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.     

Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA.

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.     

Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus

To detect Rous sarcoma virus-specific DNA in mammalian cells, we have measured the capacity of unlabeled cell DNA to accelerate the reassociation of labeled double-stranded DNA synthesized by the Rous sarcoma virus RNA directed DNA polymerase. Two populations of double-stranded polymerase products are identified by their reassociation kinetics and represent approximately 5% and 30% of the viral 70 S RNA genome. Using two strains of Rous sarcoma virus and four lines of transformed mammalian cells, we found two copies of DNA homologous to both DNA populations in Rous sarcoma virustransformed rat and mouse cells, but not in normal cells. The Rous sarcoma viruslike DNA can be demonstrated in the non-repeated fraction of transformed cell DNA and in nuclear DNA. The results are supported by evidence that the techniques employed detect the formation of extensive well-matched duplexes of cell DNA and viral polymerase products.     

Mechanism of Oncogenic Transformation by Rous Sarcoma Virus: I. Intracellular Inactivation of Cell-Transforming Ability of Rous Sarcoma Virus by 5-Bromodeoxyuridine and Light

Chick embryo fibroblasts brought into stationary phase of growth by maintenance in serum-free Eagle's MEM medium were infected with the Bryan strain of Rous sarcoma virus (B-RSV) and incubated for 18 hr in the presence of 5-bromo-deoxyuridine (BUdR). The cells were then allowed to resume growth and deoxyribonucleic acid (DNA) synthesis by addition of an enriched F12 medium containing serum and RSV antibody to prevent spread of viral infection. After 48 hr, the cultures were exposed for various periods to visible light, overlaid with solid culture medium, and observed for the appearance of foci of transformed cells. In cultures treated with BUdR at the time of infection, exposure to light resulted in a suppression of focus formation of from 50 to 90% in various experiments. Treatment with BUdR for 18 hr before infection or on the day after infection, followed by exposure to light, had no effect on focus formation. In cultures in which almost all cells were infected, treatment with BUdR followed by exposure to light did not result in cell death. This suggests that suppression of transformation is not due to selective killing of infected cells by this treatment but rather to the intracellular inactivation of the transforming ability of Rous sarcoma proviral DNA.     

Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase.

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.     

Controlled release of particle-associated RNA-dependent DNA polymerase by primary chick embryo cell cultures

Chick embryo cell cultures release a particle-associated RNA-dependent DNA polymerase into the culture medium. The release shows a characteristic time course with a maximum on the 3rd or 4th day in culture. The release of enzyme decreases when the cells are further cultivated and passaged. The enzyme was characterized as an RNA-dependent DNA polymerase by its ability to transcribe heteropolymeric RNA into DNA. It is different from the polymerase of the avian leukosis/sarcoma virus group and indistinguishable from an RNA-dependent DNA polymerase from normal embryonated chicken eggs described previously [1, 2]. The release of enzyme is independent of the genetic systems regulating the complete or partial expression of the endogenous avian leukosis virus genome. The amount of enzyme released is dependent on the age of the embryo from which the cell cultures are prepared. Cells prepared from 6-day-old embryos release maximal enzyme activity.     

Correlation of RNA binding affinity of avian oncornavirus p19 proteins with the extent of processing of virus genome RNA in cells.

We purified the p19 proteins from the Prague C strain of Rous sarcoma virus, avian myeloblastosis virus, B77 sarcoma virus, myeloblastosis-associated virus-2(0), and PR-E 95-C virus and measured their binding affinities for 60S viral RNA by the nitrocellulose filter binding technique. The apparent association constants of the p19 proteins from Rous sarcoma virus Prague C, avian myeloblastosis virus, and B77 sarcoma virus for homologous and heterologous 60S RNAs were similar (1.5 x 10(11) to 2.6 x 10(11) liters/mol), whereas those of myeloblastosis-associated virus-2(0) and PR-E 95-C virus were 10-fold lower. The sizes and relative amounts of the virus-specific polyadenylic acid-containing RNAs in the cytoplasms of cells infected with Rous sarcoma virus Prague C, myeloblastosis-associated virus-2(0), and PR-E 95-C virus were determined by fractionating the RNAs on agarose gels containing methylmercury hydroxide, transferring them to diazobenzyloxymethyl paper and hybridizing them to a 70-nucleotide complementary DNA probe. In cells infected with Rous sarcoma virus Prague C we detected 3.4 x 10(6)-, 1.9 x 10(6)-, and 1.1 x 10(6)-dalton RNAs, in PR-E 95-C virus-infected cells we detected 3.4 x 10(6)-, 1.9 x 10(6)- and 0.7 x 10(6)-dalton RNAs, and in cells infected with myeloblastosis-associated virus-2(0) we detected 3 x 10(6)- and 1.3 x 10(6)-dalton RNAs. Each of these RNA species contained RNA sequences derived from the 5' terminus of genome-length RNA, as evidenced by hybridization with the 5' 70-nucleotide complementary DNA. The ratios of subgenomic mRNA's to genome-length RNAs in cells infected with myeloblastosis-associated virus-2(0) and PR-E 95-C virus were three- to five-fold higher than the ratio in cells infected with Rous sarcoma virus Prague C. These results suggest that more processing of viral RNA in infected cells is correlated with lower binding affinities of the p19 protein for viral RNA, and they are consistent with the hypothesis that the p19 protein controls processing of viral RNA in cells.     

Antiviral Action of a Rifamycin Derivative: Formation of Rous Sarcoma Virus Particles Deficient in 60 to 70S RNA

Growth of Rous sarcoma virus-transformed cells in the presence of rifazone-8(2), a rifamycin derivative, results in the formation of noninfectious virus particles lacking 60 to 70S RNA.     

Cell Cycle-Dependent Activation of Rous Sarcoma Virus-Infected Stationary Chicken Cells: Avian Leukosis Virus Group-Specific Antigens and Ribonucleic Acid

Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary cells did not contain avian leukosis virus group-specific antigens or ribonucleic acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV endogenous RNA-directed DNA polymerase activity.     

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.     

Regulation of Rous sarcoma virus RNA-dependent DNA polymerase isoenzymes by in vitro phosphorylation-dephosphorylation

The activity of the αβ form of Rous sarcoma virus RNA-dependent DNA polymerase was stimulated upon treatment with the protein kinase purified from the same virus. This enhancement was observed for both DNA-dependent and RNA-dependent DNA polymerase activities, whereas the RNase H activity associated with the polymerase was not affected. On the other hand, the protein kinase did not induce detectable changes in the activities of the α-polymerase isoenzyme. Treatment with Escherichia coli alkaline phosphatase resulted in a reduction of the polymerase activities of the αβ isoenzyme with no effects on RNase H as well as on the α form of the DNA polymerase. Preincubations of the αβ- and α-oncornaviral polymerase isoenzymes with two other protein kinases—from avian myeloblastosis virus and from beef heart (catalytic subunit)—had no substantial effects on DNA polymerase and RNase H activities of both polymerase isoenzymes. Both α and β subunits of the polymerase isoenzymes were phosphorylated in vitro by all three protein kinases employed, although only the β subunit was shown previously to be phosphorylated in vivo.     

Inhibitory effect of alpha-methyl-1-adamantane methylamine hydrochloride (rimantadine) on RNA-dependent RNA polymerase induction in culture of cells, infected with influenza virus]

An anti-influenza preparation, rimantadine (alpha-methyl-1-adamantane methylamine hydrochloride) at concentrations of 10--25 mkg/ml depresses the RNA-dependent RNA polymerase induction in a culture of cells infected with influenza virus (fowl plague virus). The inhibitory effect is also observed 2 hours following cell infection. In vitro studies have demonstrated that rimantadine has no effect on the activity of virus-induced RNA-dependent RNA polymerase, as well as on that of RNA-dependent RNA polymerase associated with virus particles.     

Effect of p15-associated protease from an avian RNA tumor virus on avian virus-specific polyprotein precursors.

A proteolytic activity is associated with structural protein p15 in avian RNA tumor viruses. Its effect on the known intracellular viral polyprotein precursors obtained by immunoprecipitation was investigated. Cleavage of Pr76gag resulted in the sequential appearance of p15, p27, and p19. The intracellular precursor Pr180gag-pol was also cleaved by p15, whereas the intracellular glycoprotein precursors of avian RNA tumor viruses, Pr92env, remained unaffected by p15 under all conditions tested. The specificities of the antibodies used to precipitate the precursors influenced the pattern of intermediates and cleavage products obtained by p15 treatment. If virus harvested from the the Prague strain of Rous sarcoma virus, subgroup C-transformed cells at 15-min intervals was incubated at 37 degrees C for further maturation, RNA-dependent DNA polymerase activity showed an optimum of DNA synthesis with 70S viral RNA or synthetic template-primers after short incubation periods. The presence of additional p15 during incubation resulted in a shift of the enzyme activity peak toward earlier time points. Virus harvested at 3-h intervals contained significant amounts of Pr180gag-pol and Pr76gag. The addition of p15 resulted in the cleavage of Pr180gag-pol and Pr76gag, but only a few distinct low-molecular-weight polypeptides appeared. Treatment of purified RNA-dependent DNA polymerase with p15 in vitro resulted in a disappearance of the beta subunit and an enrichment of the alpha subunit. In addition, a polypeptide of 32 x 10(3) molecular weight was generated. The cleavage pattern observed differed from the one obtained by trypsin treatment.     

Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Single-stranded regions on unintegrated avian retrovirus DNA.

Using chromatography on benzoylated naphthoylated DEAE-cellulose, we found that greater than 99.5% of the unintegrated linear viral DNA species detected in quail embryo cells infected with Rous sarcoma virus contained single-stranded regions, even at 16 h after infection. These regions were distributed across the genome and, on average, were primarily of plus-strand DNA. Within most of the linear viral DNA species, the minus strand was interpreted as being of genome size with two copies of the large terminal redundancy, LTR. In contrast, the plus strands in the linear viral DNA species were exclusively subgenomic.