DNA polymerases from RNA tumor viruses and human cells: inhibition by polyuridylic acid.

Polyuridylic acid inhibited DNA polymerases purified from three species of oncornaviruses as well as three out of seven DNA polymerases purified from cells. Viral and cellular DNA polymerases could not be distinguished by polyuridylic acid inhibition, but were easily distinguished by their template preferences in the presence of magnesium.

Effects of ribonuclease and deoxyribonuclease on a murine leukemia virus (Rauscher).

Plasma pellets and femoral bone marrow from BALB/cJ mice infected with the Rauscher leukemia virus were fixed, embedded, and sectioned. The thin sections were incubated in ribonuclease and deoxyribonuclease solutions, stained, and examined in the electron microscope. Specific attention was paid to the action of the nucleases on characteristic virus particles in the plasma preparations and on viruses being produced by the "budding" phenomenon in the femoral bone marrow. Ribonuclease solutions digested the nucleoids of the virus particles in the plasma preparations from mice infected with Rauscher leukemia virus. The nucleoid portion of the "budding" virus particles in bone marrow, and the connecting cytoplasm of the stalks were also digested by ribonuclease solutions. In addition, the outer coat of the "budding" particles was affected in a nonspecific manner. The centers of the "budding" particles in the bone marrow and the nucleoids of viruses in plasma preparations were not digested by deoxyribonuclease solutions. Influenza virus, a known ribonucleic acid (RNA) virus, was used as a control for nuclease activity. The nucleoids of influenza virus particles were digested by ribonuclease but not by deoxyribonuclease solutions. After coriphosphine staining of the plasma virus preparations, the fluorescence was quenched in preparations treated with ribonuclease, but did not appear to be diminished in those treated with deoxyribonuclease. This study suggests that infection of mice with Rauscher leukemia virus produces virus particles in plasma and "budding" particles in bone marrow, both of which contain RNA.

Effects of streptovaricins and their degradation products on RNA-directed DNA polymerase of Rauscher leukemia virus.

The activities of streptovaricin complexes, streptovaricins, streptovals, and streptovarinic degradation products were elevated against RNA-directed DNA polymerases of Rauscher leukemia virus, DNA-dependent DNA polymerase of bacterial and mammalian cells, and DNA-dependent RNA polymerases of mammalian origin. The activities of streptovaricins were also listed for comparison purposes. The effects of streptovaricin complexes on viral DNA polymerases varied significantly from lot to lot, and streptovaricin complex lot 7 was the most active. All the streptovals and streptovaricin degradation products except varicinal A showed a marked improvement (twofold to tenfold) in activity against the viral enzyme over the parent streptovaricins. None of these compounds, however, displayed any significant effect on either the DNA polymerase of L1210 leukemia cells and Escherichia coli or the RNA polymerase of isolated nuclei of mouse liver. As a result of tests in these systems, some specific inhibitors of RNA-directed DNA polymerases of Rauscher leukemia virus were selected.

Effects of streptovaricins and their degradation products on infectivity of Rauscher leukemia virus.

The virucidal effects of streptovaricin (Sv) A, SvC, SvD, streptoval (Sval) C, Sval Fc, and streptovarone were evaluated by incubation of the drug with Rauscher leukemia virus (RLV) at 37 degrees C for 60 minutes prior to dillution and addition to cells (in vitro assay) or before ip injection into animals (in vivo assay). The in vitro and in vivo assays were plaque formation and splenomegaly, respectively. A dose-related effect was observed with all six compounds with the in vitro assay. On an equimolar basis, the Sv degradation products, i.e., Sval C, Sval Fc, and streptovarone were most inhibitory, followed by SvD; SvA and SvC were least active. At 0.0625 mumoles, the three Sv degradation products inactivated over 90% of the RLV. Similar results were obtained through the in vivo assay. At 0.06 mumoles, streptovarone, Sval C, and SvD showed 78,62, and 29% inhibition of splenomegaly, respectively; SvA and SvC were essentially inactive. A direct relationship was observed between inhibition on RNA-directed DNA polymrase of RLV by these compounds and their virucidal effects. No drug given at the time of injection, however, showed any significant effect on virus infective processes in vitro or in vivo. The reason for the lack of therapeutic effects of these compounds is discussed.

Inhibitory effect of 1-beta-D-arabinofuranosylthymine 5'-triphosphate and 1-beta-D-arabinofuranosylcytosine 5'-triphosphate on DNA polymerases from murine cells and oncornavirus.

This report compares the effect of the newly synthesized 1-beta-D-arabinofuranosylthymine 5'-triphosphate with 1-beta-D-arabinofuranosylcytosine 5'-triphosphate on the activity of DNA polymerases from mouse cells and oncornavirus. 1-beta-D-Arabinofuranosylthymine 5'-triphosphate inhibited all the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. The mode of inhibition of 1-beta-D-arabinofuranosylthymine 5'-triphosphate as well as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate was competitive to the deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied with changes in the combination of the inhibitor, substrate(s), and enzyme species.

Inhibitory effects of 9-beta-D-arabinofuranosylguanine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate on DNA polymerases from murine cells and oncornavirus.

The effects of the newly synthesized compound 9-beta-D-arabinofuranosylguanine 5'-triphosphate (ara-GTP) on the activity of DNA polymerases from mouse cells and oncornavirus were compared with those of 9-beta-D-arabinofuranosyladenine 5'-triphosphate. Ara-GTP did not replace deoxyguanosine 5'- triphosphate as substrate for these DNA polymerases but inhibited the activities of DNA polymerase alpha, beta, and gamma and viral DNA polymerase. DNA polymerase alpha was more sensitive than DNA polymerases beta and gamma and viral DNA polymerase to inhibition by ara-GTP. The inhibitions by ara-GTP and 9-beta-D-arabinofuranosyladenine 5'-triphosphate were due to competition or partial competition 5'-triphosphate were due to competition or partial competition with deoxynucleoside triphosphate with the same base. The inhibition constant (Ki) and the mode of inhibition of nucleotide incorporation varied depending on the combination of inhibitor, substrate(s), and enzyme species.

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.

Isolation and partial characterization of the reverse transcriptases of Rauscher murine leukemia virus, simian sarcoma virus and RD-114 virus.

Reverse transcriptases were purified from Rauscher murine leukemia virus, Simian sarcoma virus and RD-114 virus by affinity chromatography using poly(rC)-Sepharose. Molecular weight determinations of the viral enzymes gave values of 80,000 for all three enzyme preparations. Two-dimensional tryptic peptide maps of the purified enzymes showed only a few spots in similar positions.

Thymidylate synthetase of in vitro chemically and virally transformed rat cells.

Thymidylate synthetase (EC 2.1.1) from in vitro 3-methylcholanthreneor Rauscher leukemia virus-transformed rat embryo cells was studied. The enzyme from control or transformed rat cells exhibited a Km for 2'-deoxyuridylic acid of 4.5 +/- 0.2 muM, but the transformants had a higher level of enzyme activity than did control cells. Titration of the enzyme with tritiated 5-fluoro2'-deoxyuridylic acid indicated that the increased enzyme activity in the transformants was due to a greater level of cellular enzyme. While the level of enzyme activity in control cells was dependent on both the availability of nutrients in the medium and cell density, the level of enzyme activity in the transformants appeared to depend only on the availability of nutrients.

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.

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.

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.

Suramin: a potent inhibitor of the reverse transcriptase of RNA tumor viruses.

Suramin--a well-known antitrypanosomal agent--was found to exert a strong inhibitory effect on the RNA-directed DNA polymerase (reverse transcriptase) activity of several oncornaviruses such as Moloney murine leukemia virus, murine Rauscher leukemia viruses, Moloney murine sarcoma virus and avian myeloblastosis virus. Inhibition of enzyme activity was obtained with both endogenous viral RNA and (A)n . oligo(dT) as the template-primer. Suramin effected a 50% inhibition of the reverse transcriptase activity of oncornaviruses at a concentration range of 0.1--1 microgram/ml. In this aspect it compared favorably to ethidium bromide, another trypanocide drug which is considered as one of the most powerful inhibitors of oncornaviral DNA polymerases. The inhibition of reverse transcriptase activity by suramin was competitive with the template-primer, (A)n . oligo(dT), suggesting that the drug may interact with the template-primer binding site of the enzyme.

The inhibition of Rauscher leukemia virus and avian myeloblastosis virus DNA polymerases by anthracycline compounds.

Studies by other investigators have shown that adriamycin and daunorubicin exhibit antitumor and antiviral activity. A possible antiviral mechanism for the anthracycline compounds is the potent inhibition of viral DNA polymerases. Five anthracycline compounds were tested against purified Rauscher leukemia virus and avian myeloblastosis virus DNA polymerases. All compounds were found to be potent inhibitors of viral DNA polymerase activity. Inhibition was found to be primarily due to the planar ring structure (daunomycinone) common to all of these compounds. The degree of inhibition was dependent on the templates used: activated DNA, synthetic hybrids, poly(rA).dT12-18 and poly(rC).dG12-18, and the synthetic copolymer, poly(DA-dT). Alteration of the group substituent on the planar ring affected the degree of viral DNA polymerase inhibition. The inhibitory effects by anthracycline compounds appear to be relatively specific for viral polymerases.

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.

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.

In vitro inhibition of viral DNA polymerase activity by litmomycin.

Litmomycin, an antibiotic isolated from Streptomyces litmogenes, is highly active against Gram-positive bacteria and possesses antitumor activity. It inhibited viral DNA polymerase activity in vitro. The amount of litmomycin required to cause 50% inhibition of enzyme activity was 80-100 mug (180-225 nmoles)/ml of reaction mixture. The enzyme inhibition was observed when polyriboadenylate--oligodeoxythymidylate, polydeoxyadenylate-oligodeoxythymidyate, polyribocytidylate-oligodeoxyguanylate, activated DNA, and 70 S RNA were used as templates. Reaction kinetics and the mechanism of enzyme inhibition are discussed. The results suggest that litmomycin interacts with the template primer and not with the enzyme protein to stop the polymerization process.

Revistin found by screening for inhibitors of reverse transcriptase of an oncogenic virus.

Revistin, a substance that strongly inhibits the reverse transcriptase activity of murine leukemia virus in our screening system, was obtained from a cultured broth of a soil streptomyces which was closely related to Streptomyces filipinensis. The assay method for the activity was based on the inhibition by a test material of the incorporation of 3H-dTMP into DNA synthesized by the reverse transcriptase of an oncogenic RNA virus. Crude revistin was isolated by serial procedures of salting out with ammonium sulfate and precipitation with cetylpyridinium chloride. The crude material showed neither antibacterial nor antifungal activity. It exhibited against splenomegaly in mice caused by Rauscher leukemia virus infection.

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.