Deoxyribonucleic Acid Polymerases of Rous Sarcoma Virus: Kinetics of Deoxyribonucleic Acid Synthesis and Specificity of the Products

The deoxyribonucleic acid (DNA) polymerase(s) of Rous sarcoma virus synthesizes two principal products-single-stranded DNA in the form of a DNA:ribonucleic acid (RNA) hybrid and double-stranded DNA. All of the single-stranded product and 50% of the double-stranded product can be hybridized to 70S viral RNA. These results, in combination with data obtained by analysis of the kinetics of double-stranded DNA synthesis, indicate that the synthesis of double-stranded DNA is a sequel to the synthesis of single-stranded DNA and is dependent upon the latter for the provision of initial template.     

Forms of Deoxyribonucleic Acid Produced by Virions of the Ribonucleic Acid Tumor Viruses

The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.     

Deoxyribonucleic Acid Polymerase of Rous Sarcoma Virus: Studies on the Mechanism of Double-Stranded Deoxyribonucleic Acid Synthesis

The deoxyribonucleic acid (DNA) polymerase of Rous sarcoma virus synthesizes both single- and double-stranded DNA, utilizing the ribonucleic acid (RNA) of the viral genome as the initial template. Results of pulse-chase experiments indicate that the single-stranded DNA serves as unconserved template and precursor for the synthesis of double-stranded DNA. The latter reaction is apparently initiated in association with the viral RNA and may involve a partially double-stranded intermediate form.     

Reovirus: Effect of Noninfective Viral Components on Cellular Deoxyribonucleic Acid Synthesis

We determined the effects of noninfective reovirus components on cellular deoxyribonucleic acid (DNA) synthesis. Reovirus inactivated by ultraviolet light inhibited cellular DNA synthesis, whereas reovirus cores and empty capsids did not. Both cores and empty capsids were adsorbed to cells. Adenine-rich ribonucleic acid (RNA) from reovirus, adsorbed to cells in the presence of diethyl-aminoethyl-dextran, produced a partial inhibition of DNA synthesis. RNA was synthesized in the presence of actinomycin D after infection with ultraviolet light-irradiated reovirus, and this RNA synthesis was not due to multiplicity reactivation of virus infectivity. These data suggest that viral structural proteins do not inhibit DNA synthesis and that the inhibition produced by ultraviolet-irradiated virus may be mediated in part or in toto by a newly synthesized viral product.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

Deoxyribonucleic Acid polymerase from wheat embryos

A soluble DNA-dependent DNA polymerase was extracted from wheat embryos. In vitro, the incorporation of radioactive thymidine triphosphate into acid-insoluble material is dependent upon the presence of the enzyme, all four deoxyribonucleotide triphosphates, Mg²⁺, and a DNA template. Incorporation occurs on native, alkali-denatured, and strictly double-stranded DNA. The in vitro synthesized product is a polydeoxynucleotide with a chain length shorter than the template; it has the same buoyant density as wheat embryo DNA when this DNA is used as template; and it forms a double-stranded complex with the DNA template. These data suggest that the in vitro DNA synthesis catalyzed by proteins extracted from wheat embryos occurs in a semiconservative way.     

Specific Inhibition of Mammalian Ribonucleic Acid C-Type Virus Deoxyribonucleic Acid Polymerases by Rat Antisera

Inhibition of the ribonucleic acid (RNA)- and deoxyribonucleic acid (DNA)-dependent DNA polymerase activities of mammalian C-type viruses was obtained with sera from rats bearing murine leukemia virus-induced transplant tumors. Polymerase activities of nonmammalian (viper) C-type virus and murine mammary tumor virus were not inhibited by such sera nor by serum from a rat immunized with the DNA polymerase of feline leukemia virus purified by isoelectric focusing. The latter serum appeared to inhibit preferentially the DNA-dependent DNA polymerase activity of mammalian C-type viruses showing no inhibition of RNA-dependent DNA synthesis.     

Ribonucleic Acid Polymerase Catalyzing Synthesis of Double-stranded Arbovirus Ribonucleic Acid

The large-particle fraction from the cytoplasm of chick embryo fibroblasts infected with Semliki Forest virus was found to catalyze the incorporation of the 5'-triphosphates of guanosine, adenine, cytidine, and uridine into an acid-insoluble alkali-labile product. The conditions affecting the preparation and assay of this enzyme were investigated. The ribonucleic acid (RNA) polymerase was not present in uninfected cells, and it appeared in infected cells at the time of rapid viral RNA synthesis. The polymerase was found to catalyze the synthesis of a species of RNA which was resistant to ribonuclease and which exhibited the sedimentation properties, buoyant density, and thermal transition temperature of the double-stranded RNA found in vivo in chick cells infected with Semliki forest virus. Attempts to demonstrate that the reaction product of this enzyme also included single-stranded viral RNA were not successful. Although other interpretations are possible, these results give some support to the suggestion that more than one enzyme may be involved in the replication of viral RNA.     

Intracellular Forms of Adenovirus Deoxyribonucleic Acid I. Evidence for a Deoxyribonucleic Acid-Protein Complex in Baby Hamster Kidney Cells Infected with Adenovirus Type 12

The total intracellular deoxyribonucleic acid (DNA) from baby hamster kidney cells abortively infected with (3)H-adenovirus type 12 was analyzed in dye-buoyant density gradients. Between 10 and 20% of the cell-associated radioactivity derived from viral DNA bands in a density position which is 0.043 to 0.085 g/cm(3) higher than that of viral DNA extracted from purified virions. The DNA in the high-density region (HP-fraction) is almost completely absent when DNA, ribonucleic acid (RNA) or protein synthesis is chemically inhibited in separate experiments. The HP-fraction is not found when the virus does not adsorb to and enter the cell. The DNA in the HP-fraction appears as early as 2 hr after inoculation. At 2 hr after infection, the HP-fraction is present both in the nucleus and the cytoplasm. This DNA hybridizes exclusively with viral DNA and sediments at approximately the same rate in both neutral and alkaline sucrose density gradients. Electron microscopy has revealed no circular DNA molecules in this fraction. Evidence indicates that the viral DNA in the HP-fraction exists in a complex with protein and possibly RNA. The protein component of the complex is resistant to enzymatic digestion, whereas the complex is susceptible to ribonuclease treatment. Digestion with deoxyribonuclease reduces the amount of DNA found in the HP-fraction. The structure and biological function of this complex are currently being investigated.     

Size and Composition of Marek''s Disease Virus Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) extracted from purified nucleocapsids of Marek's disease herpesvirus (MDV) was cosedimented with T4 and with herpes simplex virus (HSV) DNA in neutral sucrose density gradients and with T4 DNA in alkaline sucrose density gradients. These experiments indicated that the intact MDV DNA had a sedimentation constant of 56S corresponding to a molecular weight of 1.2 x 10(8) daltons. In the alkaline gradients, the largest and most prominent band contains a DNA sedimenting at 70S corresponding to 6.0 x 10(7) daltons in molecular weight. The DNA is therefore double-stranded and not cross-linked. Isopycnic sedimentation of the MDV DNA molecules with SPO1, Micrococcus lysodeikticus, and HSV DNA gave a density of 1.705 g/cm(3) corresponding to 46 guanine plus cytosine moles per cent. Lastly, in hybridization tests the DNA hybridized with RNA of infected cells but not with that of uninfected cells supporting the conclusion that it is viral.     

Ribonuclease-Sensitive Deoxyribonucleic Acid Polymerase Activity in Uninfected Rat Cells and Rat Cells Infected with Rous Sarcoma Virus

Rat cells infected with the B77 strain of avian sarcoma virus [R(B77) cells] produced no virus-like particles but contained information for the production of infectious B77 virus. (3)H-labeled deoxyribonucleic acid (DNA) product of the B77 virus endogenous DNA polymerase system was used to determine the relative amounts of B77 virus-specific ribonucleic acid (RNA) in B77 virus-infected chicken and R(B77) cells. R(B77) cells were found to contain much less B77 virus RNA than did B77 virus-infected chicken cells. Ribonuclease-sensitive DNA polymerase activity was present in high-speed pellet fractions from Nonidet extracts of B77 virus-infected rat cells. Similar preparations from some uninfected rat cells contained lesser amounts of a similar ribonuclease-sensitive DNA polymerase activity. The endogenous template for the DNA polymerase activity in high-speed pellet fractions from R(B77) cells was not related to B77 virus RNA or to RNA of a rat C-type virus. The DNA product of the endogenous DNA polymerase in high-speed pellet fractions of R(B77) cells hybridized to a small extent with RNA from the same fraction and to a similar extent with RNA from uninfected rat cells.     

Sensitivity of Ribonucleic Acid and Deoxyribonucleic Acid Viruses to Different Species of Interferon in Cell Cultures

Although two deoxyribonucleic acid (DNA) viruses, pseudorabies (PsRV) and vaccinia, are as susceptible as a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), to interferon when tested in chicken or mouse cells, they are refractory to inhibition in interferon-treated primary rabbit kidney cells and in a continuous line (RK-13) of rabbit kidney cells. Superinfection with VSV of RK-13 cells first infected with PsRV completely blocks the replication of PsRV with no effect on VSV yield. When the same experiment is carried out in RK-13 cells pretreated with 1,000 units of interferon, VSV replication is inhibited, which permits PsRV to replicate normally. These findings demonstrate that in the same cell one virus (PsRV) can be refractory to interferon and a second virus (VSV) can be susceptible. These experiments show that rabbit kidney cell cultures are deficient in the synthesis of resistance factors active against the DNA viruses tested and raise the possibility that separate resistance factors may exist for RNA and DNA viruses. In the case of sequential infection of interferon-treated RK-13 cells with vaccinia and VSV, it was found that not only was vaccinia replication refractory to inhibition by interferon, but also that prior infection with vaccinia was able to partially reverse the effect of the inhibitor on the replication of the VSV used for superinfection. On the basis of these and other data it is postulated that a vaccinia virion component or a replication product of vaccinia virus, or both, enables VSV to escape the inhibiting action of interferoninduced resistance factors.     

Properties of Maedi Nucleic Acid and the Presence of Ribonucleic Acid- and Deoxyribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase in the Virions

Maedi virus contains a ribonucleic acid (RNA) which can be resolved into three major components, namely, 62S, 33S, and 13S, by sucrose gradient centrifugation. The presence of RNA- and deoxyribonucleic acid (DNA)-dependent DNA polymerase in virions of maedi virus was demonstrated. The enzyme product could be converted into acid-soluble form by pancreatic deoxyribonuclease, but was resistant to digestion by pancreatic ribonuclease and to hydrolysis by NaOH.     

Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase Activity in Cells Infected with Influenza Virus

Deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase activity was assayed on nuclear preparations of chick embryo fibroblast cells at various times after infection with an influenza A virus (fowl plague virus) and was compared with the activity of uninfected cells. Polymerase activity was increased by about 60% by 2 hr after infection, and this increase coincided with an increase in RNA synthesis in infected cells, as determined by pulse-labeling with uridine. No difference could be detected between the polymerases of infected and uninfected cells as to their requirements for DNA primer, divalent cations, and nucleoside triphosphates, and they were equally sensitive to addition of actinomycin D to the reaction mixture. It is possible that host cell DNA-dependent RNA polymerase is involved in the replication of influenza virus RNA.     

Acquisition of Sequences Homologous to Host Deoxyribonucleic Acid by Closed Circular Simian Virus 40 Deoxyribonucleic Acid

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.     

Deoxyribonucleic Acid Synthesis in Permeabilized Spheroplasts of Saccharomyces cerevisiae

Osmotically shocked spheroplasts from Saccharomyces cerevisiae incorporated deoxynucleoside triphosphates specifically into double-stranded nuclear and mitochondrial deoxyribonucleic acid (DNA). Results with this in vitro system for cells with and without mitochondrial DNA were compared. Strains lacking mitochondrial DNA were used to study nuclear DNA replication. With a temperature-sensitive mutant defective in DNA replication in vivo, DNA synthesis in vitro was temperature sensitive as well. The product of synthesis with all strains after very short labeling times consisted principally of short fragments that sedimented at approximately 4S in alkali; with longer pulse times or a chase with unlabeled nucleotides, they grew to a more heterogenous size, with an average of 6 to 8S and a maximum of 15S. There was little, if any, integration of these DNA fragments into the high-molecular-weight nuclear DNA. Analysis by CsCl density gradient centrifugation after incorporation of bromodeoxyuridine triphosphate showed that most of the product consisted of chains containing both preexisting and newly synthesized material, but there was also a small fraction (ca. 20%) in which the strands were fully synthesized in vitro. (32)P-label transfer ("nearest-neighbor") experiments demonstrated that at least a part of the material synthesized in vitro contained ribonucleic acid-DNA junctions. DNA pulse-labeled in vivo in a mutant capable of taking up thymidine 5'-monophosphate, sedimented in alkali at 4S, as in the case of the in vitro experiments.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Effect of Chloramphenicol and Rifampin

Conjugal replication of R64-11 deoxyribonucleic acid (DNA) and the concomitant transfer of R64-11 DNA to DNA-deficient minicells are dependent upon processes that are inhibited by rifampin and chloramphenicol. The rifampin-sensitive product is not present in vegetatively growing cells and is needed to initiate both conjugal DNA replication in donor cells and DNA transfer to recipient minicells. If the rifampin-sensitive product is a ribonucleic acid (RNA) molecule (rather than RNA polymerase itself), our data indicate that this RNA species required for initiation of conjugal activity does not need to be translated into a protein product. The chloramphenicol-sensitive product(s) is present in vegetatively growing cells in sufficient quantity to permit most donor cells to carry out one round of plasmid conjugal replication and transfer. The initiation of second and subsequent rounds of conjugal replication and transfer are dependent on the synthesis of both the rifampin-sensitive and chloramphenicol-sensitive products. Our results demonstrate a correspondence between the amount of conjugal DNA replication in the donor and the amount of DNA transferred to recipient minicells under all conditions, and therefore suggest but do not prove that plasmid transfer is dependent on conjugal DNA replication. The results also add additional proof that R64-11 transfer to minicells is discontinuous. All of these results are discussed in regard to further refinements of old models for the mechanism of conjugal transfer as well as a more radical departure from current dogma.