Inhibition of the DNA Polymerase of Rauscher Leukemia Virus by Single-Stranded Polyribonucleotides

Abstract

The DNA polymerase of Rauscher murine leukemia virus is strongly and specifically inhibited by nontemplate, single-stranded polyribonucleotides with either the resident viral RNA, native calf-thymus DNA, or poly[d(A-T)l as templates. These inhibitory homo-polymers are apparently bound to the template site of the polymerase, since they interact competitively with the template. The strength of the inhibition depends on the particular homopolymer used: poly(U) > poly(G) >> poly(A) > poly(C). The Ki for poly(U) was 0.08 Mg/ml, which represents an apparent affinity six times greater than that observed for viral RNA. No such inhibition was observed with a highly purified DNA polymerase from mouse embryos or the Escherichia coli enzyme.There are now many reports of RNA-dependent DNA polymerases in oncogenic RNA viruses (1-8). Although similar activities have been detected in partially purified preparations from normal (9, 10) and leukemic (11-13) cells, a satisfactory assay for the specific determination of these enzymes in cell extracts has not been described. The required specificity can only be achieved when properties that are unique to the viral enzymes are clearly understood. The work described here concerns one possible unique property.The published findings of Spiegelman et al. (5) show that synthetic, single-stranded homopolyribonucleotides do not serve as templates for the DNA polymerase of the avian myeloblastosis virus. The present communication describes the effects of these homopolymers on the polymerization of deoxyribonucleotides by the enzyme of the Rauscher murine leukemia virus. The enzyme-polynucleotide interactions described here may provide a basis for a specific assay for viral polymerases. MATERIALS AND METHODS Virus purificationRauscher murine leukemia virus (RLV) was purified from two different sources. One source was a 10-fold mouse (strain CFW-S) plasma concentrate (lot number RPV-HL-68-4 of Hazleton Laboratories) that contained 5.48 log units (spleen enlarging)/ml. The viruses from this source were purified according to published methods (3). Rauscher virus from JLS-V5 tissue culture fluids was also purified by this procedure after an initial sedimentation at 78,000 X g. DNA polymerase preparationsCrude Rauscher virus DNA polymerase was prepared by detergent lysis of purified RLV at 0-4°C for 10 min in a solution containing 0.05 M Tris . HCl (pH 7.5), 0.1 M NaCl, 1 mM EDTA, 8 mM MgCl2, 4 mM dithiothreitol, 0.1% (v/v) Nonidet P-40, and 150-300 ,ug of viral protein per ml.DNA polymerase was repeatedly purified from 16-to 18-day-old BALB/c mouse embryos by a method similar to that described by Alberts et al. (14) for the purification of DNA polymerase I from Escherichia coli. ln the procedure, decapitated embryos were homogenized at 4°C in a pH 8.1 buffer containing 5 M NaCl-0.05 M MgCl2-5 mM 2-mercaptoethanol-0.02 M Tris HCl. A 105,000 X g supernatant was prepared from the homogenate after it had been subjected to sonication (Bronwill Biosonik with a 12-mm diameter probe at maximum o...

Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1

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Effects of Polyadenylic Acids on Functions of Murine RNA Tumor Viruses

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Effect of the Fv‐1 gene on leukemia virus in mouse cell heterokaryons

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