Synthesis of VSV RNPs in vitro by cellular VSV RNPs added to uninfected HeLa cell extracts: VSV protein requirements for replication in vitro

Viral ribonucleoprotein (RNP) particles isolated from vesicular stomatitis virus (VSV)-infected cells synthesized genome-length, complementary viral RNA, in addition to viral messenger RNA, in the presence of uninfected HeLa S10 extracts. The newly synthesized viral RNA was assembled into an RNP-like structure. RNA replication in vitro ceased when protein synthesis was blocked with pactamycin. Antibody raised against VSV NS protein inhibited in vitro RNA replication as well as mRNA synthesis. Anti-N protein also inhibited RNA replication, although it has no effect on the synthesis of mRNAs. Anti-G and anti-M IgG had no effect on either reaction. Anti-L IgG stimulated RNA replication 1.5- to 2-fold, lthough the synthesis of mRNA was inhibited.     

Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.     

Studies on the synthesis of viral RNA-polymerase-template complexes in BHK 21 cells infected with Semliki Forest virus.

Two main types of virus-specific RNA sedimenting at about 42 and 26 S, respectively, on sucrose density gradients are synthesized in animal cells infected with Semliki Forest virus (SFV). Two and one-quarter hours after infection of BHK 21 cells with SFV only about 10% of the total amount of virus-specific RNA that accumulates during the 8-hr growth cycle of the virus have been synthesized. If at this time an inhibitor of protein synthesis, cycloheximide, is added to infected cells, both of these RNA species are synthesized during the viral growth cycle with the same time course and in amounts similar to those in untreated cultures. This result suggests that the RNA polymerase-template complexes present at 2.25 hr post infection (p.i.) are stable and synthesize all virus-specific 42 and 26 S RNA accumulating during the viral growth cycle. These polymerase-template complexes are synthesized between 45 min and 2.25 hr p.i.During multiplication of SFV in BHK 21 cells the amount of radioactivity incorporated from radioactively labeled amino acids into protein during a series of labeling intervals decreases, and the ratio of synthesis of virus-specific proteins to cellular proteins increases. Both of these effects are not expressed to a significant extent at 2.25 hr post infection (p.i.). Sodium dodecyl sulfate-polyacrylamide-gel electrophoretic (SDS-PAGE) analysis of the polypeptides synthesized in infected cultures shortly after removal of cycloheximide, which had been present from 2.25 – 6 hr p.i. shows that cycloheximide does not block the development of these changes in cellular protein synthesis. Some implications of these findings concerning the synthesis of virus-specific RNA and the interference of virus multiplication with cellular protein synthesis are discussed.     

Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus: I. Rescue in cell cultures from different species

The replication of two DNA viruses, vaccinia and pseudorabies (PsRV), was not inhibited in three cell lines of rabbit origin (RK-13 and RK-1337, rabbit kidney; and RC-60, rabbit cornea) which had been pretreated with rabbit interferon. In contrast, the replication of vesicular stomatitis virus (VSV), an RNA virus, was susceptible to interferon-induced resistance in rabbit cell lines. These results reinforced the possibility that there are separate interferon-induced resistance factors for RNA and DNA viruses and that cultured rabbit cells are deficient in the synthesis of the resistance factors needed to inhibit DNA viruses.When cell cultures of rabbit origin were pretreated with homologous interferon and doubly infected with vaccinia and VSV, a variety of responses was observed. Vaccinia was able to rescue VSV from the inhibitory effects of interferon-induced resistance in two rabbit cell lines (RK-13 and RC-60), but not in RK-1337 cells. Similar experiments were carried out in mouse L cells and in primary chick embryo (CE) cell cultures; both RNA and DNA viruses were susceptible to inhibition by interferon-induced resistance in these cells. In L cells, double infection with vaccinia was able to rescue VSV; however, in CE cell cultures superinfection with vaccinia did not rescue VSV from the inhibitory effect of interferon. In these cells the synthetic pathways or virion factors furnished by vaccinia which are required for the rescue of VSV are sensitive to the action of interferon.     

the influence of the host cell on the inhibition of virus protein synthesis in cells double infected with vesicular stomatitis virus and mengovirus

The ability of mengovirus to inhibit the synthesis of vesicular stomatitis virus (VSV) proteins and of VSV to inhibit the synthesis of mengovirus proteins during double infection in three different cell lines was investigated. Although cellular protein synthesis was inhibited after infection of cells by each virus, the ability of one virus to decrease translation of the mRNA species of the co-infecting virus varied with the cell type. Superinfection of mengovirus-infected L-929 cells by VSV resulted in essentially no inhibition in the synthesis of either mengovirus or VSV proteins. In HeLa cells and CHO cells the synthesis of both VSV and mengovirus proteins was inhibited under conditions of simultaneous or sequential infection. The inhibition of VSV protein synthesis after infection of HeLa cells by mengovirus was not a result of a modification or inactivation of virus mRNAs. When extracted from double infected cells, the VSV mRNAs manifested normal biological activity, as determined by their ability to stimulate the synthesis of VSV proteins in a micrococcal nuclease-treated cell-free system from L cells. The interference of non-interference of one virus by another in different cell lines was also measured by quantifying the number of infectious particles produced in each cell line. The results were similar to those reported above for protein synthesis inhibition. These experiments suggest that the interference of mengovirus with VSV mRNA translation in HeLa cells is not necessarily reflective of the mechanism by which mengovirus inhibits cellular protein synthesis. Also, the host cell appears to influence the extent or nature of the interference of one virus by the other.     

Relation between the levels of mRNA abundance and kinetics of protein synthesis in pseudorabies virus-infected cells

Virus proteins synthesized by pseudorabies virus-infected cells can be classified into five groups on the basis of the kinetics of their synthesis at various stages of the infective process; virus mRNAs can similarly be classified into four groups. To determine whether the kinetics of synthesis of specific proteins are determined solely by the level of abundance in the cells of their mRNAs, we have compared at various times after infection the relative synthesis of these proteins with the relative abundance of their mRNAs. We have focused on two proteins: the 142K major capsid protein, an early-late protein, and the 136K major DNA binding protein, an early protein. The mRNAs encoding these proteins were identified. The relative abundances of these mRNAs in the cytoplasms of the infected cells were found to be the same as those associated with the polysome fractions. The relative amount of the proteins synthesized by the infected cells at a given stage of the infective process closely reflected the relative amount of the mRNA encoding these proteins that was present in the cells at that stage of the infective process. Most virus mRNA species that are present in the cytoplasm of infected cells were represented on polysomes to approximately an equal extent. Some RNA species were, however, significantly underrepresented under certain conditions in the polysomal fractions. We conclude that whereas the amount of many virus proteins synthesized by the infected cells is determined mainly by the level of the abundance of their mRNAs, additional controls operate in the cells that determine the relative rates of synthesis of some other virus proteins.     

Regulation of the synthesis of Sindbis virus-specified RNA: role of the virion core protein

Cells infected with seven different RNA+ mutants of Sindbis virus were found to accumulate a virus-specified polypeptide of mol. wt. 144000 (p144) during incubation at the non-permissive temperature, while at the same time synthesis of the virus structural proteins was drastically reduced. Mapping of the tryptic peptides of p144 showed that it contained the amino acid sequences of all the virus structural proteins. At the non-permissive temperature cells infected with the same seven mutants (out of 28 examined) also showed increased synthesis of 26S RNA, the mRNA for the virus structural proteins, relative to 42S RNA, and the virus genome, compared with infections by wild-type virus. We propose that both these phenotypic effects are the results of a single mutational step and that the primary defect in the processing of the virus structural protein precursor induces the relatively increased rate of synthesis of structural protein mRNA. Temperature-shift experiments with mutant-infected cells showed that p144 itself is not the agent of this effect. The failure of exposure to zinc ions to alter the RNA ratio in wild-type virus-infected cells suggested that the virus envelope proteins are not involved either, since their synthesis is preferentially inhibited under these circumstances. It is possible that it is the failure to synthesize the proper quantity of core protein in the mutant-infected cells which causes the shift of RNA synthesis in favour of structural protein mRNA.     

Replication of standard and defective Ross River virus in BHK cells: Patterns of viral RNA and polypeptide synthesis

Summary Virus-specific macromolecule synthesis has been examined in BHK cells infected with Ross River virus. Unpassaged virus (R-0) and tenth-passage virus (R-10) have been compared. In infected cells R-0 generates i) 45S, 38S, 33S and 26S viral RNAs, ii) virus-specific precursor polypeptides of mol. wt. 127,000, 95,000 and 61,000 and iii) viral envelope proteins (mol. wts. 52,000 and 49,000) and nucleocapsid protein (mol. wt. 32,000). Thus in terms of virus-specific RNA and polypeptide synthesis, the replication of standard RRV is analogous to that of Semliki Forest virus and Sindbis virus.R-10 interferes with the replication of standard Ross River virus and generates large amounts of 19S and 24S defective RNA species; 45S and 26S RNA synthesis was not markedly affected. Defective RNAs are associated with RNAse-sensitive, 50S cytoplasmic particles which contain a variety of (mainly host) proteins but no nucleocapsid protein. No evidence for translation of defective RNAs was obtained.R-10 infection is also characterized by a relatively early shut down of host protein synthesis and by a reduction in virus-specific polypeptide synthesis and nucleocapsid formation. The data suggest that defective Ross River virus interferes primarily at the translational level.With 11 Figures     

Studies of temperature sensitive mutants of bacteriophage Qbeta, defective in both replication and translation.

Temperature sensitive mutants of bacteriophage Qbeta have been isolated which fail in the synthesis of their virus RNA at the non-permissive temperature (42 degrees C). Nine mutants have been studied in some detail. Cells infected with these mutants at 37 degrees C and incubated long enough to produce substantial amounts of Qbeta RNA cease Qbeta RNA replication when shifted to 42 degrees C. The mutants can be classified into 3 groups according to the amount of Qbeta RNA replicase activity exhibited in extracts from infected cells isolated at various times after shift to 42 degrees C: in group 1 mutants, enzyme activity is the same, regardless of the time of isolation after shift; in group 2 mutants enzyme activity increases with time of isolation after shift; in group 3 mutants, enzyme activity decreases with time of isolation after shift. Synthesis of all virus proteins is suppressed at 42 degrees C in cells infected with group 2 of group 3 mutants. In cells infected with group 2 mutants, synthesis of Qbeta RNA replicase subunit beta is increased, but synthesis of other virus proteins is depressed at 42 degrees C. The inhibition of virus RNA and protein synthesis is reversible. A detailed analysis of these experiments suggests that a defective Qbeta RNA replicase is involved in the inhibition of both virus RNA and protein synthesis.     

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.