Analyses of the mRNA transcription processes of Punta Toro phlebovirus (Bunyaviridae)

The time course of the syntheses of Punta Toro (PT) phlebovirus (Bunyaviridae) small (S)-size viral RNA (S vRNA), viral complementary RNA (S vcRNA), and messenger RNA (S mRNA) species has been analyzed using single-stranded DNA probes representing the two S-coded gene products. The data obtained support the conclusion that PT S RNA has an ambisense coding strategy (T. Ihara, H. Akashi, and D. H. L. Bishop, Virology 136, 293-306, 1984) with the viral nucleocapsid protein, N, encoded in a viral-complementary, subgenomic, mRNA species and a putative nonstructural protein, NSs, encoded in a viral-sense, subgenomic, second S mRNA species. In the absence of puromycin (or cycloheximide) full-length S vRNA, S vcRNA, and subgenomic N mRNA and putative NSs mRNA species were identified in PT virus-infected cell extracts. In the presence of inhibitors of protein synthesis (puromycin or cycloheximide) newly synthesized N mRNA species were detected, but not full-length S vcRNA, nor S vRNA, nor the S coded NSs mRNA species. The mRNA species recovered from drug-treated cells have been translated in vitro to synthesize viral N protein. Analyses of the 5' ends of the N and NSs mRNA species have shown them to be heterogeneous in sequence and some 11-18 bases longer than the ends of the genomic RNA species, indicating that they represent nonviral primer sequences like those identified for bunyavirus mRNA species (D. H. L. Bishop, M. E. Gay, and Y. Matsuoka, Nucleic Acids Res. 11, 6409-6418, 1983). The presence of such additional sequences on mRNA derived from representatives of two Bunyaviridae genera appears by these analyses to be a more conserved feature than the S RNA coding arrangement of the respective viruses.     

Replication in vitro of the influenza virus genome: selective dissociation of RNA replicase from virus-infected cell ribonucleoprotein complexes

Summary Replication of the influenza virus genome involves two discrete step reactions: vRNA-directed primer-independent (unprimed) synthesis of cRNA; and cRNA-directed unprimed synthesis of vRNA. Nuclear extracts from both MDCK and HeLa cells infected with influenza virus A/PR8/34 exhibited unprimed synthesis of both cRNA and vRNA strands (a parameter of RNA replication). Ribonucleoprotein (RNP) complexes with the replication activity were isolated from these nuclear extracts by glycerol gradient centrifugation in the presence of 0.1 M KCl. At 0.5 M KCl, however, these complexes were dissociated into stripped RNP and soluble protein fractions. The soluble fraction contained the activity of exogenous template-dependent unprimed RNA synthesis, indicating that the RNA replicase is dissociated from RNP upon exposure to high salt concentrations. On the other hand, the high salt-treated RNP catalyzed only primer-dependent RNA synthesis, but regained a low level activity of exogenous template-dependent unprimed RNA synthesis by adding nuclear extracts from uninfected cells, suggesting that host factor(s) is involved in the functional interconversion of influenza virus RNA polymerase.     

Virus-specific RNA species present in the cytoplasm of Rous sarcoma virus-infected chicken cells

Polyadenylated and nonpolyadenylated cytoplasmic RNA from chicken cells infected with wild-type (wt) and transformation-defective (td) Rous sarcoma virus (RSV) were fractionated on sucrose density gradients and hybridized with a [3H]cDNA probe complementary to RSV-RNA to determine the size distribution of virus-specific RNA. Two major species of poly (A) -containing viral RNA sedimenting at 35 and 21 S were detected in wt and td virus-infected cells. Both RNA classes were released from polyribosomes with puromycin, suggesting that each RNA species serves as messenger in vivo. Another minor size class of virus-specific RNA, 10-12 S, was found in both the polyadenylated and nonpolyadenylated RNA fractions.     

Barley stripe mosaic virus in sperm and vegetative cells of barley pollen

We have developed a method that allows us to isolate with high yield RNP particles containing intact RNA from BHK21 cells infected with Semliki Forest virus. Two types of virus-specific RNA sedimenting with about 42 S and 26 S on sucrose gradients are found in the gradient fractions containing the large and intermediate-sized polyribosomes. That the 26 S RNA and at least 50% of the 42 S RNA are presumably part of the polyribosomes has been shown by CsCl density gradient analysis of the fixed ribonucleoprotein particles. We suggest therefore that both molecules are virus specific mRNA. From the quantitative aspects of our results, we conclude that they are the main species of virus specific polyribosome associated RNA in the infected cells. The total virus-specific RNA synthesized during different 2 hr intervals, which is incorporated into polyribosomes, is greater than 20% during the 0 to 2 hr p.i. interval and decreases later. We suggest therefore that especially during the first hours after infection, a major part of the total RNA synthetic capacity of the virus is directed toward mRNA production. Both polyribosome-associated molecules do not hybridize to RNA extracted from the SFV particle.     

Inhibition of protein synthesis by vaccinia virus. II. Studies on the role of virus-induced RNA synthesis

Cytoplasmic RNA synthesis can be detected in vaccinia virus-infected HeLa cells in the presence of 2 micrograms/ml but not 20 micrograms/ml of actinomycin D. When RNA synthesis is observed protein synthesis is inhibited in infected, treated cells. We had previously noted that such a correlation may also be observed in infected, cycloheximide-treated cells. If actinomycin D (20 micrograms/ml) is added to these cells at various times after infection and treatment, the inhibition of protein synthesis seen upon removal of cycloheximide does not continue beyond the point to which it had developed before the actinomycin D was added. These results indicate that the inhibition of protein synthesis can be correlated with the amount of cytoplasmic RNA synthesized in infected cells and that this RNA synthesis and the subsequent inhibition of protein synthesis can be prevented by sufficiently high concentrations of actinomycin D. The cytoplasmic RNA which is synthesized does not appear to consist of double-stranded RNA nor of extensive self complementary regions. The cytoplasmic RNA synthesized in infected, cycloheximide treated cells appears to consist of early virus mRNA which can function as mRNA in vitro in a cell-free system derived from normal cells. An examination of the phosphorylation of ribosomal proteins shows six additional phosphoproteins in infected cells, two of which may be observed in infected cycloheximide-treated cells, suggesting that phosphorylation of ribosomal proteins cannot be directly correlated with the inhibition of overall protein synthesis seen in infected cycloheximide-treated cells.     

Sorting vector producer cells for high transgene expression increases retroviral titer

Vector producer cells are derived from helper cell lines expressing viral proteins that have been transduced to express a transgene-carrying retroviral genome. Vector producing cells express two relevant forms of RNA in their cytoplasm: vector RNA (vRNA) that is packaged as the actual gene transfer agent, and messenger RNA (mRNA) from which transgene is translated. Two premises underlie this study: (1) vRNA is limiting for virus production and (2) mRNA is proportional to vRNA. Together, these premises predict that transgene expression in the vector producing cells will be predictive of the viral titer from those cells. In this case, sorting the vector producing cells for high transgene expression should select for more virus production in vector producing cell supernatants. This prediction was supported, with a greater than fivefold benefit in viral titer. This demonstrates a rapid and simple method by which to obtain significantly increased viral titers from the same vector producing cell preparation.     

Sequence complementarity of sonchus yellow net virus RNA with RNA isolated from the polysomes of infected tobacco

Polyribosomal RNA from tobacco infected with sonchus yellow net virus (SYNV) contained sequences which hybridized to 125I-labeled SYNV RNA and which were complementary to 80 to 100% of the viral RNA genome. The poly(A)-containing RNA from polyribosomes was complementary to over 90% of the viral genome but the polyribosomal RNA lacking poly(A) hybridized to approximately 40-60% of the genome. The kinetics of hybridization of all three fractions are best explained by the presence of a single abundance class of viral-complementary RNA. However, titration hybridization of poly(A)+ RNA to an excess of SYNV RNA suggested that viral-complementary sequences which contain poly(A) may vary in concentration over a factor of about fivefold. About 1.5 to 4.6% of the fraction containing poly(A), 0.02 to 0.06% of the fraction lacking poly(A) and 0.04 to 0.18% of the total polyribosomal RNA was complementary to viral RNA as estimated from the kinetics of hybridization. The viral complementary RNA(vcRNA) was heterogeneous in size with a modal sedimentation coefficient of 12 S and a profile in sucrose density gradients similar to the polyadenylated polyribosomal RNA.     

Subcellular distribution of RNA sequences complementary to sonchus yellow net virus RNA

RNA isolated from free and membrane-bound polyribosomes of sonchus yellow net virus (SYNV)-infected tobacco was hybridized to SYNV RNA. RNA from free polyribosomes was shown to be complementary to nearly 100% of the SYNV genome, whereas RNA from membrane-bound polyribosomes was complementary to only 40% of the SYNV RNA. When RNA derived from the two classes of polyribosomes was fractionated by chromatography on oligo(dT)-cellulose, sequences complementary to SYNV RNA were present in both bound and unbound fractions. Neither nuclear RNA nor poly(A)-containing nuclear RNA from SYNV-infected plants hybridized to SYNV RNA. The results suggest that SYNV messenger RNAs are selectively partitioned in the cytoplasm and that hybridizing sequences are not abundant in the nuclei of infected cells.     

Translation of 26 S virus-specific RNA from Semliki Forest virus-infected cells in vitro

Two types of virus-specific RNA are associated with polyribosomes of BHK-21 cells 3 hr after infection with Semliki Forest virus. These RNA species sediment at about 42 S and 26 S, respectively, in sucrose density gradients. Addition of the 26 S RNA after isolation from polyribosomes to an in vitro protein-synthesizing system derived from Krebs II ascites cells stimulated the incorporation of [³⁵S]methionine into one protein. This protein has the same electrophoretic mobility on SDS polyacrylamide gels and an identical fingerprint pattern after tryptic digestion as the core protein of purified Semliki Forest virus. 26 S RNA prepared by extraction of total RNA with phenol from infected cells and repeated sucrose density-gradient centrifugation also stimulated protein synthesis in vitro. A possible role of the 26 S RNA as mRNA for all viral structural proteins is discussed.