In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.     

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.     

Method for reproducible large-volume production and purification of Rauscher murine leukemia virus.

Rauscher murine leukemia virus was produced in roller-bottle cultures of chronically infected JLS-V9 cells. Virus from this culture fluid was concentrated and purified by two semi-isopycnic bandings in sucrose gradients. Virus material obtained from young, nonconfluent cultures (early-harvest virus) yielded products characteristically containing endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with high specific activity (400 to 1,000 pmol of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour). Fluids obtained from older confluent cultures (late-harvest virus) yielded products with endogenous ribonucleic acid-dependent deoxyribonucleic acid polymerase with little or no specific activity (200 pmol or less of [3H]thymidine 5'-triphosphate incorporated per milligram of protein per hour), but with higher virus particle counts and greater amounts of protein and gs antigen than the early-harvest products.     

Adaptation and Infection of Mouse Bone Marrow (JLS-V9) Cells in Suspension Culture for Production of Rauscher Leukemia Virus

JLS-V9 mouse bone marrow cells were readily adapted to suspension culture, chronically infected with Rauscher leukemia virus (RLV), and subsequently grown in 7.5- and 14-liter New Brunswick fermentors. The suspension-type cell system can be modified to produce virus with clearly defined properties, such as high ribonucleic acid-dependent deoxyribonucleic acid polymerase (RDDP) activity, high particle count, and high infectious particle count. Biological and biophysical properties of suspension-produced RLV were not affected by concentration and purification employing continuous-flow and rate-zonal centrifugation procedures. The RDDP assay was standardized and showed a linear incorporation of (3)H-thymidine 5'-monophosphate ((3)H-TMP) up to 30 min. Further characterization indicated that a high percentage of (3)H-TMP incorporation was due to RDDP.     

Alterations of protein synthesis in arbovirus-infected L cells

Lust, George (Fort Detrick, Frederick, Md.). Alterations of protein synthesis in arbovirus-infected L cells. J. Bacteriol. 91:1612-1617. 1966.-Cellular protein synthesis and ribonucleic acid (RNA) synthesis in mouse L cells were markedly depressed 1 hr after infection with Venezuelan equine encephalomyelitis virus. Host RNA and protein synthesis were inhibited more rapidly by the virus infection than by actinomycin D. In cells infected 4 hr, a cytoplasmic RNA polymerase was demonstrated which was absent in uninfected cells. At this time, deoxyribonucleic acid-directed RNA synthesis catalyzed by the nuclear RNA polymerase was inhibited in vitro in enzyme preparations from nuclei of virus-infected cells. For optimal activity, the cytoplasmic RNA polymerase required the four nucleoside triphosphates, Mg(++), and RNA. The enzyme was insensitive to actinomycin D and deoxyribonuclease, indicating that it catalyzed RNA-directed RNA synthesis. Attempts to purify the induced polymerase further were unsuccessful. Fresh preparations had to be used because the enzymatic activity was unstable.     

Reversible Inactivation of the Deoxyribonucleic Acid Polymerase of Rauscher Leukemia Virus

Rauscher leukemia virus deoxyribonucleic acid polymerase is reversibly inactivated by 6 m guanidine-hydrochloride. Gel filtration in 6 m guanidine-hydrochloride reveals that the viral deoxyribonucleic acid polymerase consists of a single polypeptide chain of approximately 70,000 molecular weight.     

Polyuridylic Acid-directed Phenylalanine Incorporation in Minicell Extracts

Cell-free extracts of miniature Escherichia coli cells deficient in deoxyribonucleic acid (DNA) and DNA-dependent ribonucleic acid polymerase have been shown to be capable of polyuridylic acid-directed [(14)C]phenylalanine incorporation.     

RNA-Dependent DNA Polymerase Activity of RNA Tumor Viruses II. Directing Influence of RNA in the Reaction

The role of ribonucleic acid (RNA) in deoxyribonucleic acid (DNA) synthesis with the purified DNA polymerase from the avian myeloblastosis virus has been studied. The polymerase catalyzes the synthesis of DNA in the presence of four deoxynucleoside triphosphates, Mg(2+), and a variety of RNA templates including those isolated from avian myeloblastosis, Rous sarcoma, and Rauscher leukemia viruses; phages f2, MS2, and Qbeta; and synthetic homopolymers such as polyadenylate.polyuridylic acid. The enzyme does not initiate the synthesis of new chains but incorporates deoxynucleotides at 3' hydroxyl ends of primer strands. The product is an RNA.DNA hybrid in which the two polynucleotide components are covalently linked. Free DNA has not been detected among the products formed with the purified enzyme in vitro. The DNA synthesized with avian myeloblastosis virus RNA after alkaline hydrolysis has a sedimentation coefficient of 6 to 7S.     

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.     

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.     

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.     

Electron Microscopic Observations on the Ribonucleic Acid of Murine Leukemia Virus

The ribonucleic acid (RNA) of murine leukemia virus (MLV) Rauscher strain was observed by the aid of electron microscopy with the use of the protein monolayer technique. RNA was observed directly after release from virus particles or after isolation by sedimentation in sucrose density gradients. Molecules were found in an extended linear form. Many of the RNA filaments released by detergent treatment contained curled regions, suggesting the linear filaments were originally coiled within the virus particle. The relationship of the curled areas to the containment of the RNA within the virus particle is discussed, and a mechanism for the inclusion of RNA in the budding virion is proposed. Treatment of the extended MLV-RNA with dimethyl sulfoxide resulted in the collapse of the molecule forming a tangled complex. Treatment with urea or heating at 50 C in 3 mm NaCl also produced this effect. Also under the conditions in which MLV-RNA was linear, RNA from Rous sarcoma virus also was linear, but Newcastle disease virus RNA and ribosomal RNA of rat liver had collapsed structures. The results indicated that the RNA of MLV, and perhaps other RNA-containing tumor viruses, has a specific unique conformation dependent upon hydrogen bonds.     

Isolation and properties of a testicular ribonucleic acid polymerase

A procedure is described for isolation in a soluble form of a ribonucleic acid polymerase from rat testes. Evidence is presented that this enzyme catalyzes three distinct reactions: (a) deoxyribonucleic acid-directed synthesis of RNA in the presence of all four major ribonucleoside triphosphates; (b) DNA-primed formation of polyadenylic acid and other ribohomopolymers in the presence of single ribonucleoside triphosphate substrates; (c) synthesis of complementary polyribonucleotides in the presence of various ribohomopolymer primers. The properties of these reactions are discussed with respect to metal ion requirements, affinities for ribonucleoside triphosphates and primer polynucleotides, heat denaturation of DNA primers, and the effects of ionic strength, beta-mercaptoethanol, polyamines, temperature, and inhibitors on the rates and extent of the reactions. The testicular ribonucleic acid polymerase is a very unstable enzyme that can be stabilized by high concentrations of glycerol.     

Physical and Biochemical Properties of Progressive Pneumonia Virus

The physical and biochemical characteristics of progressive pneumonia virus were found to be remarkably similar to those of the ribonucleic acid (RNA) tumor viruses. Significant findings included the presence of a 60 to 70S RNA genome, RNA-dependent deoxyribonucleic acid polymerase activity, and common morphological properties. This information correlates with previously reported biological observations and supports the provisional inclusion of enveloped RNA-containing "slow viruses" within the RNA tumor virus group.     

The Influence of Syringomycin on Ribonucleic Acid Synthesis

Syringomycin, a wide-spectrum antibiotic produced by strains of Pseudomonas syringae which cause bacterial canker of peach, was able to bind to salmon sperm and calf thymus deoxyribonucleic acid but not to calf thymus histone; it also inhibited ribonucleic acid polymerase activity. These abilities to bind to deoxyribonucleic acid and to inhibit ribonucleic acid polymerase were inactivated when the phytotoxic and antibiotic properties of syringomycin were inactivated.     

Effect of Rifamycins and Related Antibiotics on the Deoxyribonucleic Acid-Dependent Ribonucleic Acid Polymerase of Vaccinia Virus Particles

A number of compounds related to rifampin which act as expected in the Escherichia coli system have been tested for their ability to inhibit the vaccinia particle deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase in vitro. Some compounds are inactive even at concentrations of 500 mug/ml, others are able to produce partial inhibition, and others strongly inhibit the enzyme activity at 150 mug/ml or less. The inhibition, where present, operates immediately but appears to be at least partially reversible. At least one compound which is without effect against bacterial RNA polymerase is a potent inhibitor of the viral RNA polymerase. As the enzyme activity of rifampin-resistant mutants of vaccinia virus is inhibited to the same extent as that of the wild type, the observed in vitro effect on vaccinia virus RNA polymerase is not identical with the in vivo effect specifically directed against a vaccinia-specified protein.     

In Vitro System for Production of Mouse Mammary Tumor Virus

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C(3)H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 10(10) particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.