Separation of the Infectious Ribonucleic Acid of Potato Spindle Tuber Virus from Double-Stranded Ribonucleic Acid of Plant Tissue Extracts

The infectious ribonucleic acid (RNA) of potato spindle tuber virus (PSTV) can be separated by hydroxyapatite chromatography from double-stranded RNA detectable in low amounts in both infected and uninfected plant tissue extracts. The chromatographic behavior of ribonuclease-sensitive PSTV RNA resembles that of transfer RNA.     

Incorporation of In Vitro Synthesized Reovirus Double-Stranded Ribonucleic Acid into Virus Corelike Particles

When reovirus double-stranded ribonucleic acid (dsRNA) was synthesized in vitro by using a large-particulate fraction (LP-fraction) from reovirus-infected L cells, a significant amount of the (3)H-labeled dsRNA product was incorporated into reovirus corelike particles bound to the LP fraction. These corelike particles were found to be indistinguishable from virus core derived by chymotryptic digestion of virions when compared on the basis of their (i) resistance to chymotryptic digestion, (ii) buoyant density in CsCl, (iii) particle size as determined by agarose chromatography, (iv) elution characteristics from diethylaminoethyl-Sephadex, and (v) resistance of the incorporated (3)H-dsRNA to ribonuclease digestion in 0.01 m NaCl. When the replicase reaction was partially inhibited by NaCl, there was an accumulation of particles that were less dense than the virus core. All of the results indicate that some virus core assembly takes place during the in vitro replicase reaction.     

Reovirus Replicase-Directed Synthesis of Double-Stranded Ribonucleic Acid

After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand (³H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of ³H-cytidine triphosphate (or ³H-uridine triphosphate) and adenine triphosphate-α-³²P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3′-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of ³H to ³²P in these fragments indicated that RNA synthesis proceeded from the 5′ to 3′ terminus.     

Mechanism of Synthesis of Vaccinia Virus Double-Stranded Ribonucleic Acid In Vivo and In Vitro

The synthesis of vaccinia virus double-stranded ribonucleic acid (RNA) in infected HeLa cells was sensitive to actinomycin D, suggesting that a deoxyribonucleic acid dependent reaction is involved. Some double-stranded RNA was made in the presence of cytosine arabinoside in infected cells. Double-stranded and complementary RNA were synthesized in vitro by using vaccinia cores. These two observations indicate that some of the double-stranded RNA is read from "early" genes. The double-stranded RNA synthesized in vitro had the same properties as that made in vivo. At least 70% of the double-stranded RNA made in vivo was in ribonuclease-resistant form prior to sodium dodecyl sulfate-phenol extraction. In addition, there was a complementary RNA in infected cells which could be converted to double-stranded RNA by annealing.     

Double-Stranded Ribonucleic Acid in Viruses of Penicillium citrinum

[This corrects the article on p. 811 in vol. 34.].     

Homologous Interference by Incomplete Sendai Virus Particles: Changes in Virus-Specific Ribonucleic Acid Synthesis

Incomplete Sendai virus particles (I particles) interfered with the replication of several strains of infectious Sendai virions (standard virus) but not with the replication of Newcastle disease virus, mumps virus, or Sindbis virus. I particles did not induce interferon, and ultraviolet irradiation of I particles abolished their ability to interfere. Protein synthesis was not necessary to establish interference. The degree of interference depended on the interval between exposure of cells to the I particles and challenge by standard virus, and this was reflected in the degree of inhibition of virus-specific ribonucleic acid (RNA) synthesis in infected cells. The most dramatic change was decreased accumulation of 50S virus-specific RNA in infected cells. RNA species sedimenting slower than 50S were not as markedly reduced in total amount, but hybridization experiments showed that a substantial portion of these slowly sedimenting RNA species were plus strands, presumably representing replicas of the RNA species in I particles. When I particles in insufficient numbers to interfere were added to cells as late as 8 hr after standard virus, there were no obvious changes in virus-specific RNA species in the cells; however, significant amounts of 19 and 25S RNA species, representing progeny of the I particles, appeared in the culture medium. It was concluded that interference was an intracellular event affecting an early step in virus replication. Competition by I particles for cell sites or substrates needed by standard virus seemed a less likely mechanism of interference than competition for enzymes specified by standard virus.     

Fermentation Process for Double-Stranded Ribonucleic Acid, an Interferon Inducer

Double-stranded ribonucleic acid (ds-RNA) isolated from Escherichia coli infected with bacteriophage MS2 is a potent interferon inducer. High levels of ds-RNA are formed in nonpermissive cells infected with MU9, an amber coat protein mutant of MS2. This mutant has been used to develop a process for large-scale ds-RNA production. Preparation of quantities of MU9 lysate sufficient for ds-RNA production in fermentors is described. Over 300 mug of ds-RNA/ml can be accumulated after MU9 infection of cultures grown to high density in corn steep liquor medium. This is approximately 300 times the amount of ds-RNA made by MS2 infection of cells grown in tryptone medium. Maximum ds-RNA formation requires only 3 hr. The ds-RNA is stable and remains inside nonaerated cells for at least 17 hr.     

Isolation and Characterization of a Double-Stranded Ribonucleic Acid from Penicillium chrysogenum

Double-stranded ribonucleic acid has been obtained from cells of the fungus Penicillium chrysogenum. This ribonucleic acid appears to be associated with mycophage and is an efficient inducer of interferon Its. extraction and partial purification are discussed, and evidence for its double-stranded and ribosidal nature is reviewed. The implications of viral nucleic acid in the life processes of fungi are considered.     

Characterization of Bluetongue Virus Ribonucleic Acid

An improved purification procedure yielded bluetongue virus free from any single-stranded ribonucleic acid (RNA) component. Double-stranded RNA obtained from purified virus or isolated from infected cells was fractionated into 5 components by means of sucrose gradient sedimentation analysis, and into 10 components by electrophoresis on polyacrylamide gels. The size of these components vary from 0.5 x 10(6) to 2.8 x 10(6) daltons, with a total molecular weight estimate of about 1.5 x 10(7) for the viral nucleic acid. The denaturation of the genome and separation of the resulting fragments are also discussed.     

Mechanism of Reovirus Double-Stranded Ribonucleic Acid Synthesis In Vivo and In Vitro

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.     

Characterization of Ribonucleic Acid from Visna Virus

A single-stranded ribonucleic acid(s) has been isolated from purified virions of visna virus. It consists of two major components, namely 63S and "4S," under the conditions employed for ribonucleic acid (RNA) extraction. The 63S component can be converted to subunits by heat and dimethylsulfoxide treatments. Analyses by base composition indicate that the "4S" RNA isolated from visna virus is not a random breakdown product of the 63S component as a result of extraction, nor is it randomly derived from cellular RNA.     

Release of Rubella Virus Ribonucleic Acid from Ribonucleoprotein by Polyanions

Rubella virus ribonucleoprotein was accessible to pancreatic ribonuclease, Pronase, and certain polyanions. Most of the ribonucleic acid (RNA) label was made acid-soluble by ribonuclease, whereas Pronase and the polyanions liberated 40S RNA from the ribonucleoprotein.     

Association of Viral Ribonucleic Acid with Cellular Membranes in Chick Embryo Cells Infected with Sindbis Virus

Membranes from cells infected with Sindbis virus had associated with them viral ribonucleic acid (RNA) polymerase and about 60 to 70% of the viral RNA labeled when short pulses were used. This RNA contained most of the replicative intermediate and replicative form of viral RNA found in the infected cells. The use of "Mg(2+) sarkosyl crystals" permitted the isolation of membrane-bound nucleic acids and allowed the demonstration that Sindbis virus RNA was synthesized on a membrane-viral RNA complex. Viral RNA from the infecting virions first became associated with the membranes during the latent period and, subsequently, slowly detached. The attachment of the viral RNA to the membranes did not require active viral RNA polymerase, since RNA from ts6, an RNA(-) temperature-sensitive mutant of Sindbis virus, associated with cellular membranes at a nonpermissive temperature. However, the subsequent detachment of the RNA from the membranes was restricted in the absence of viral RNA synthesis. The results indicate that association of viral RNA with cellular membranes may represent an early step occurring during the replication of Sindbis virus RNA.     

Ribonucleic Acid-Dependent Deoxyribonucleic Acid Polymerase in Visna Virus

A ribonucleic acid-dependent deoxyribonucleic acid polymerase was found in virions of visna virus. The enzyme product was resistant to ribonuclease and alkaline hydrolysis but susceptible to the digestion of deoxyribonuclease.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

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.     

Granulosis Virus Deoxyribonucleic Acid: a Closed, Double-Stranded Molecule

Studies on deoxyribonucleic acid purified from the granulosis virus of Trichoplusia ni have revealed the presence of a closed, double-stranded superhelix which sediments at 95S relative to relaxed circles (74S) and linear (60S) forms. Molecular weight estimates show that this insect virus deoxyribonucleic acid has a size of 100 x 10(6) daltons.     

Inhibition of Host-Cell Protein and Ribonucleic Acid Synthesis by Newcastle Disease Virus

The mechanisms of Newcastle disease virus-(NDV) induced inhibition of cell protein and ribonucleic acid (RNA) synthesis were investigated. It was observed that the ability of NDV to inhibit cell RNA synthesis is dependent on the virus strain. The inhibitors, azauridine and cycloheximide, were added to cell cultures at different times after infection to study the roles of protein and RNA synthesis in the viral inhibition process. Viral inhibition of cell RNA synthesis and viral inhibition of cell protein synthesis become resistant to cycloheximide at a different time after infection than that in which they become resistant to azauridine. The results indicate that the inhibition of cell RNA synthesis by the Texas strain involves the synthesis of inhibitory proteins which are coded by the viral genome. The Texas and Beaudette strains of NDV appear to employ different mechanisms for the inhibition of host-cell protein synthesis. Viral inhibition of cell protein synthesis does not appear to cause, or be the result of, viral inhibition of cell RNA synthesis.