Inhibition of reverse transcriptase by polyvinyl sulfate (PVS).

The effect of the ribonuclease inhibitor polyvinyl sulfate on the activity of retroviral DNA polymerase and terminal c transferase was examined. This substance was found to be a potent inhibitor of these enzymes by virtue of competition between the sulfated sidechains of the molecule and the template primer for a site on the enzyme. The significance of these findings in relation to searching for reverse transcriptase is discussed.

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.

Quantitative structure--activity correlations of rifamycins as inhibitors of viral RNA-directed DNA polymerase and mammalian alpha and beta DNA polymerases.

Twenty-two 3-substituted rifamycins were tested for inhibition of mammalian alpha and beta DNA polymerase and viral RNA-dependent DNA polymerase ("reverse transcriptase"). Quantitative structure--activity relationships (QSAR) were formulated for the three systems. Inhibition is linearly dependent on the partition coefficient and is highly favored by the presence of bulky hydrazones or oximes. None of these agents proved to be a selective or specific inhibitor of reverse transcriptase. A correlation in terms of log P and (log P)2 was obtained from data on a more closely related set of analogues from a published study. For murine reverse transcriptase, log P0 = 5.1.

Phosphonoformate inhibits reverse transcriptase.

The new antiviral substance phosphonoformate (PFA) has been tested in a cell-free system for its effect on reverse transcriptases from an avian retrovirus (avian myeloblastosis virus, AMV) and from mammalian retroviruses (Rauscher leukaemia virus, RMuLV; bovine leukaemia virus; baboon endogenous virus; simian sarcoma virus; visna virus). The observed inhibitory effect of PFA has been compared with that of a structurally related substance, phosphonoacetate (PAA). Phosphonoformate, at a concentration of 100 microM, reduced the activities of all the above mentioned polymerases by 90% when (rA)n.(dT)10 was used as a template/primer. The dose-response curves for AMV and RMuLV polymerases primed with (rA)n.(dT)10 showed PFA to be a 1000-fold more active than PAA; the RMuLV polymerase activity was reduced to 50% after incubation with 0.7 microM-PFA and 0.7 mM-PAA, respectively. There was no difference in PFA inhibition of virus-associated and purified reverse transcriptase activity. Results with various synthetic templates showed that both the RNA- and the DNA-dependent polymerase activities of reverse transcriptase were inhibited by PFA. The endogenous polymerase activity of AMV was inhibited to 50% at 100 microM-PFA, while PAA had no effect. The PFA inhibition was dependent on whether Mg2+ or Mn2+ was used as divalent cation in the assay. Phosphonoformate arrested DNA synthesis immediately after being added to the assay system. The mechanism of inhibition of the AMV polymerase was non-competitive with respect to substrate and template and the apparent inhibition constants were 16 microM and 9 microM, respectively.

Inhibition of deoxyribonucleic acid polymerases from human cells and from simian sarcoma virus by pyran.

Pyrans are co-polymers of divinyl ether and maleic anhydride. Four pyrans of various molecular weights more potently inhibited terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) from a human cell line of acute lymphoblastic leukemia origin (Molt-4) than they did DNA polymerases alpha, beta and gamma from these cells and DNA polymerase from simian sarcoma virus. For example, the concentrations of one pyran required for 50% inhibition of terminal deoxynucleotidyltransferase, DNA polymerases alpha, beta and gamma and viral DNA polymerase were 0.9, 110, 125, 35 and 47 microgram/ml respectively. Quantitatively similar results were obtained with the other pyrans. Inhibition of these enzymes by pyran was dependent on the concentrations of both the bivalent cation and template/primer or initiator in assay mixtures, but not on the concentrations of the substrate (deoxyribonucleoside 5'-triphosphate), enzyme, or bovine serum albumin. These results suggested that pyran inhibited these enzymes by complexing bivalent cations, which caused a decreased affinity of template/primer or initiator for each enzyme and a decrease in enzyme activity.

Binding of tRNA to reverse transcriptase of RNA tumor viruses.

The interaction of tRNA with the reverse transcriptase (RNA-dependent DNA polymerase) of mammalian RNA viruses, such as Moloney murine leukemia virus and simian sarcoma virus, has been studied. Whereas the purified reverse transcriptase of mammalian viruses sedimented in glycerol gradients as a globular protein with a molecular weight of 70,000, after interaction with tRNA the enzyme cosedimented with a protein of 150,000 molecular weight. The twofold increase in molecular weight could be a result of either two reverse transcriptase molecules complexed with a tRNA or, alternatively, several tRNA molecules bound to a single enzyme polypeptide. The enzyme complexes were dissociated in part upon degradation of the tRNA moiety by pancreatic RNase A. The reverse transcriptase released from virions of Moloney murine leukemia virus, simian sarcoma virus, and avian myeloblastosis virus, by nonionic detergent, migrated faster on glycerol gradients than purified enzyme preparation. This phenomenon was probably due to complex formation between part of the virion enzyme and the tRNA, which is endogenous in virions. Addition of exogenous tRNA was needed, however, to quantitatively complex all the virion reverse transcriptase of Moloney murine leukemia virus and simian sarcoma viruses. The reverse transcriptase of Moloney murine leukemia virus did not show tRNA species specificity in the binding reaction when glycerol gradients were used for assay. Thus, several tRNA species of Escherichia coli, yeast, chicken, and rat origin were able to complex with the enzyme. The species specificity in the interaction between tRNA and avian myeloblastosis virus reverse transcriptase was also examined. We demonstrated that under our experimental conditions, this enzyme binds different tRNA species of E. coli and yeast as well as tRNA of chicken origin.

Inhibition of deoxynucleotide-polymerizing enzyme activities of human leukemia lymphoblasts and simian sarcoma virus by tilorone and thirteen of its analogs.

Tilorone, which is 2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one dihydrochloride, and 13 of its analogs inhibited human cellular DNA polymerases alpha and beta assayed with activated DNA as template and also cellular DNA polymerase gamma and DNA polymerase from simian sarcoma virus assayed with poly(A) (dT)12-18 as template. Terminal deoxynucleotidyltransferase (TdT), which has no template requirement, was not inhibited by any of the 14 compounds when d(A)12-18 or d(G)12-18 was used as initiator. Three compounds did not inhibit TdT assayed with activated DNA as initiator, but 11 compounds did, and these 11 compounds were generally less inhibitory to TdT than to the other DNA polymerases. The three compounds that did not inhibit TdT assayed with activated DNA but did inhibit the other DNA polymerases will be useful in the characterization of TdT activity. Modifications of the polycyclic ring structure of tilorone and the kinds of substituent groups attached to the ring structures influenced the degree of inhibition of all enzymes.

Differential synthesis of mammalian type C viral gene products in infected cells.

Radioimmunological techniques were applied to the quantitation of the translational products of the gag, pol, and env genes of mammalian type C viruses. Analysis of the viral proteins associated with simian sarcoma-associated virus (SSA V) and SSA V-infected cells revealed in each that the level of reverse transcriptase was less than 1% of that of the major viral structural protein, p30. The rate of intracellular degradation of reverse transcriptase in SSA V-infected cells was found to be no greater than that of several viral structural proteins, indicating that the lower levels of viral enzyme resulted from its decreased synthesis. By screening individual cells infected at limiting SSA V dilution, it was possible to isolate a clone (clone 16), which demonstrated levels of viral p12, p30, and gp70 similar to those found in wild-type SSA V-infected cells, and which released noninfectious virions in large quantity. The noninfectious virions and clone 16 cells were shown to lack immunologically or enzymologically detectable reverse transcriptase. With serial passage of clone 16 cells, reverse transcriptase activity became spontaneously detectable in tissue culture fluids, concomitant with the appearance of infectious virus. The reverse transcriptase associated with this virus was indistinguishable from SSA V polymerase, indicating that the genetic alteration restricting SSA V pol gene expression in clone 16 cells was reversible. These results further demonstrate the strict requirement of reverse transcriptase for establishment of type C virus infection. Possible mechanisms to account for the patterns of type C viral gene expression detected in SSA V-infected cells are discussed.

Interruption of oncornavirus replication by modified rifamycin antibiotics.

Thirteen rifamycin SV derivatives containing 3'-alkylaminomethyl substituents fail to inhibit the activities of the simian sarcoma virus Type 1 DNA polymerase, and of cellular DNA, RNA, and poly(A) polymerases prepared from NIH Swiss mouse embryos. These compounds show a range in their toxicities for NIH Swiss mouse 3T3 cells and in their capacities to inhibit production of foci of morphologically altered cells by murine sarcoma virus (MSV). Three compounds--the N-methyl-N-hydroxyethylaminomethyl, the N,N-dimethyl-aminomethyl, and the N4-methylpiperazinomethyl rifamycin derivatives--are comparable to adenine arabinoside and ribavirin in their toxicity for 3T3 cells, but these compounds show superior focus inhibition. These compounds inhibit oncornavirus production apparently by exacerbation of a delay in growth that results from infection of 3T3 cells with MSV.

Replication of primate oncornavirus SSV-1 on MRC-5 human diploid cells.

MRC-5 human diploid cells infected with Simian Sarcoma Virus from woolly monkey (SSV-1) were not transformed but an efficient replication of non transforming SiLV was demonstrated. Increase of virus reverse transcriptase activity paralleled cell replication during successive passages. Preliminary results concerning the influence of viral infection on the life span and the karyotype of MRC-5 diploid cells will be reported and several implications of these findings discussed.