Cytoplasmic Fractions Associated with Semliki Forest Virus Ribonucleic Acid Replication

When actinomycin D-treated chick fibroblasts were labeled with (3)H-uridine for varying periods during the log phase of Semliki Forest virus infection, radioactivity was found associated with different cytoplasmic fractions. After a 1-min period of labeling, it appeared in a large cytoplasmic structure which was seen in electron micrographs of infected cells. Sediments of sucrose density gradients of cytoplasmic extracts of these cells also contained these structures. Three forms of viral ribonucleic acid (RNA) were associated with this cytoplasmic structure: a ribonuclease-sensitive 42S form identical to the RNA of the mature virus, a ribonuclease-sensitive 26S form, and a ribonuclease-resistant 20S form. After a 5- to 10-min labeling period, radioactivity was associated with a ribonuclease-sensitive 65S cytoplasmic fraction which contained only the 26S RNA form. Finally, after a 1-hr labeling period, a 140S ribonuclease-resistant particle was the most prominent radioactive structure in the cytoplasm. This particle contained only 42S viral RNA. Negative-contrast electron micrographs of the 140S particle and the virion demonstrated structural differences between them. The base compositions of the 42S and 26S viral RNA forms were not significantly different. The base composition of the 20S form differed significantly from that of the other two viral RNA forms, but the values obtained for the mole fractions of the bases present in the 20S form differed, and depended on the period during the virus growth cycle in which (32)P was present. These results suggested that viral RNA originated in the large cytoplasmic body. The 20S RNA appeared to be a structure engaged in viral RNA replication and the 140S particle appeared to be a virus precursor.     

Growth Kinetics of Yaba Tumor Poxvirus After In Vitro Adaptation to Cercopithecus Kidney Cells

Yaba tumor poxvirus has been adapted to continuous in vitro cultivation in monolayers of cercopithecus kidney cells. At 35 C, the minimum replicative cycle, after synchronous infection of CV-1 cells with multiplicity of infection of 135 focusforming units per cell, was 35 hr; however, maximum virus yields were not obtained until 75 hr postinfection (PI). Cytoplasmic incorporation of (3)H-thymidine [viral deoxyribonucleic acid (DNA) synthesis] was detected 3 hr PI and was preceded by synthesis of nonstructural associated antigens (YS). Synthesis of YS antigens was not inhibited by the DNA inhibitor, arabinofuranosyl cytosine (ARA-C). Synthesis of at least two virion structural antigens, although not detected by immunofluorescence until 2 hr after the onset of DNA synthesis, occurred in the presence of ARA-C, indicating potential translation of these structural antigens from parental DNA. The first progeny DNA was completed by 20 hr PI but was not detected in infectious form until 35 hr PI. The maximum rate of progeny DNA completion occurred between 20 and 30 hr PI. DNA synthesis continued 45 to 50 hr PI. The adapted virus retained its oncogenicity and, like the wild type, replicated better at 35 C than at 37 C. A synthetic step associated with viral DNA synthesis appears to be temperature-sensitive.     

Electrophoretic Characterization of Foot-and-Mouth Disease Virus-Specific Ribonucleic Acid

Foot-and-mouth disease virus (FMDV)-specific ribonucleic acid (RNA) was analyzed by electrophoresis on 0.5% agarose gels. Four classes of RNA were resolved as a function of mobility in agarose: two classes of slowly migrating multistranded RNA, the infectious viral RNA with intermediate mobility, and a minor fast-moving class of lower-molecular-weight single-stranded RNA. The major RNA species were infectious viral RNA and the slowest migrating class of multistranded RNA. The latter RNA was polydisperse when analyzed by sucrose gradient centrifugation, it was partially ribonuclease resistant, and it was the predominant RNA species labeled during the initial period of (3)H-uridine triphosphate incorporation in the cell-free system. Heat treatment studies indicated that part of the slowest-moving RNA was degraded at 60 C and almost complete degradation was detected at 100 C. It was concluded that this RNA is the replicative intermediate in viral RNA synthesis. The second class of multistranded RNA contained both a ribonuclease-resistant RNA and a second RNA peak which was detected only after heat treatment at temperatures above 75 C. Fractions of FMDV-specific RNA isolated by sucrose gradient centrifugation were analyzed by agarose-gel electrophoresis. Infectious viral RNA was detected only in the 37S zone and was the major species of RNA in this part of the gradient. The ribonuclease-resistant RNA (the 20S zone) contained about equal amounts of multistranded RNA (both classes) and the low-molecular-weight single-stranded RNA. All sucrose gradient fractions between 20 and 40S were found to contain the replicative intermediate, although the major portion was detected in the 20 to 25S region.     

THE SYNTHESIS OF 5S RIBOSOMAL RNA DURING POLLEN DEVELOPMENT

Tradescantia paludosa 5S ribosomal RNA (rRNA) has been characterized with respect to its base composition and relative electrophoretic mobility in comparison with that of E. coli . The period of 5S rRNA synthesis during pollen grain development was determined by pulse labeling the RNA synthesized during a 24 hr period of development with ³²P and then chasing in cold medium until pollen maturity. The period of highest 5S rRNA synthesis was found to occur prior to microspore mitosis. During and following mitosis over a period of 4 days there was a sharp decrease in the amount of 5S RNA synthesized and during the last 48 hr of pollen maturation, no 5S rRNA was synthesized.     

Basis for Variable Response of Arboviruses to Guanidine Treatment

The effect of guanidine on the replication of the group A arboviruses, Sindbis virus, and Semliki Forest virus (SFV) was studied. Guanidine rapidly, but reversibly, inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of viral RNA was inhibited, but that of ribonuclease-resistant forms was least affected. This inhibition occurred when the drug was added at any point during the log phase of virus growth. The growth of SFV was also markedly inhibited, but Sindbis virus growth was unimpaired. Infection of guanidine-treated cells with the viruses together resulted in a significant inhibition of the yields of both. It appears that, in the case of Sindbis virus, viral RNA is ordinarily produced in such excess that inhibition of its synthesis does not reduce virus yields. In the case of SFV, guanidine also markedly distorts the pattern of RNA synthesis by greatly decreasing the production of the 26S interjacent RNA form. This may account for the observed inhibition of SFV growth in the presence of guanidine.     

Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [³H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 10⁶ and 1.45 × 10⁶ daltons, respectively, in comparison with molecular weights of 1.29 × 10⁶ and 0.68 × 10⁶ daltons for the completed ribosomal RNA's.     

Effect of Actinomycin D on Virus-induced Ribonucleic Acid Polymerase Formation in Foot-and-Mouth Disease Virus-Infected Baby Hamster Kidney Cells

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.     

TRANSPORT OF VIRAL RNA IN KB CELLS INFECTED WITH ADENOVIRUS TYPE 2

Messenger RNA transport was studied in KB cells infected with the nuclear DNA virus adenovirus type 2. Addition of 0.04 µg/ml of actinomycin completes the inhibition of ribosome synthesis normally observed late after infection and apparently does not alter the pattern of viral RNA synthesis: Hybridization-inhibition experiments indicate that similar viral RNA sequences are transcribed in cells treated or untreated with actinomycin. The polysomal RNA synthesized during a 2 hr labeling period in the presence of actinomycin is at least 60% viral specific. Viral messenger RNA transport can occur in the absence of ribosome synthesis. When uridine-³H is added to a late-infected culture pretreated with actinomycin, viral RNA appears in the cytoplasm at 10 min, but the polysomes do not receive viral RNA-³H until 30 min have elapsed. Only 25% of the cytoplasmic viral RNA is in polyribosomes even when infected cells have been labeled for 150 min. The nonpolysomal viral RNA in cytoplasmic extracts sediments as a broad distribution from 10S to 80S and does not include a peak cosedimenting with 45S ribosome subunits. The newly formed messenger RNA that is ribosome associated is not equally distributed among the ribosomes; by comparison to polyribosomes, 74S ribosomes are deficient at least fivefold in receipt of new messenger RNA molecules.     

Interferon Action on Parental Semliki Forest Virus Ribonucleic Acid

Actinomycin D-treated chick fibroblasts were infected with purified (32)P-labeled Semliki forest virus, and ribonucleic acid (RNA) was extracted after 1 or 2 hr. Within 1 hr, viral RNA forms sedimenting in sucrose gradients at 42S, 30S, and 16S were present. The 42S form corresponded to the RNA of the virion. The 16S form appeared to be a double-stranded template for the formation of new viral RNA, since nascent RNA was associated with it and the molecule could be heat-denatured and subsequently reannealed by slow cooling. Interferon treatment before infection, or puromycin (50 mug/ml) or cycloheximide (200 mug/ml) added at the time of virus infection, had no effect on the formation of the 30S RNA but inhibited the production of the 16S form. Several findings made it unlikely that these results were due to breakdown of parental RNA and reincorporation of (32)P into progeny structures. The results suggested that the mechanism of interferon action involves inhibition of protein synthesis by parental viral RNA, since a specific viral RNA polymerase had previously been demonstrated to be necessary for production of 16S RNA. No protein synthesis appears necessary for formation of 30S RNA from parental virus RNA.     

Reovirus-directed Ribonucleic Acid Synthesis in Infected L Cells

Reovirus replication in L-929 mouse fibroblasts was unaffected by 0.5 mug of actinomycin per ml, a concentration which inhibited cell ribonucleic acid (RNA) synthesis by more than 90%. Under these conditions of selective inhibition, the formation of both single-stranded and double-stranded virus-specific RNA was detected beginning at 6 hr after infection. The purified double-stranded RNA was similar in size and base composition to virus RNA and presumably was incorporated into mature virus. The single-stranded RNA formed ribonuclease-resistant duplexes when annealed with denatured virus RNA but did not self-anneal, thus indicating that it includes copies of only one strand of the duplex. The single-stranded RNA was polyribosome-associated and may function as the virus messenger RNA. Production of both types of virus-induced RNA required protein synthesis 6 to 9 hr after infection. At later times in the infectious cycle, only double-stranded RNA synthesis was dependent on continued protein formation.     

Ribonucleic Acid Transcriptases in Sendai Virions and Infected Cells

Sendai virions contain an enzyme which catalyzes the incorporation of ribonucleotides into ribonucleic acid (RNA). Enzyme activity was optimal at pH 8.0 and 28 C; otherwise conditions were similar to those reported for Newcastle disease virion (NDV) RNA polymerase. The initial rate of RNA synthesis by the Sendai virion enzyme was about 10 pmoles per mg of protein per hr, but after 3 hr of incubation the rate increased about fivefold. The virion enzyme was compared with an RNA polymerase in the microsomal fraction of infected cells. Both enzymes made predominantly single-stranded RNA which was complementary in base sequences to 50S virion RNA. Most of the RNA synthesized by the virion polymerase sedimented at 16S, but the product of the microsomal enzyme sedimented at about 8S.     

Method of examining viral RNA metabolism in cells in culture: metabolism of vesicular stomatitis virus RNA.

A method is described for radioactively labeling viral RNA and then quickly halting further incorporation of radioactive precursor into RNA so that the fate of the labeled RNA can be followed. Small complementary RNAs synthesized during vesicular stomatitis virus infection in the presence of cycloheximide do not metabolize to virion length molecules when protein synthesis inhibition is reversed.     

Initiation sites for translation of sindbis virus 42S and 26S messenger RNAs.

Sindbis virus 26S RNA is the principal species of virus-specific RNA found in the infected cell; it is derived from a one third segment of virion 42S RNA. When translated in cell-free extracts from mouse ascites cells or rabbit reticulocytes, 26S RNA directed the synthesis primarily of the 33,000 dalton virus capsid protein, and the protein products were in the form of free peptides rather than peptidyl-tRNA. In contrast, the polypeptides synthesized in either extract in response to Sindbis virus 42S RNA were heterogeneous, ranging in molecular weight from 33,000 to 190,000, and were largely in the form of peptidyl-tRNA. The number of independent initiation sites on the 26S and 42S RNAs was determined by analyzing a tryptic digest of reaction products labeled with yeast N-formyl-35S-methionyl-tRNAFmet. The 26S RNA appeared to contain a single initiation site, and this site could also be found in varying amounts in different preparations of 42S RNA. However, a second initiation site, distinct from that of 26S RNA, was the major site in 42S virion RNA. These results suggest that 42S virion RNA contains two potential sites for initiation of protein synthesis. Only one of these may be active, however, and it is postulated that the second site functions primarily, if not exclusively, in the subgenomic 26S RNA species. In this regard, Sindbis virus 42S RNA may represent a novel form of a eucaryotic messenger RNA.     

RNA SYNTHESIS IN THE DIFFERENT PHASES OF CELL CYCLE OF CHICK EMBRYO CELLS

RNA synthesis was studied at different phases of the cell cycle of chick embryo fibroblasts, which were synchronized by medium replacement in the confluent phase. The synthesis of DNA started at 4 hr and continued for 8 hr. RNA synthesis increased with time after medium change. The ratio of total amount of radioactivity in nuclear RNA prepared at 0, 2 and 8 hr was 1.0:1.03:5.05. The distribution of radioactive RNA in the sedimentation pattern was similar, showing remarkable incorporation in 45S region of ribosomal precursor RNA. The base composition of newly synthesized RNA, however, varied at different time intervals after medium replacement. Even within the G₁ phase, the molar percentage of G and C was quite different. Treatment with actinomycin D at a concentration of 0.02 μg/ml for 1 hr specifically inhibited ribosomal RNA synthesis. At 2 hr after medium change, ribosomal and AU-rich RNA including larger than 28S were synthesized in about equal amounts.     

Characterisation of rinderpest virus RNA and the action of actinomycin D on its replication

RNA extracted from purified rinderpest virus was characterised by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. The predominant virion RNA species had a sedimentation constant of 46S and its estimated molecular weight was 4.8 × 10⁶ daltons. Consistently high amounts of UMP and AMP were detected. The melting-temperature profile of the virion RNA suggested absence of secondary structure. The effect of actionomycin D on the replication of rinderpest virus in Vero cells was studied by following the viral RNA synthesis using labelled uridine as well as by infectivity titration. The viral RNA synthesis was not affected until 12 h following infection and was inhibited thereafter between 18 and 48 h to an extent of 25% at 5 and 10 Μg levels of the drug. A 100 to 1000-fold reduction in the infectivity titres was observed in the presence of the drug. These results suggest that actinomycin D inhibits rinderpest viral RNA replication. Sedimentation analysis of viral RNA extracted from drug-treated cultures showed inhibition of the genome RNA of rinder-pest virus.     

Defective Interfering Passages of Sindbis Virus: Nature of the Intracellular Defective Viral RNA

BHK cells infected with defective-interfering passages of Sindbis virus accumulate a species of RNA (20S) that is about half the molecular weight of the major viral mRNA (26S). We have performed competitive hybridization experiments with these species of RNA and have established that 20S RNA contains approximately 50% of the nucleotide sequences present in 26S RNA. Our further studies, however, demonstrate that 20S RNA is unable to carry out the messenger function of 26S RNA. We found very little of the defective RNA associated with polysomes in vivo. In addition, it was unable to stimulate protein synthesis in vitro under conditions in which 26S RNA was translated. We have also examined viral RNA synthesis in BHK cells infected with standard or defective-interfering passages of Sindbis virus. This comparison suggests that defective partioles do not synthesize a functional replicase.     

Dexamethasone Induction of the Intracellular RNAs of Mouse Mammary Tumor Virus

We have studied the kinetics of dexamethasone induction of mouse mammary tumor virus (MMTV) RNAs and proteins in virus-infected rat XC cells and GR mouse mammary tumor cells. A detectable increase in viral RNA in infected XC cells was present within 10 min after hormone addition, and half-maximal induction was achieved in less than 2 h. The increase in viral RNA concentration was apparent first in nuclear RNA and later in the cytoplasm. Within the first 15 min of induction, only genome-sized RNA (35S, 7.8 kilobases) was present in augmented amounts, whereas the major subgenomic RNA (24S, 3.8 kilobases) did not appear until at least 30 to 60 min postinduction. The sequential appearance of these RNAs, the probable mRNA's for the gag and env proteins, paralleled the order of appearance of the gag and env proteins, respectively, after hormone treatment. An additional species of viral RNA (20S, 2.5 kilobases) was detected during these induction experiments, but the role of this RNA is not known. Both subgenomic RNAs contain sequences derived from both the 5′ and 3′ termini of genomic RNA and are presumably spliced. After dexamethasone induction of infected XC cells, we detected two smaller env-related proteins which were not found in full hormone induction. The functional role of these smaller proteins is not known. A previously reported smaller species of RNA (13S, 1.0 kilobase) did not appear to be induced and was shown to be cellular rather than viral in origin. In the fully induced infected XC and GR mammary tumor cells, the only viral RNAs present were the 35S and 24S RNAs. In addition, mammary tumors contained only these two viral RNAs. Thus, tumor cells appear to contain only the viral RNAs which direct the synthesis of the gag, pol, and env proteins of the virion.     

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes.     

Morphogenetic pathway of bacteriophage φ6: A flow analysis of subviral and viral particles in infected cells

Three types of virus-specific particles of double-stranded RNA bacteriophage φ6 were isolated and characterized by pulse-label and pulse-chase experiments on φ6-infected Pseudomonas phaseolicola. The first particle was “previrion I”, which consisted of early proteins P1, P2, P4 and P7, and had no RNA. It was detected immediately after labeling of proteins and the radioactivity was chased into the second structure, designated previrion II, after ten minutes. Previrion II contained three segments of double-stranded RNA in addition to the component of previrion I, and had RNA polymerase activity that produced messenger RNA species coding for late proteins. The RNA polymerase activity in the cell extract emerged nearly in parallel with the synthesis of late proteins, and this activity of previrion II was supposed to be responsible for late protein synthesis in infected cells. Via previrions I and II, the third radioactive particle was observed in infected cells after late protein synthesis started. This particle was identified as the intact virion, because it had infectivity as well as all of the viral components, including lipids. This intact virion was accumulated in the infected cell before bursting the cell.