Virus-specific RNA Synthesis Research Articles

Persistent infection of cells in culture by measles virus. 3. Comparison of virus-specific RNA synthesized in primary persistent infection in HeLa cells.

The pattern of actinomycin D-resistant RNA synthesis was examined during primary infection of HeLa cells by virulent Edmonston measles virus and in two HeLa clones persistently infected by the same strain of virus. One of these clones, K11, produces infectious virus of low virulence for HeLa cells, and the other, K11A-HG-1, has thus far failed to yield infectious virus. The patterns of virus-specific RNA synthesized in these three types of infection are qualitatively similar to each other and to the patterns of virus-specific RNA synthesis in other paramyxovirus infections. There were, however, quantitative differences. In addition, virions of the virulent Edmonston strain of measles virus were found to contain high-molecular-weight RNA with a sedimentation constant identical to that of Newcastle disease virus.

Transcriptive functions of Sendai virus nucleocapsid in infected cells

The synthesis of virus-specific RNA in the cytoplasm of Ehrlich tumor cells infected with Sendai virus started within parental nucleocapsid structures which had a buoyant density in CsCl gradients of 1.31 g/ml and 1.38 g/ml. Two hours after infection the 1.31 nucleocapsid structure disappeared, and the 1.38 nucleocapsid structure was converted into a complex of density 1.45 g/ml which contained polysomes. The synthesis of virus-specific RNA proceeded within this complex. Cycloheximide did not prevent the formation of the 1.45 complex and the synthesis of virus-specific RNA, suggesting that parental nucleocapsid within the complex contained transcriptase. The 1.45 complex dissociated 3 hr after infection, and nascent RNA separated from the parental RNA. Our findings suggest that the 1.45 complex is a transcriptive complex which contains the parental nucleocapsid, and that genome RNA is transcribed within the nucleocapsid by a virion transcriptase.

The synthesis of virus-specific single-stranded RNA in larvae of Bombyx mori infected with a cytoplasmic polyhedrosis virus.

RNA synthesis in larvae of Bombyx mori was examined by theincorporation of 32P into purified single- and synthesis double-stranded RNAs. In larvae infected with a cytoplasmic polyhedrosis virus (CPV), both single- and double-stranded RNA were synthesized after pre-treatment with 10 µg actinomycin D. Approximately 70 % of the single-stranded RNA synthesized in these larvae re-annealed with heat-denatured RNA extracted from purified virions of B. mori CPV. It did not self-anneal, nor did it anneal significantly with RNA from Arctia caja CPV, or from B. morilaτvae. This suggested that the single-stranded RNA is largely virus-specific and represents viral messenger RNA.

Characterization of three complementation groups of vesicular stomatitis virus

The complementation properties of 25 temperature-sensitive mutants of VSV have been tested by measuring the yield of virus in mixedly infected cells at the nonpermissive temperature, 38°C. Two of the mutants, ts10W and ts16BW, complement all the mutants except each other and therefore form one distinct complementation group, A. Two other mutants, ts12W and ts29W do not complement each other but complement nearly all the other mutants and have been assigned to a second complementatation group, B. A group of six mutants, all of which show an increased heat lability of the virion, show no complementation within the group and have been assigned to a third group, C. Most of the remaining mutants complement at least one member of each of groups A, B, and C but have not yet been assigned to other groups since many of the crosses of these mutants with group C mutants fail to show complementation. Virus-specific RNA synthesis in L cells infected with the various mutants was investigated by measuring the incorporation of 14C-labeled uridine. All the group C mutants show little or no virus-specific RNA synthesis at 38°C, even at high multiplicities, suggesting that in these mutants the defect is in the structural RNA polymerase. The two mutants of group A show little or no virus-specific RNA synthesis at 38°C at low input multiplicities but considerable RNA synthesis at high multiplicities. Since this synthesis is not reduced by the presence of puromycin it would appear that these mutants have a structural polymerase which is functional at 38°C but are defective in some other type of RNA synthesis. Mutants of group B show RNA synthesis at 38°C comparable to that of the wild type at all multiplicities but fail to produce infectious virus after a shift from 30 to 38°C at any time during the growth cycle. The defect in these mutants therefore appears to be in some function such as the synthesis or assembly of structural proteins.

Effects of nucleoside analogs on transcription of simian virus 40

Brief incubation of BSC-1 cells with 5-fluorouridine (FUrd) produces an irreversible inhibition of RNA synthesis. The drug has no effect on the appearance of nuclear T-antigen or on the rate of viral DNA synthesis during lytic infection of BSC-1 cells. The ultimate yield of infectious virus is inversely related to the length of exposure to FUrd before infection. We have also followed the synthesis of virus-specific RNA in 3T3 cells transformed by SV40. The relative amount of hybridizable, virus-specific RNA in the cytoplasm of 3T3 cells transformed by SV40 and treated with FUrd is at least equal to or greater than in control, untreated cells. A strong inhibition of virus-specific RNA is found in cells treated with 5-bromotubercidin. These findings are consistent with previous conclusions that FUrd selectively inhibits rRNA but not mRNA synthesis, whereas 5-bromotubercidin reversibly inhibits both mRNA and rRNA.

Interaction of viral RNA with Escherichia coli: III. Synthesis of poliovirus-specific RNA directed by isolated poliovirus RNA

Poliovirus RNA directs the synthesis of virus-specific RNA in E. coli as reported previously for poliovirus-induced double-stranded RNA. Synthesis of viral RNA can be followed by conversion of viral RNA into a double-stranded RNase-resistant state, by increase in infectivity and by hybridization of newly synthesized RNA to viral RNA. Virus-specific RNA synthesis occurs also in the presence of inhibitors of protein synthesis indicating that an enzyme is present in E. coli which can use RNA as a template.

RNA polymerase activity in virions of parainfluenza 1 virus (Sendai).

Preparations of Sendai virus purified from infected allantoic fluid catalysed the incorporation of3H- or32P-labelled ribonucleoside triphosphates into an acid-insoluble product during incubation in an RNA polymerase reaction mixture containing the nonionic detergent Nonidet P-40. The requirements for the enzyme were established, and the product was shown to be ribonucleic acid. Comparison of the RNA synthesised by the enzyme with UNA formed in infected cells during inhibition of virus-induced protein synthesis by cycloheximide suggests that the virion enzyme is involved in early virus-specific RNA synthesis in infected cells.

Effects of arginine starvation on macromolecular synthesis in infection with type 2 adenovirus: II. Synthesis of virus-specific RNA and DNA

The effects of arginine starvation on DNA and RNA synthesis and on polyribosomes in KB cells infected with type 2 adenovirus were studied. Synthesis of viral DNA in arginine-starved (Arg −) cells was demonstrated by CsCl equilibrium density gradient centrifugation, zonal velocity sedimentation in alkaline sucrose gradients, and DNA-DNA hybridization. However, the amounts of cellular and especially viral DNA in Arg − cells were significantly lower than in cells maintained in complete medium (Arg +). No evidence was obtained to suggest that this quantitative difference was due to degradation of newly synthesized DNA in Arg − cells. Up to 12 hr after infection, no quantitative or qualitative difference was found between Arg + and Arg − cells with regard to viral mRNA transcribed. Thereafter (to 17 hr p.i.), the rate of viral RNA synthesis decreased but the same species continued to be synthesized. At 17.5 hr p.i., the lowering of the rate of RNA synthesis was reflected both in the nucleus and in the cytoplasm of Arg − cells and affected cellular as well as viral RNA. However, the relative distribution of viral mRNA between cytoplasmic fractions (polyribosomal and nonpolyribosomal) was unaffected by arginine deprivation. Polyribosomes decreased progressively after infection in Arg − cells while they increased in Arg + cells. The change in polyribosomal profile was not dependent on Ad2 infection, but also occurred in uninfected Arg − cells. Under both conditions, the changes induced by arginine deprivation were reversible by arginine restoration as late as 20 hr after infection and/or arginine deprivation. It is postulated that failure of virion assembly to occur in Arg − cells is not due to failure of a major structural viral constituent to be synthesized ( see also Rouse and Schlesinger, 1972); rather it must involve lack of an exquisitely arginine-dependent factor (or factors) needed for normal DNA synthesis and, especially, for enabling the DNA that is made to associate with the structural proteins that are also synthesized. This conclusion is supported by the earlier finding that the DNA preformed in Arg − cells does, but the preformed viral proteins do not, participate in virion assembly which occurs upon arginine restoration.

Studies on the biosynthesis and characterization of rhinovirus ribonucleic acid

The synthesis of rhinovirus type 20 specific RNA in infected HeLa cells was examined in the presence of actinomycin D and uridine-5- 3H. The virus induced in the infected cells the synthesis of two major components of RNA sedimenting at 32 S and 18 S, respectively, and a heterogeneous component with a sedimentation coefficient of 24–26 S. Treatment of virus-specific RNA components with ribonuclease resulted in the complete degradation of 32 S and 24–26 S components, while the 18 S component showed partial resistance to ribonuclease. Poliovirus type 1 under identical conditions induced the synthesis of two components of RNA sedimenting at 35 S and 18 S, respectively. Rhinovirus specific RNA in infected HeLa cells was first detectable between 4 and 5 hr postinfection at about the same time that infectivity first appeared intracellularly. The maximal synthesis of virus specific RNA took place between 6 and 7 hr postinfection. The purification of rhinovirus in cesium chloride resulted in the loss of infectivity and appearance of phosphotungstic acid-penetrable virus particles. RNA extracted from the purified virions sedimented with a major peak at 32 S but with a heterogeneous skewed type of sedimentation along the trailing edge.

The response of BHK21 cells to infection with type 12 adenovirus: VI. Synthesis of virus-specific RNA

When G1-arrested BHK21 cells are infected with type 12 adenovirus (Ad12), some of the newly synthesized RNA is virus specific. RNA-DNA hybridization-competition experiments have demonstrated that this RNA corresponds to up to 60% of those “early” mRNA sequences which are transcribed in productively infected human embryonic kidney (HEK) cells. No “late” Ad12 mRNA was detected in BHK21 cells. Comparison of Ad12-specific RNA sequences transcribed in Ad12-infected BHK21 cells and in Ad12-induced hamster tumor cells (line HT 2) by hybridization-competition reaction indicated that no virus-specific sequences are transcribed in HT 2 cells which are not also present in BHK21 cells abortively infected with Ad12. Attempts to detect any Ad12 DNA synthesis in BHK21 cells by DNA-DNA hybridization, under conditions of minimal background cellular DNA synthesis, were unsuccessful. Correspondingly, immunofluorescent staining experiments failed to detect Ad12 capsid protein synthesis in over 10 4 infected cells.

Synchronization of Rous sarcoma virus production in chick embryo cells

The growth of chick embryo fibroblasts transformed by and producing the Schmidt-Ruppin strain of Rous sarcoma virus was synchronized by exposure to serum-free medium. It was found that virus-specific RNA and protein synthesis and virus release are synchronized in these cells and occur in the early S and G 1 phases of the cell cycle, respectively. These findings support the hypothesis that RSV synthesis is dependent upon specific host cell processes. The duration of the phases of the cell cycle in transformed cells could not be distinguished from the pattern in uninfected cells. Evidence is presented which indicates that the synchrony of transformed cells is achieved by the low pH of the serum-free medium rather than by the absence of serum.

Fate of parental reovirus in infected cell

Parental reovirus is not completely uncoated after infection to yield free genome RNA. Instead it is converted into a subviral particle which contains besides 10 segments of double-stranded RNA and A-rich RNA, all core polypeptides namely λ1, λ2, μ1, and σ2 and a polypeptide with a molecular weight of 65,000 which is derived from the outer capsomere protein μ2 by cleavage. Isolated partially uncoated particles catalyze the synthesis of virus specific messenger RNA. Late during the replication cycle partially uncoated particles are converted into particles with a slightly higher density in CsCl than the virus by the addition of newly synthesized polypeptide σ3.

Virus-specific RNA formed in Newcastle disease virus-infected cells after suppression of protein synthesis by cycloheximide

In Newcastle disease virus-infected cells the accumulation of virus-specific RNA is prevented if protein synthesis is inhibited by cycloheximide at an early stage of infection. At a later stage cycloheximide enhances the synthesis of smaller (18–35 S) virus-specific RNA while the synthesis of 49 S RNA is suppressed. This is accompanied by a corresponding change in the relative amounts of “plus” and “minus” RNA strands: the proportion of “plus” strand is sharply decreased. A possible significance of the phenomenon for the regulation of virus-specific RNA synthesis is discussed.

Interferon Production by Double-stranded RNA: A Comparison of Induction by Reovirus to that by a Synthetic Double-stranded Polynucleotide

SUMMARY Treatment of chick cells with reovirus resulted in the production of an inhibitor of virus replication which was characterized as an interferon. No infectious virus was released, nor could any virus-specific RNA synthesis be detected in cells receiving virus. The ability of reovirus to induce interferon formation in chick cells was less sensitive to irradiation with ultraviolet light than was its ability to produce infectious virus in L cells. These data suggest that the interferon was induced by the double-stranded RNA of the inoculum virus. Comparison of this system with that induced by a synthetic double-stranded polynucleotide showed that the latter was considerably less sensitive to the effects of metabolic inhibitors, suggesting that the two inducers do not stimulate interferon formation by the same mechanism. The effect of addition of two metabolic inhibitors simultaneously to cells previously treated with a synthetic polynucleotide suggests that their effect is not a direct one on the formation of an interferon messenger RNA and its translation.

Fine structure of Sendai virus nucleocapsid.

Japanese Journal of MicrobiologyVolume 15, Issue 6 p. 549-551 NoteFree Access Fine Structure of Sendai Virus Nucleocapsid Yasuji Amano, Yasuji Amano Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this authorTakeshi Takano, Takeshi Takano Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this authorKazuko Takahashi, Kazuko Takahashi Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this authorNakao Ishida, Nakao Ishida Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this author Yasuji Amano, Yasuji Amano Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this authorTakeshi Takano, Takeshi Takano Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this authorKazuko Takahashi, Kazuko Takahashi Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this authorNakao Ishida, Nakao Ishida Department of Bacteriology, Tohoku University School of Medicine, SendaiSearch for more papers by this author First published: June 1971 https://doi.org/10.1111/j.1348-0421.1971.tb00619.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES 1Bikel, I., and Duesberg, P. H. 1969. Proteins of Newcastle disease virus and of the viral nucleocapsid. J. Virol. 4: 388– 393. 2Blair, C. D., and Robinson, W. S. 1968. Replication of Sendai virus 1. Comparison of the viral RNA and virus-specific RNA synthesis with Newcastle disease virus. Virology 35: 537– 549. 3Hosaka, Y. 1968. Isolation and structure of the nucleocapsid of HVJ. Virology 35: 445– 457. 4Klug, A., and Caspar, D.L.D. 1960. The structure of small viruses. Advances Virus Res. 7: 225– 325. 5Markham, R., Frey, S., and Hills, G. J. 1963. Methods for the enhancement of image detail and accentuation of structure in electron microscopy. Virology 20: 88– 102. 6Markham, R., Hitchborn, J. H., Hills, G. J., and Frey, S. 1964. The anatomy of the tabacco mosaic virus. Virology 22: 342– 359. 7Thon, F. 1966. On the defocusing development of phase contrast in electron microscopical images. Zschr. Naturforsch. 21a: 476– 478. 8Yada, K. 1967. Study of chrysolite asbestos by a high resolution electron microscope. Acta Cryst. 23: 704– 707. Citing Literature Volume15, Issue6June 1971Pages 549-551 ReferencesRelatedInformation

Replication and synthesis of Japanese encephalitis virus ribonucleic acids in Vero cells.

Neither replication nor ribonucleic acid synthesis of Japanese encephalitis virus (JEV) in Vero cells was affected by actinomycin D at a concentration of 0.5 μg/ml, but both were markedly inhibited by formycin A, an inhibitor of RNA synthesis, at a concentration of between 10 and 25 μg/ml. Synthesis of virus-specific RNA's was then studied in the presence of actinomycin D. The viral RNA synthesis, as estimated from the eventual virus yields in cells treated with formycin A at different intervals from infection, was found to proceed 6 to 7 hr in advance of mature virus formation which began at 10 hr after infection. The newly synthesized viral RNA detected by a 6-hr pulse-labeling with 3H-uridine was observed at the 43S region in a sucrose gradient centrifugation. With a shorter pulse, however, a rapidly labeled virus-specific RNA was detected first in the 10 to 15S region, which was soluble in 2 m NaCl, and this label moved to the ribonuclease-resistant 26S RNA during a subsequent 6-hr period. Further studies of this 26S RNA by pulse-chase experiments demonstrated that a minor 20S double-stranded form accumulated as a by-product in the viral 43S RNA replication. Thus the two ribonuclease-resistant forms of virus-specific RNA, 26S and 20S, were identified in JEV-infected Vero cells. Results of these experiments suggest that the 43S single-stranded viral RNA and the 20S double-stranded minor RNA might be formed through the intermediate type 26S RNA which is partially ribonuclease-resistant.

Synthesis of virus-specific RNA in cells infected with two different variants of measles virus.

Vero cells were infected with the DP (diluted passage) and UP (undiluted passage) measles virus variants at input multiplicities of 0.01 and 0.002, respectively. The amount and sedimentation characteristics of virus-specific RNA synthesized was determined by pulse-labelling with H3-uridine for periods of 4 hours in the presence of actinomycin D. During the first two days after infection the quantity and quality of RNA synthesized by the two variants was comparable. Hereafter the rate of RNA synthesis in DP virus-infected cells increased and reached maximum after another three days, at which time there were signs of a general cytopathic degeneration. In contrast hereto the rate of RNA synthesis in UP virus-infected cells decreased markedly during the period between two and five days after infection and remained at a low level during an additional observation period of ten days. Cytopathic degeneration of UP virus infected cultures did not become pronounced until between 10 and 14 days after infection.

Evidence for an RNA replicative intermediate in cells infected with Newcastle disease virus.

SUMMARY Cells infected with Newcastle disease virus contained significant amounts of base-paired RNA. Base pairing in this RNA was suggested by its resistance to ribonuclease and by the sharp thermal transition from ribonuclease resistance to sensitivity at 83° in 0.01 m-NaCl. The base-paired RNA sediments heterogeneously with a peak at 35 s. Preferential labelling of the base-paired RNA after short periods of incubation with [3H]uridine suggests that it may serve as an intermediate in the synthesis of single-stranded virus-specific RNA. The kinetics of [3H]uridine incorporation into the various species of virus-specific RNA are discussed.

Inhibition of rubella virus-specific RNA synthesis by interferon.

SummaryThe effect of interferon (IF) on rubella virus (RV) replication in Vero tissue culture cells was investigated. Viral-specific RNA components were synthesized in RV-infected Vero cells in the presence of actinomycin D. The synthesis of these RNA species was shown to be inhibited by IF.

Actinomycin D and influenza virus multiplication in the chick embryo fibroblast

The effects of actinomycin D on the replication of the WSN strain of influenza virus were studied in the chick embryo fibroblast. No virus is produced when actinomycin is added to cells within the first hour of infection. When added at later times, the yields of infectious particles are reduced. Actinomycin blocks the synthesis of virus-specific RNA at any time it is added. Synthesis of viral proteins is not detected if cells are exposed to actinomycin within the first hour of infection, but if the drug is added after the second hour, viral proteins continue to be synthesized and incorporated into virus particles for at least 17 hours, even though synthesis of viral RNA has been blocked. The effect on the synthesis of viral RNA is not reversed by removal of the drug.