DNA Polymerase of Murine Sarcoma-Leukemia Virus: Lack of Detectable RNase H and Low Activity With Viral RNA and Natural DNA Templates

Kirsten murine sarcoma-leukemia virus (Ki-MSV[MLV]) was found to contain less RNase H per unit of viral DNA polymerase than avian Rous sarcoma virus (RSV). Upon purification by chromatography on Sephadex G-200 and subsequent glycerol gradient sedimentation the avian DNA polymerase was obtained in association with a constant amount of RNase H. By contrast, equally purified DNA polymerase of Ki-MSV(MLV) and Moloney [Mo-MSV(MLV)] lacked detectable RNase H if assayed with two homopolymer and phage fd DNA-RNA hybrids as substrates. On the basis of picomoles of nucleotides turned over, the ratio of RNase H to purified avian DNA polymerase was 1:20 and that of RNase H to purified murine DNA polymerase ranged between <1:2,800 and 5,000. Based on the same activity with poly (A).oligo(dT) the activity of the murine DNA polymerase was 6 to 60 times lower than that of the avian enzyme with denatured salmon DNA template or with avian or murine viral RNA templates assayed under various conditions (native, heat-dissociated, with or without oligo(dT) and oligo(dC) and at different template enzyme ratios). The template activities of Ki-MSV(MLV) RNA and RSV RNA were enhanced uniformly by oligo(dT) but oligo(dC) was much less efficient in enhancing the activity of MSV(MLV) RNA than that of RSV RNA. It was concluded that the purified DNA polymerase of Ki-MSV(MLV) differs from that of Rous sarcoma virus in its lack of detectable RNase H and in its low capacity to transcribe viral RNA and denatured salmon DNA. Some aspects of these results are discussed.     

Characterization of endogenous RNA-directed DNA polymerase activity of reticuloendotheliosis viruses.

Reticuloendotheliosis viruses (REV) contain an endogenous RNA-directed DNA polymerase activity. The endogenous DNA polymerase activity can be elicited in purified preparations of REV by treatment with nonionic detergents. The enzyme activity has a strong preference for manganous ions. Therefore, appreciable endogenous DNA polymerase activity can be demonstrated only if the reaction mixture contains appropriate concentrations of manganous ions. Enzyme activity can be inhibited by pretreatment with RNase or deletion of one or more deoxyribonucleoside triphosphates from the reaction mixture. In contrast, actinomycin D has little effect in initial DNA synthesis. The results from both velocity and equilibrium centrifugation indicate that the nascent chains of product DNA are associated with 60S viral RNA. The DNA product of the endogenous DNA polymerase reaction is hybridizable to REV RNA, but not to avian leukosis virus RNA.     

Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

Mechanistic Independence of Avian Myeloblastosis Virus DNA Polymerase and Ribonuclease H

Differential inhibition conditions were established for the DNA polymerase and RNase H activities of avian myeloblastosis virus (AMV) with ether-disrupted AMV and a purified enzyme preparation. The RNase H activity of ether-disrupted AMV with (rA)(n).(dT)(n) and (rA)(n).(dT)(11) as substrates was inhibited 80 to 100% by preincubation with NaF at a final reaction concentration of 27 to 30 mM. Under these conditions, the DNA polymerase activity was inhibited only 0 to 20%. Similar inhibitions were found with exogenous Rous sarcoma virus 35S and 70S RNA.DNA hybrid and phiX174 DNA.RNA hybrid as substrates. Studies were also performed with a purified enzyme preparation, in which the two activities essentially co-purified. The RNase H activity was inhibited >80% by 150 mM KCl with three different hybrid substrates, whereas the DNA polymerase activity was uninhibited. The DNA polymerase was completely inactivated by heat denaturation at 41 C or by omission of the deoxytriphosphates from the reaction mixture; the RNase H remained active. These differential inhibition conditions were used to compare the size of the DNA product synthesized with and without simultaneous RNase H action and to examine the effect of inhibition of the DNA polymerase on the size of the RNase H products. The size of the products of one activity was not affected by inhibition of the other activity. These results suggest that the AMV DNA polymerase and RNase H are not coupled mechanistically.     

In vitro transcription of 70S RNA by the RNA-directed DNA polymerase of Rouse sarcoma virus: lack of influence of RNase H.

The influence of Rous sarcoma virus (RSV)-associated RNase H on the in vitro synthesis of DNA by the RSV RNA-directed DNA polymerase was determined under conditions whereby RNase H activity was selectively inhibited with NaF. Not only were we unable to detect any effect on the size, structure, or genetic complixity of the DNA product synthesized in the absence of RNase H activity, but the displacement of DNA from the 70S RNA:DNA hybrid structures was also unaffected. The suitability of 70S RNA:DNA hybrid structures synthesized in vitro to serve as a substrate for RNase H is discussed.     

RNase H and RNA-directed DNA polymerase: associated enzymatic activities of murine mammary tumor virus.

The RNA-directed DNA polymerase of murine mammary tumor virus, a type B RNA tumor virus, was purified sequentially through DEAE-cellulose, phosphocellulose (step gradient), and phosphocellulose (linear salt gradient) chromatography followed by glycerol sedimentation centrifugation. During all stages of purification, coincident peaks of RNA-directed DNA polymerase activity, templated by polyribocytidylate-oligodeoxyguanidylate, and RNase H digestion of [3H]polyriboadenylate-polydeoxythymidylate were observed, and both enzymatic activities displayed a cation preference for magnesium. Under conditions that removed adventitiously associated nucleases, RNase H activity was found to co-purify with polymerase. The specificity of this nuclease was assayed with various prepared substrates, which indicated that the polymerase-associated RNase H activity was directed only against the RNA strand of an RNA-DNA hybrid. It is highly probable that RNase H (RNA-DNA hybrid: ribonucleotide-hydrolase, EC 3.1.4..34) and RNA-directed DNA polymerase of type B viruses are associated enzymatic activities analogous to those observed for avian and mammalian type C RNA tumor viruses.     

In Vitro Transcription of 70S RNA by the RNA-Directed DNA Polymerase of Rous Sarcoma Virus: Lack of Influence of RNase H

The influence of Rous sarcoma virus (RSV)-associated RNase H on the in vitro synthesis of DNA by the RSV RNA-directed DNA polymerase was determined under conditions whereby RNase H activity was selectively inhibited with NaF. Not only were we unable to detect any effect on the size, structure, or genetic complexity of the DNA product synthesized in the absence of RNase H activity, but the displacement of DNA from the 70S RNA:DNA hybrid structures was also unaffected. The suitability of 70S RNA:DNA hybrid structures synthesized in vitro to serve as a substrate for RNase H is discussed.     

Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses

Immunoglobulin G directed against the DNA polymerase of Rauscher murine leukemia virus (R-MuLV) could bind to 125I-labeled DNA polymerase of spleen necrosis virus (SNV), a member of the reticuloendotheliosis virus (REV) species. Competition radioimmunoassays showed the specificity of this cross-reaction. The antigenic determinants common to SNV and R-MuLV DNA polymerases were shared completely by the DNA polymerases of Gross MuLV, Moloney MuLV, RD 114 virus, REV-T, and duck infectious anemia virus. Baboon endogenous virus and chicken syncytial virus competed partially for antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. DNA polymerases of avian leukosis viruses, pheasant viruses, and mammalian type B and D retroviruses and particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. We also present data about a factor in normal mammalian immunoglobulin G that specifically inhibits the DNA polymerases of REV and mammalian type C retrovirus DNA polymerases.     

The presence of polyriboadenylic acid sequences in calf lens messenger RNA

The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T₁-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [³H]adenosine, are located at the 3′ termini of mRNA as determined by 3′ exoribonuclease digestion. Limited digestion of the [³H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)-filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3′-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.     

Purification and characterization of the DNA polymerase and RNase H activities in Moloney murine sarcoma-leukemia virus.

Two RNase H (RNA-DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) activities separable by Sephadex G-100 gel filtration were identified in lysates of Moloney murine sarcoma-leukemia virus (MSV). The larger enzyme, which we have called RNase H-I, represented about 10% of the RNase H activity in the virion. RNase H-I (i) copurified with RNA-directed DNA polymerase from the virus, (ii) had a sedimentation coefficient of 4.4S (corresponds to an apparent mol wt of 70,000), (iii) required Mn-2+ (2 mM optimum) for activity with a [3-h]poly(A)-poly(dT) substrate, (iv) eluted from phosphocellulose at 0.2 M KC1, and (v) degraded [3-H]poly(A)-poly(dT) and [3-H]poly(C)-poly(dG) at approximately equal rates. The smaller enzyme, designated RNase H-II, which represented the majority of the RNase H activity in the virus preparation, was shown to be different since it (i) had no detectable, associated DNA polymerase activity, (ii) had a sedmimentation coefficient of 2.6S (corresponds to an apparent mol wt of 30,000), (iii) preferred Mg-2+ (10 to 15 mM optimum) over Mn-2+ (5 to 10 mM optimum) 2.5-fold for the degradation of [3-H]poly(A)-poly(dT), and (iv) degraded [3-H]poly(A)-poly(dT) 6 and 60 times faster than [3-H]poly(C)-poly(dG) in the presence of Mn-2+ and Mg-2+, respectively. Moloney MSV DNA polymerase (RNase H-I), purified by Sephadex G-100 gel filtration followed by phosphocellulose, poly(A)-oligo(dT)-cellulose, and DEAE-cellulose chromatography, transcribed heteropolymeric regions of avian myeloblastosis virus 70S RNA at a rate comparable to avian myeloblastosis virus DNA polymerase purified by the same procedure.