Transcription of 70S RNA by DNA polymerases from mammalian RNA viruses.

DNA polymerases purified by the same procedure from four mammalian RNA viruses, simian sarcoma virus type 1, gibbon ape lymphoma virus, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus are capable of transcribing heteropolymeric regions of viral 70S RNA without any other primer. In this reconstituted system the enzymes from simian sarcoma virus type 1, Mason-Pfizer monkey virus, and Rauscher murine leukemia virus transcribe viral 70S RNA almost as efficiently as the DNA polymerase from the avian myeloblastosis virus, but gibbon ape lymphoma virus DNA polymerase is approximately three-to fivefold less efficient. Although there is a substantial difference among the sizes of these DNA polymerases (160,000 daltons for the avian myeloblastosis virus enzyme, 110,000 daltons for the Mason-Pfizer monkey virus enzyme, and 70,000 daltons for the mammalian type C viral polymerases), the ability to transcribe viral 70S RNA is a characteristic common to these enzymes.     

Purification and characterization of gibbon ape leukemia virus DNA polymerase.

An RNA directed DNA polymerase was purified over 2500 fold from gibbon ape leukemia virus by successive column chromatography on Sephadex G100, DEAE cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 7.5, a Mn2+ optimum of 0.8 mM, and KCl optimum of 80 mM. The purified enzyme transcribes heteropolymeric regions of viral 60-70 S RNA isolated from avian myeloblastosis virus, Rauscher murine leukemia virus and simian sarcoma virus and it is inhibited by antiserum prepared against either gibbon ape leukemia virus or simian sarcoma virus DNA polymerases.     

Cellular factors required for papillomavirus DNA replication.

In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.     

Characterization of an RNA-directed DNA-polymerase from a cell line derived from a radiation-induced lymphoma in mice.

An RNA-directed DNA polymerase was purified from a cell line derived from a radiation-induced lymphoma in NIH Swiss mice which produced non-infectious type C virus particles. The enzyme was isolated from a high speed particulate fraction which bands at a density of 1.16--1.19 g/ml in a sucrose gradient, and purified by successive chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 7.5, a KCl optimum of 50 mM, and a Mn2+ optimum of 0.25 mM. It prefers (dT)15 . (A)n to (dT)15 . (dA)n as the primer template and transcribes the poly(C) strand of (dG)15 .(C)n and (dG)15 . (OMeC)n. It transcribes heteropolymeric regions of avian myeloblastosis virus 70 S RNA, and is inhibited by antiserum to Rauscher murine leukemia virus DNA polymerase. Comparison of the properties of DNA polymerase purified from radiation-induced lymphoma cells with the DNA polymerase purified from non-defective murine type C RNA tumor viruses shows that the mouse lymphoma enzyme is both biochemically and immunologically related to murine leukemia virus DNA polymerases.     

A comparison of the enzymatic responses of the DNA polymerases from four RNA tumor viruses.

DNA polymerases from avian, feline, murine and simian RNA tumor viruses exhibit substantial differences in optimal assay conditions and vary widely in their template-primer preferences. Avian DNA polymerase utilizes both natural and synthetic template-primers efficiently in the presence of Mg⁺⁺ as well as Mn⁺⁺. By contrast, the mammalian viral DNA polymerases are much more responsive to poly(A)·oligo(dT) than to other template-primers, and exhibit up to 20-fold greater activity with Mn⁺⁺ than with Mg⁺⁺. In addition, simian sarcoma virus DNA polymerase shows no detectable response to poly(C)·oligo(dG) over a wide variety of conditions stimulatory to the other viral enzymes.     

Deoxyribonucleic Acid Polymerase Activities in Normal and Leukovirus-Infected Chicken Embryo Cells

Chicken embryo cells normally contain, in addition to deoxyribonucleic acid (DNA)-dependent DNA (D-DNA) polymerases, a novel "R-DNA-polymerase" which specifically copies polyriboadenylic acid strands. This R-DNA polymerase cannot copy natural ribonucleic acid or polyribocytidylic acid strands to a significant extent. Infection of cells with the leukovirus RAV-2 leads to the intracellular formation of large amounts of the viral RNA-dependent DNA polymerase whose properties differ from the cell R-DNA polymerase. Chicken cells transformed by a Rous sarcoma virus mutant which produce noninfectious alpha-type Rous sarcoma virus (f), a leukovirus known to be deficient in the viral RNA-dependent DNA polymerase, do not contain detectable viral RNA-dependent DNA polymerase, whereas the cellular R-DNA polymerase is found in normal amounts. There seems to be no relationship between the cellular R-DNA polymerase and the RNA-dependent DNA polymerase of the avian leukoviruses.     

Comparison of Rous Sarcoma Virus-Specific Deoxyribonucleic Acid Polymerases in Virions of Rous Sarcoma Virus and in Rous Sarcoma Virus-Infected Chicken Cells

Labeled virions of Rous sarcoma virus (RSV) were disrupted with detergent and analyzed on equilibrium sucrose density gradients. A core fraction at a density of approximately 1.24 g/cc contained all of the (3)H-uridine label and about 30% of the (3)H-leucine label from the virions. Endogenous viral deoxyribonucleic acid (DNA) polymerase activity was only found in the same location. Additional ribonucleic acid (RNA)- and DNA-dependent DNA polymerase activities were found at the top of the gradients. RNA-dependent and DNA-dependent DNA polymerase activities were also found in RSV-converted chicken cells. Particles containing these activities were released from cells by detergent and were shown to contain viral RNA. These particles were analyzed on equilibrium sucrose density gradients and were found to have densities different from virion cores.     

Simian virus 40 DNA extracted from infected cells with sodium deoxycholate no longer reflects its in vivo superhelix density.

When simian virus 40 DNA is extracted from infected cells with low concentrations of sodium deoxycholate, which selectively extract non-encapsidated simian virus 40 DNA, the DNA has a lower average number of superhelical turns than the DNA extracted from purified viral particles. During extraction, a partial deproteinization of the DNA by a concentration of detergent that did not inactivate a nicking-closing activity led to the removal of some superhelical turns. The DNA extracted in this way no longer reflected its in vivo number of superhelical turns.     

Lack of Serological Relationship Among DNA Polymerases of Avian Leukosis-Sarcoma Viruses, Reticuloendotheliosis Viruses, and Chicken Cells

Antibodies against a large and a small DNA polymerase isolated from chicken embryos and against avian myeloblastosis virus DNA polymerase were used to study the serological relationships of the DNA polymerase activities of three avian systems with RNA and a DNA polymerase-avian leukosis-sarcoma viruses, reticuloendotheliosis viruses, and a fraction from uninfected chicken cells. The DNA polymerase activity of disrupted virions of all avian leukosis-sarcoma viruses tested was neutralized to the same extent by antibody against avian myeloblastosis virus DNA polymerase and was not neutralized by the antibodies against chicken cellular DNA polymerases. The viruses tested included induced leukosis viruses and Rous-associated virus-O. The DNA polymerase activity of disrupted virions of all of the reticuloendotheliosis viruses was not neutralized by any of the antibodies. The chicken endogenous RNA-directed DNA polymerase activity was neutralized partially or completely, in different experiments, by antibody against the small DNA polymerase isolated from chicken embryos, but was not neutralized by the other two antibodies.     

Deoxyribonucleic Acid Polymerase(s) of Rous Sarcoma Virus: Effects of Virion-Associated Endonuclease on the Enzymatic Product

Purified preparations of Rous sarcoma virus (RSV) contain ribonuclease which is either a constituent of the virion surface or an adsorbed contaminant. Treatment of the virus with nonionic detergent to activate ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase renders the viral genome susceptible to hydrolysis by the external ribonuclease. The extent of this susceptibility can be substantially reduced by the use of limited amounts of detergent. At a concentration of detergent which provides a maximum initial rate of DNA synthesis, the degradation of endogenous viral RNA results in a reduced yield of high molecular weight DNA: RNA hybrid from the polymerase reaction. Attempts to detect virion-associated deoxyribonuclease, by using a variety of double helical DNA species as substrates, have been unsuccessful, but small amounts of nuclease activity directed against single-stranded DNA may be present in purified virus.     

Inhibition of reverse transcriptase activity of RNA-tumor viruses by fagaronine+.

Fagaronine, a benzophenanthridine alkaloid from roots of $\text{Fagara zanthoxyloides}$ (Rutaceae), has been reported to possess anti-leukemic activity. It inhibited RNA-directed DNA polymerase activity from avian myeloblastosis virus, Rauscher leukemia virus and simian sarcoma virus. With poly rA·oligo dT, the alkaloid concentration for 50% inhibition of the enzyme activity from these viruses was in the range of 6–12 μg (15 – 31 nmoles) per ml of reaction mixture. The enzyme reaction was also inhibited with activated DNA and 70S RNA as templates; however, with poly rC·oligo dG no inhibition of enzyme activity was obtained. These results suggest that fagaronine inhibits enzyme activity by interaction with the A:T templateprimer.     

Purification of RNA-directed DNA polymerase from mouse spleen infected with Rauscher leukemia virus.

RNA-directed DNA polymerase was purified from spleens of Balb/c and NMRI mice infected with Rauscher murine leukemia virus. The method includes cell fractionation and lysis of microsomal fraction, chromtography on Sephadex G-200 and phosphocellulose. Estimation of molecular weight from the sedimentation rate of the purified enzyme in a glycerol gradient was consistent with a structure containing one polypeptide with a molecular weight of 70,000. Purified RLV DNA polymerase from spleen could transcribe purified DNA polymerase from purified virions. This simple preparation method offers a procedure for large scale preparation of the RNA-directed DNA polymerase which can be used for synthesis of DNA complementary to mRNA.     

Purification and characterization of baboon endogenous virus DNA polymerase.

An RNA-directed DNA polymerase was purified from baboon endogenous type-C virus by successive column chromatography on DEAE cellulose, phosphocellulose and hydroxyapatite. The purified DNA polymerase has a molecular weight of 68 000, a pH optimum of 8.0, a Mn2+ optimum of 1 mM, and a KCl optimum of 40 mM. The purified enzyme transcribes heteropolymeric regions of viral 60--70 S RNA isolated from different type-C viruses. The purified enzyme is immunologically related to a similarly purified polymerase from the cat endogenous type-C virus RD114.