Development of an improved product enhanced reverse transcriptase assay.

A PCR based reverse transcriptase (RT) assay was developed that has 10(4)-fold higher sensitivity than conventional nucleotide incorporation assays and allows discrimination between false positive results generated by cellular polymerases and positives resulting from authentic RT activity. Recently, several reverse transcriptase (RT) assays have been developed where a reverse transcriptase reaction is performed on an RNA template/DNA primer combination. A specific region of the cDNA product is then amplified by the polymerase chain reaction to increase the sensitivity of cDNA detection. These reverse transcriptase assays up to 10(6)-fold more sensitive at detecting retroviruses than conventional methods. The drawback to these assays with increased sensitivity is the increased incidence of false positive results generated by cellular polymerases that can reverse transcribe. The MS2 bacteriophage RNA template and primers from one of the recently developed assays were used as the basis to develop the assay. A simple high resolution agarose gel was used as the endpoint for the assay without compromising sensitivity. In addition, the pH of the RT reaction was lowered to pH 5.5, the RT incubation was 1 h, and protease inhibitors were added to the RT reaction components. These modifications yield an assay that can discriminate between authentic RT activity and contaminating cellular polymerases.

Comparative kinetic analyses of interaction of inhibitors with Rauscher murine leukemia virus and human immunodeficiency virus reverse transcriptases.

The inhibitory effects of several nucleoside triphosphate analogs on Rauscher murine leukemia virus (RMuLV) and human immunodeficiency virus (HIV) type 1 reverse transcriptases (RTs) were studied. With RNA as the template, the apparent K(m) and apparent K(i) values of HIV RT toward its substrates and inhibitors are 12 to 500 times lower than the corresponding values for RMuLV RT. However, the k(i)/k(m) ratios (inhibition efficiencies) for HIV and RMuLV RTs'are similar for AZTTP (zidovudine triphosphate), d4TTP [3'-deoxythymidine-2'-ene-(3'-deoxy-2',3'-didehydrothymidine) triphosphate], PMEADP [9-(2-phosphonylmethoxyethyl)adenine diphosphate], FIAUTP [1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-iodouracil triphosphate], and HPMPCDP [(S)-1-(3-hydroxy-2-phosphylmethoxypropyl) cytosine diphosphate]. With DNA as the template, the K(m) values are similar for HIV and RMuLV RTs. However, the K(i)/K(m) values of HIV and RMuLV RTs are significantly different for ddCTP, ddATP, and 3TCTP (2',3'-dideoxy-3'-thiacytidine). The RTs of RMuLV and HIV are sufficiently different from one another that the kinetic inhibition constants for a particular antiviral compounds should be determined to indicate whether anti-RMuLV activity is likely to be predictive for the anti-HIV activity of the compound. This information, in conjunction with species-specific drug metabolism differences and tissue culture antiviral activity, is important in determining the suitability of a particular animal model.

Inhibition of mouse retroviral disease by bioactive glutaminase-asparaginase.

Glutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viraemic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compared favourably with those of azidothymidine given intraperitoneally at 30 mg/kg/day. Western blots performed on splenic tissue from control and treated animals indicated that glutamine depletion prevented readthrough of an amber codon at the gag-pol junction, stopping translation of viral mRNA at that point. Treatment of RLV-infected animals with PGA resulted in nearly a 200% increase in mean survival time even when therapy was initiated late in the course of the disease. To our knowledge, this is the first demonstration that a nutrient required for viral replication can be enzymically depleted in vivo to inhibit viral replication.

Differential inhibition of reverse transcriptase and various DNA polymerases by digallic acid and its derivatives.

Digallic acid (gallic acid 5,6-dihydroxy-3-carboxyphenyl ester) [4] was found to be a potent inhibitor of the activities of the reverse transcriptases from murine leukemia virus (MLV) and human immunodeficiency virus (HIV). Under the reaction conditions specified for each of MLV and HIV reverse transcriptases, both enzymes were inhibited by approximately 90% in the presence of 0.5 micrograms/ml digallic acid. Under the same conditions, however, gallic acid had no effect on the reverse transcriptase activity. The mode of the inhibition by digallic acid was partially competitive with respect to the template.primer, (rA)n.(dT)12-18', and noncompetitive to the triphosphate substrate, dTTP. The Ki value of digallic acid for HIV-reverse transcriptase was determined to be 0.58 microM. Examination of several derivatives of digallic acid have shown that all three hydroxyl groups at the 3, 4, and 5 positions seem to be required for the inhibitory activity of these compounds. Besides reverse transcriptase, DNA polymerases alpha and beta were moderately inhibited by digallic acid, whereas DNA polymerase gamma, terminal deoxynucleotidyltransferase, and E. coli DNA polymerase I were virtually insensitive to inhibition by this compound.

Differential inhibition of the activities of reverse transcriptase and various cellular DNA polymerases by a traditional Kampo drug, sho-saiko-to.

A traditional Kampo drug, Sho-saiko-to, composed of several herb extracts, differentially inhibited the activities of reverse transcriptase and human cellular DNA polymerase alpha and beta. Reverse transcriptases from murine leukemia virus and human immunodeficiency virus were inhibited by over 80% and 50%, respectively, in the presence of 100 micrograms/ml Sho-saiko-to, whereas DNA polymerase alpha was much less sensitive to inhibition by this drug than were the reverse transcriptases. DNA polymerase gamma was not inhibited by this drug at concentrations of up to 500 micrograms/ml. Only DNA polymerase beta was moderately inhibited by Sho-saiko-to. Thus, it has been shown that the inhibition by Sho-saiko-to is relatively specific for reverse transcriptase and that the drug contains as yet unidentified inhibitory substance(s) for reverse transcriptase.

Inhibition of reverse transcriptase activity by a flavonoid compound, 5,6,7-trihydroxyflavone.

5,6,7-Trihydroxyflavone (baicalein) is a potent inhibitor of the activities of reverse transcriptases from murine leukemia viruses (MLV) (Rauscher and Moloney strains) and human immunodeficiency virus (HIV). Under the reaction conditions specified for each of the MLV- and HIV-reverse transcriptases, both enzyme activities were inhibited by more than 90% in the presence of 2 micrograms/ml baicalein. The mode of the inhibition by baicalein was competitive with respect to the template.primer, (rA)n.(dT)12-18, and noncompetitive to dTTP substrate. Ki value of baicalein for the MLV-reverse transcriptase was determined to be 0.37 microM.

Hemin inhibits virion-associated reverse transcriptase of murine leukemia virus.

The virion-associated reverse transcriptase activity of Rauscher murine leukemia virus was inhibited by freshly prepared hemin at a concentration of 10(-4) M. When the hemin solution was aged at room temperature for 5 days, the concentration of 50% inhibition decreased to as low as 10(-7) M. Removal of O2 from the solution partially prevented the aging. The hemin inhibition was reversible and appears to be directed against the enzyme rather than the template. Hemin did not inhibit the activity of reverse transcriptase purified from avian myeloblastosis virus.

Polyamines antagonize both the antileukemic activity and the reverse transcriptase stimulatory activity of 4,4'-diacetyldiphenylurea bis(guanylhydrazone) (DDUG).

(Diacetyldiphenylurea)bis(guanylhydrazone) (DDUG) functions as a cationic trypanocide antagonized in vivo by exogenous concomitant addition of the biologically active polyamine, spermine. It also inhibits the DNA polymerases of L1210 murine leukemia cells. We have found that DDUG stimulates Rauscher murine leukemia virus DNA polymerase activity in a manner similar to polyamines. Such stimulation does not occur if DNA synthesis is carried out on spermine + activated DNA complexes. We also show that the in vivo antileukemic activity of DDUG in the L1210 ascites mouse model is antagonized by biologically active polyamines. These studies suggest a new intracellular target for the antileukemic activity of DDUG: interference with polyamine function.

Inhibition of reverse transcriptase activity by 2',3'-dideoxythymidine 5'-triphosphate and its derivatives modified on the 3' position.

Inhibitory effects of 2',3'-dideoxythymidine 5'-triphosphate (ddTTP) and its three derivatives modified on the 3' position of ribose moiety [3'-azido-2',3'-dideoxythymidine 5'-triphosphate (3'-N3-ddTTP), 3'-amino-2',3'-dideoxythymidine 5'-triphosphate (3'-NH2-ddTTP) and 2'-deoxyxylo-furanosylthymine 5'-triphosphate (dXTP)] on the activity of the reverse transcriptase purified from Rauscher murine leukemia virus were examined and compared with each other. When (rA)n X (dT)12-18 was used as the template X primer in the presence of manganese ion, all these compounds except 3'-NH2-ddTTP inhibited the reverse transcriptase activity in competitive fashion with respect to the dTTP substrate. The inhibition potentials of these compounds are ordered as follows: 3'-N3-ddTTP (Ki = 1.8 microM) greater than ddTTP (Ki = 9.3 microM) greater than dXTP (Ki = 16.3 microM), and the Ki values of these inhibitors are smaller than the Km of dTTP (30 microM). The observed inhibitions were mainly due to competition between the dTTP substrate and inhibitor rather than chain-termination of the elongating DNAs caused by incorporation of these dideoxy compounds.

The effect of murine interferon-alpha/beta on an established Rauscher murine leukemia virus-induced erythroleukemia in BALB/c mice.

Rauscher murine leukemia virus (R-MuLV) induces a rapidly developing erythroleukemia in BALB/c mice. Previously, we have shown that mouse interferon-alpha/beta (Mu IFN-alpha/beta) applied shortly after virus inoculation efficiently inhibits the leukemic process (Hekman et al., 1981). Here we describe the effect of Mu IFN-alpha/beta on an established leukemia. Varying doses of Mu IFN-alpha/beta were injected over 3 days, starting 8 to 12 days after virus inoculation. The effect of Mu IFN-alpha/beta on the leukemic process was monitored by measuring the spleen weight, reverse transcriptase activity in the serum and, in selected experiments, by microscopic examination of sections of the spleen using standard histological and immunological staining techniques. Depending on the spleen weight at the start of its application (maximal about 450 mg), Mu IFN-alpha/beta caused a dramatic reduction in the number of virus-infected erythroleukemic cells in the spleen. Also, R-MuLV disappeared from the serum within 3 days. If Mu IFN-alpha/beta was injected into R-MuLV-infected mice with an already 10-fold enlarged spleen, it could only stop further development of leukemia. Results obtained with crude Mu IFN-alpha/beta preparations were confirmed with absolutely pure Mu IFN-beta.

Stimulation of Rauscher leukemia virus DNA polymerase DNA-directed DNA synthesis by cationic trypanocides and polyamines.

Activated DNA-directed DNA synthesis catalyzed by Rauscher leukemia virus (RLV) and other type C mammalian retroviral DNA polymerases is uniquely stimulated by biologically active polyamines. Cationic trypanocides may act as antagonists of polyamine function. As described here, several cationic trypanocides stimulate RLV polymerase-catalyzed DNA-directed DNA synthesis at concentrations significantly inhibiting eukaryotic DNA polymerases. Such stimulation is negated by polyamines. Kinetic analysis of the stimulation of RLV DNA polymerase by three structurally dissimilar cationic trypanocides (Antrycide, Burroughs-Wellcome Compound 64A, and Bayer Compound 1694) suggests that such stimulation is, in part, due to a drug:DNA structural interaction resembling the polyamine:DNA structural complex recognized by the RLV DNA polymerase.

Characterization of a p30 fraction from Rauscher leukemia virus which has an associated ATPase activity.

The p30 antigen from Rauscher leukemia virus (R-MuLV) was separated into two fractions by chromatography on either phosphocellulose or DEAE-cellulose. The p30-I and p30-II were indistinguishable immunologically or by isoelectrofocusing and gel electrophoresis. An ATPase activity was tightly associated with p30-II that could not be separated by ion-exchange chromatography, isoelectrofocusing, or glycerol velocity gradient sedimentation. The ATPase hydrolyzed the gamma phosphate from only ATP or dATP. Immunoglobulin directed against R-MuLV p30 completely inhibited the p30-II associated ATPase. Glycerol velocity gradient analysis showed that p30-I sedimented as a 30-kDa species while the p30-II and its associated ATPase sedimented as a 60-kDa species. The p30-II was converted entirely to a 30-kDa form by treatment with 0.2% (w/v) lithium dodecyl sulfate, suggesting that it represented a complexed species of p30. Finally, p30-II was found to stimulate the activity of R-MuLV reverse transcriptase, but p30-I had no effect on the activity of the enzyme. These results suggested the existence of at least two different forms of p30 in R-MuLV.

[Isolation of reverse transcriptase from the avian myeloblastosis virus in preparative quantities]. / Vydelenie obratnoi transkriptazy virusa mieloblastoza ptits v preparativnykh kolichestvakh.

Inverse transcriptase of bird myeloblastosis virus is a unique instrument for artificial synthesis of structural genes of viruses, plants, animals. Methods for the virus production in preparative amounts are developed due to selection of the corresponding line of chickens, conditions of their maintenance, diet infection methods and myeloblastosis diagnostics. Main demands to the inverse transcriptase preparations (their high activity, absence of nuclease impurities, high concentration of the enzyme preparation solutions and their stability in storage) are ensured by zonal centrifugation purification of the virus in a sucrose density gradient, described methods of inverse transcriptase isolation and purification as well as conditions of its storage.

Optimal conditions for detection of reverse transcriptase activity in human placentas.

Optimal conditions for detecting reverse transcriptase activity in human placental extracts are described. They vary with the state of the placenta at birth and are influenced by relative amounts of detergent, monovalent cation, and protein in the reaction mixture. Demonstrating activity of the placental enzyme requires detergent, but the enzyme is sensitive to high detergent concentrations. This sensitivity can be be altered by lowering the monovalent cation concentration from 0.154 to 0.034 M and by adding protein to the reaction mixture. The detection of reverse transcriptase in Rauscher murine leukemia virus and baboon endogenous type C virus, but not in the Mason-Pfizer monkey type D virus, shows similar requirements.

Purification of a specific inhibitor of reverse transcriptase from human placenta.

Human placental extracts contain a factor which specifically and reversibly inhibits the reverse transcriptase of mammalian retroviruses. This placental inhibitor has been partially purified and characterized. It elutes at 0.1-0.2 M phosphate on hydroxyapatite chromatography and can be further purified by phosphocellulose chromatography where it elutes at 0.4 M KCl. By these purification procedures, specific activities of 40-70,000 units of inhibitor per mg of protein were obtained. The size of the inhibitor is about 60-65,000 daltons as estimated by velocity sedimentation. The inhibitor purified by these techniques selectively inhibits the activity of purified reverse transcriptase from Rauscher murine leukemia virus and baboon endogenous virus. It is substantially less active against the reverse transcriptase of avian myeloblastosis virus. The specificity of this inhibitor for mammalian enzymes and particularly for the human placental reverse transcriptase suggests that it plays a role in the regulation of DNA synthesis in human placental development.

Effects of exogenous polyamine and trypanocides on the DNA polymerase activities from Trypanosoma brucei brucei, mouse thymus and murine leukemia virus.

The effects of exogenously added spermine on activated (gapped) DNA-directed and poly(dC) . (dG)12-18-directed DNA synthesis were tested on the chromatographically separated DNA polymerase activities of Trypanosoma brucei brucei. Activated DNA-directed DNA synthesis by the Peak I (eluting from DNA-agarose at 0.15 M KCl) and Peak II (eluting at 0.3 M KCl) polymerase was consistently inhibited or stimulated, respectively, by exogenous spermine. Kinetic analysis revealed that inhibition of the Peak I enzyme with respect to template DNA occurred by a mixed mechanism, while a major factor in the stimulation of the Peak II enzyme by spermine appeared to be the polyamine-mediated reversal of "substrate inhibition' by DNA at concentrations above 10 micrograms/ml. The apparent Km values of Peak I and Peak II DNA polymerase for activated DNA were determined to be 5 and 0.5 microgram/ml, respectively. In contrast to the results observed with activated DNA, activation of Peak II-enzyme-catalyzed poly(dC)-directed DNA synthesis was similar at all template-primer concentrations. Peak I enzyme-catalyzed poly(dG) synthesis was either inhibited or slightly stimulated by spermine, depending upon the presence or absence of heteropolymeric DNA, respectively. Dose-dependent inhibition of DNA-directed DNA synthesis catalyzed by T. b. brucei DNA polymerases, murine thymus DNA polymerase alpha, and Rauscher murine leukemia virus reverse transcriptase by trypanocides was examined to determine a possible mechanism of selective toxicity by such agents. The drugs Antrycide (quinapyramine), pentamidine, imidocarb, Berenil (diminazene aceturate), WR-199-385-[2,5-bis(4-guanylphenyl)furan . 2HCl] and isometamidium inhibited DNA polymerases of the eucaryotic cells at approximately the same degree, and at similar concentrations. The presence of spermine in reaction mixtures did not spare any drug inhibition. Stimulation of reverse transcriptase activity was observed in the presence of Antrycide and imidocarb, however, this could be negated by stimulatory amounts of spermine present in the reaction mixture. The results, obtained using an activated DNA-directed assay system, suggest that trypanosomal DNA polymerases are not the selective target of trypanocidal drugs currently available.

Inhibition of RNA dependent DNA polymerases by anthracycline antibiotics.

In our experimental work we intended to study the effect of certain anthracycline antibiotics, like Adriamycin, Daunomycin, Carminomycin on the RNA dependent DNA polymerase, i.e. reverse transcriptase (RT) system. Over the direct effect on the RT our aim was to find out the rate of selectivity of above antibiotics on the RT. For doing this we compared the above effects to those found on natural nucleic acid polymerases. These experiments were confirmed using synthetic polynucleotid template poly(rA)n(dT)12-18 for the Rauscher RT enzyme and poly(dA)n . poly(dT)n for E. coli DNA polymerase I. In our work we have shown that Carminomycin, in contrast to Adriamycin and Daunomycin, possesses a highly specific inhibitory effect on the RT enzyme system.

Divalent cation-dependent pyridoxal 5'-phosphate inhibition of Rauscher leukemia virus DNA polymerase: characterization and mechanism of action.

We have shown that pyridoxal 5'-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn2+, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.

Polyamines stimulate DNA-directed DNA synthesis catalyzed by mammalian type C retroviral DNA polymerases.

In the presence of optimal concentrations of Mg2+, rates of activated (gapped) DNA-directed DNA synthesis by purified mammalian type C retroviral DNA polymerases are stimulated greater than 10-fold by the polyamines spermine and spermidine. Such stimulation was not observed using either similar concentrations of the polyamines cadaverine or putrescine or exogenously provided salt or ammonium ions. Avian type C as well as mammalian type B and type D retroviral DNA polymerases, in contrast to the mammalian type C enzyme, were found to be relatively insensitive to spermine and spermidine stimulation. Kinetic analysis of the polyamine stimulation of activated DNA-directed DNA synthesis carried out using spermine and purified Rauscher leukemia virus DNA polymerase revealed at least two distinct mechanisms of activation of DNA synthesis. 1) At DNA concentrations below 2.5 micrograms/ml, spermine appears to interact with the enzyme-DNA complex in order to stimulate synthesis. 2) At DNA concentrations above 2.5 micrograms/ml, increased spermine stimulation is observed which appears to be due to its direct interaction with the activated DNA template resulting in either selective limitation of the formation of "dead-end" enzyme-DNA complexes or its ability to convert such nonproductive enzyme binding sites into productive sites for the initiation of synthetic activity. The addition of spermine to reaction mixtures was found to increase both the apparent Km and Vmax of the activated (gapped) DNA-directed reaction with regard to template concentration.