azidothymidine triphosphate.

HIV-1 reverse transcriptase can catalyze the addition of either azidothymidine monophosphate (AZTMP) or thymidine monophosphate (dTMP) to a primer strand opposite template adenosine bases. The ratio of incorporation of AZTMP to dTMP as catalyzed by HIV-1 reverse transcriptase has been determined to be 0.4 using an RNA-DNA duplex substrate prepared from oligonucleotides with sequences taken from the HIV-1 genome sequence. Slight variations are found for the incorporation ratio of the two nucleotides on other substrates. Substrates containing more than one adenosine in the single-stranded part of the template allow for more chances to incorporate AZTMP and less full-length product. Variations in the intensity of bands on an autoradiograph of a DNA sequencing gel corresponding to different positions of incorporation of AZTMP suggest that not all template adenosine positions offer the same level of discrimination against incorporation of AZTMP. A reverse transcriptase containing a set of four mutations (D67N, K70R, T215Y, K219Q) known to cause resistance to AZT in cell culture assays has a ratio of incorporation that is 0.77 +/- 0.03 times the ratio for the wild-type reverse transcriptase opposite one specific template adenosine. In contrast, a hybrid mutant containing the same four mutations that cause resistance to AZT and an additional mutation, Y181C, which by itself causes resistance to the non-nucleoside inhibitor L-697,661 [Sardana et al. (1992), J. Biol. Chem. 267, 17526-17530], has a ratio of incorporation that is 1.34 +/- 0.01 times that of the wild-type, indicating that the hybrid mutant enzyme is more susceptible to inhibition by AZTTP than the wild-type reverse transcriptase.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiviral Agents↗

PubMed1996

Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.

We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.

DNA Primers↗

PubMed1993

Reverse transcription of the Mauriceville plasmid of Neurospora. Lack of ribonuclease H activity associated with the reverse transcriptase and possible use of mitochondrial ribonuclease H.

The Mauriceville mitochondrial plasmid of Neurospora encodes a reverse transcriptase that synthesizes a full-length cDNA copy of the major plasmid transcript beginning directly opposite the 3' end of the template RNA (Kuiper, M. T. R., and Lambowitz, A. M. (1988) Cell 55, 693-704). Here, we show that the Mauriceville plasmid reverse transcriptase has no detectable RNase H activity and that cDNAs synthesized either by the column-purified reverse transcriptase or by the endogenous reverse transcriptase in purified ribonucleoprotein particles remain in a full-length duplex with the template RNA. The column-purified Mauriceville plasmid reverse transcriptase initiates cDNA synthesis by using short DNA primers, which remain attached to the 5' end of the (-) strand DNA (Wang, H., Kennell, J. C., Kuiper, M. T. R., Sabourin, J. R., Saldanha, R., and Lambowitz, A. M. (1992) Mol. Cell. Biol. 12, 5131-5144). We find that these primer DNAs can be precisely removed by S1 nuclease digestion of the initial cDNA.RNA duplex, suggesting a mechanism whereby this structure may contribute to primer removal in vivo. Finally, we show that Neurospora mitochondria contain an endogenous RNase H activity, which is present in mitochondrial ribonucleoprotein particle preparations prior to their purification. This mitochondrial RNase H can degrade the endogenous plasmid transcript in ribonucleoprotein particles in vitro and could play a similar role in vivo. The finding that the Mauriceville plasmid reverse transcriptase, which is believed to be a primitive enzyme, has no detectable RNase H activity is consistent with the hypothesis that retroviral reverse transcriptases acquired their RNase H domains from a gene encoding a cellular RNase H.

Amino Acid Sequence↗

PubMed1975

Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus.

We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus. Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing. Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein. After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template. A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP. Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity. When the transcriptase preparation was incubated with protein kinase and [gamma-32P]ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity, suggesting 32P incorporation into transcriptase or transcriptase-associated proteins. A 20-60% decrease of reverse transcriptase activity was observed after incubation of reverse transcriptase with phosphatase. The results suggest that phosphorylative modification of reverse transcriptase may be critical in the regulation of reverse transcriptase-catalyzed DNA synthesis.

Alkaline Phosphatase↗

PubMed1989

Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching.

We have analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.

DNA, Single-Stranded↗

PubMed1990

Template switching by reverse transcriptase during DNA synthesis.

The ability of reverse transcriptase to make template switches during DNA synthesis is implicit in models of retrovirus genome replication, as well as in recombination and oncogene transduction. In order to understand such switching, we used in vitro reactions with purified nucleic acids and enzymes. The assay system involved the use of an end-labeled DNA primer so as to allow the quantitation of elongation on a donor template relative to the amount of elongation achieved by template switching (by means of sequence homology) when an acceptor template RNA was added. We examined several variables that affected the efficiency of the reaction: (i) the reaction time, (ii) the relative amounts of acceptor and donor template, (iii) the extent of sequence overlap between the donor and acceptor templates, and (iv) the presence or absence of RNase H activity associated with the reverse transcriptase. The basic reaction, with RNA templates and normal reverse transcriptase, yielded as much as 83% template switching. In the absence of RNase H, switching still occurred but the efficiency was lowered. Also, when the donor template was changed from RNA to DNA, there was still switching; not surprisingly, this was largely unaffected by the presence or absence of RNase H. Finally, we examined the action of the RNase H on RNA templates after primary transcription but prior to template switching. We found that in most cases, both ends of the original RNA template were able to maintain an association with the DNA product. This result was consistent with the work of others who have shown that RNase H acts as an endonuclease.

Chromosome Deletion↗

PubMed2000

Immunomodulating effect of 2,3,4,5-tetrahydro-1H-3-benzazepines (a new class of non-nucleoside inhibitors of reverse transcriptase).

The effects of newly synthesized, reverse transcriptase inhibitors, 3-benzazepines, for their effects on natural killer (NK) cell and blast transformation in human peripheral blood mononuclear cells were investigated. The most effective reverse transcriptase inhibitors were KF1, KF2 and KF3, which primarily suppressed immunological functions. Besides the inhibition of T cell proliferation, the benzazepines also show inhibitory effect on NK cell functions, in particularly, against large granular lymphocytes and monocytes. The B lymphocytes and Fc mediated killer functions were less inhibited.

Adjuvants, Immunologic↗

PubMed1982

Differential inhibition of DNA polymerase and RNase H activities of the reverse transcriptase by phosphonoformate.

Three potential inhibitors of reverse transcriptase activities, phosphonoformate (PF), phosphonoacetate (PAA), and ethyl-diethyl phosphonoformate (Et-PF), were compared in this study. Only PF was found to inhibit the DNA polymerase activity of the purified reverse transcriptase of Moloney murine leukemia virus (M-MuLV) and avian myeloblastosis virus (AMV). The degree of DNA polymerase inhibition was linear with PF concentration; 50% inhibition was achieved at 10 muM. Whereas PF inhibited both the RNA and DNA dependent DNA polymerase activities, the RNase H activity of the reverse transcriptase was unaffected. Both the endogenous DNA polymerase activity in detergent disrupted virus and the activity of the purified enzyme with the isolated virus genome 70S RNA were inhibited by PF. However, higher concentrations of PF were needed to inhibit the endogenous reaction. The inhibition by PF appeared to be reversible and noncompetitive with respect to the substrate deoxythymidine triphosphate (dTTP). Addition of PF after the initiation of DNA synthesis immediately arrested the reaction.

Animals↗

PubMed1990

Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases.

Four flavonoids, 5,6,7-trihydroxyflavone (baicalein), 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,3',4',5,6,7-hexahydroxyflavone (quercetagetin) and 3,3',4',5,5',7-hexahydroxyflavone (myricetin), were found to be potent inhibitors of reverse transcriptases from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV). Under the reaction conditions employed, any one of these flavonoids almost completely inhibited the activity of RLV reverse transcriptase at a concentration of 1 microgram/ml. HIV reverse transcriptase was inhibited by 100%, 100%, 90% and 70% in the presence of 2 micrograms/ml quercetin, myricetin, quercetagetin and baicalein, respectively. The mode of inhibition of these flavonoids was competitive (RLV reverse transcriptase) or partially competitive (HIV reverse transcriptase) with respect to the template.primer complex, (rA)n.(dT), and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values for RLV reverse transcriptase were found to be 0.37 microM and 0.08 microM for baicalein and quercetin, respectively and those for HIV reverse transcriptase were 2.52 microM, 0.52 microM, 0.46 microM and 0.08 microM for baicalein, quercetin, quercetagetin and myricetin, respectively. Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones and flavanones) carried out to clarify the structure/activity relationships, revealed that the presence of both the unsaturated double bond between positions 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, (i.e. baicalein) were a prerequisite for the inhibition of reverse transcriptase activity. Removal of the 6-hydroxyl group of baicalein required the introduction of three additional hydroxyl groups at positions 3, 3' and 4' (quercetin), to afford a compound still capable of inhibiting the reverse transcriptase activity. Quercetagetin which contains the structures of both baicalein and quercetin, and myricetin which has the structure of quercetin with an additional hydroxyl group on the 5' position also proved strong inhibitors of reverse transcriptase activity. The inhibition by baicalein of reverse transcriptase is highly specific, whereas quercetin and quercetagetin were also strong inhibitors of DNA polymerase beta and DNA polymerase I, respectively. Myricetin was also a potent inhibitor of both DNA polymerase alpha and DNA polymerase I.

Animals↗

PubMed1975

Specific binding of tryptophan transfer RNA to avian myeloblastosis virus RNA-dependent DNA polymerase (reverse transcriptase).

The ability of tryptophan tRNA (tRNATrp) to initiate reverse transcription of the 70S RNA of avian RNA tumor viruses suggested that the reverse transcriptase (RNA-dependent DNA polymerase; deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase; EC 2.7.7.7) might have a specific binding site for the tRNA. A complex of tRNATrp and the avian myeloblastosis virus reverse transcriptase has been demonstrated using chromatography on Sephadex G-100 columns. Of all the chicken tRNAs, only tRNATrp and a tRNA4Met bind to the enzyme with high enough affinity to be selected from a mixture of the chicken cell tRNAs. The ability of tRNATrp to change the sedimentation rate of the enzyme indicates that tRNATrp is not binding to a contaminant in the enzyme preparation. Treatment of the enzyme with monospecific antibody to reverse transcriptase prevented binding of tRNA as well as inhibited the DNA polymerase activity of the enzyme. The ability of reverse transcriptase to utilize tRNATrp aa a primer for DNA synthesis, therefore, appears to involve a highly specific site on the enzyme.

Animals↗

PubMed1987

Inhibition of viral reverse transcriptase by 2',5'-oligoadenylates.

Viral reverse transcriptase activity was inhibited in a concentration dependent manner by 2',5'-oligoadenylate. Kinetically this inhibition was of a mixed type where 2',5'-oligoadenylate was not strictly competitive with dTTP. The potency of inhibition was more marked in the absence than in the presence of sulfhydryl agents. 2',5'-oligoadenylate had no effect on DNA-dependent E. coli DNA polymerase and was much less active against mammalian DNA polymerases. This is the first report of reverse transcriptase inhibition by an inducible constitutive natural ligand.

Adenine Nucleotides↗