The intracellular half-lives of nonreplicating nucleocapsids of DI particles of wild type and mutant strains of vesicular stomatitis virus

We have used defective interfering (DI) particles purified free of all infectious virus to determine the intracellular biological stability of nonreplicating nucleocapsids of vesicular stomatitis virus (VSV). Following infection of BHK-21 cells or tc-7 cells with a low multiplicity of pure DI particles, we superinfected them at varying times afterward with a high multiplicity of infectious helper virus to allow replication of those DI particle nucleocapsids retaining biological activity. Careful quantitation of DI particles in the yields from each time point showed that the biological half-life of intracellular VSV Indiana wild type DI particle nucleocapsids was 6 hr in BHK-21 cells and 5.3 hr in tc-7 cells (not significantly different). However, a DI particle from a temperature-sensitive mutant (tsG31) of VSV exhibited a biological nucleocapsid half-life of 12.5 hr and a DI particle isolated following 5 years of persistent infection had a half-life of 18 hr. These findings have significance for the stability of DI particle activity in vivo during acute infections where virus and DI particles are not always present together in the same cells due to cycling interactions. The increased half-life of DI nucleocapsids after years of persistent infection contrasts with the decreased stability and debilitation generally observed in infectious virus from persistent infection. Finally, transcapsidation studies showed that the intracellular half-life differences between DI particles are due mainly to their RNA genomes rather than to the N protein which encapsidates them.     

Susceptibility of Marek's disease lymphoblastoid cell lines to infection with influenza and pseudorabies viruses and the protective effect of immunization with influenza virus-infected lymphoblastoid cells

Summary The susceptibility of 3 lymphoblastoid cell lines (MDCC-MSB1, MDCC-HP1 and MDCC-RP1) derived from Marek's disease tumours to infection with influenza (AoPR₈) and pseudorabies viruses was studied. MSB1 and HP1 were more susceptible to infection with influenza virus than was RP1. It was shown in the case of HP1, which was studied in greater detail, that although the majority of the cells synthesized influenza virus haemagglutinin when infected at high multiplicity, only a small proportion produced mature infectious particles. The results obtained from both high and low multiplicities of infection suggested that infection with AoPR₈ was abortive in the majority of the cells of all 3 cell lines. Infected cultures remained viable when subcultured over several days. All 3 cell lines were resistant to pseudorabies virus. The block in pseudorabies virus replication probably occurred after adsorption. Immunization of chickens with HP1 lymphoblastoid cells superinfected with influenza virus AoPR₈ and inactivated with glutaraldehyde gave better protection against Marek's disease than immunization with uninfected HP1 cells.With 2 Figures     

Reversible restriction of vesicular stomatitis virus in permissive cells treated with inhibitors of prostaglandin biosynthesis

Indomethacin, a potent nonsteroidal inhibitor of prostaglandin synthetase (cyclooxygenase) reduced yields of infectious vesicular stomatitis virus in HEp-2 cells more than 99% if added to cultures at levels of 10^?3M either before or after infection. Other permissive cell lines differed according to the treatment period and drug level required for restricting productive infections. The inhibitory effect of indomethacin was progressively reduced if infection of cells was delayed for increasing times after drug removal. Strong inhibition of viral replication also occurred in cells treated with the cyclooxygenase antagonists naproxen, phenylbutazone, and oxyphenylbutazone whereas phenacetin, which does not block cyclooxygenase function, was inactive. Enhanced viral replication occurred in indomethacin-treated HEp-2 cultures when these cells were subsequently exposed to such substances as prostaglandin El, cyclic AMP, or insulin. Conversely, indomethacin-treated cells remained restrictive for VSV if they were subsequently exposed to metabolic inhibitors of functional DNA (actinomycin D or mitomycin C), messenger RNA synthesis (α-amanitin), or protein synthesis (cycloheximide) at concentrations that normally do not compromise viral replication. Pretreatment of HEp-2 cells with mitomycin C markedly shifted the dose response for indomethacin-mediated inhibition of VSV from a 90% inhibitory dose of about 10^?4 M to one of 10^?9 M or lower. These findings suggest that preexisting host factors essential for replication of VSV, although rendered nonfunctional by the drug indomethacin, can be replenished unless their synthesis is blocked by various classes of metabolic inhibitors.     

Defective interfering particles of Semliki Forest virus generated in BHK cells do not interfere with viral RNA synthesis in Aedes albopictus cells

Defective interfering (DI) particles of Semliki Forest virus (SFV) were generated by serial high multiplicity passage of plaque-purified virus on BHK cells. Defective interfering passages of SFV depressed the synthesis of 42 and 26 S viral RNA and induced the formation of two new single-stranded RNA forms (molecular weight, 1.2 × 10⁶ and 0.56 × 10⁶) in BHK and Vero cells but not in Aedes albopictus cells. These results suggest that the invertebrate cells restricted replication of the alphavirus DI particles.     

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.     

Interferon production and virus replication in lymphoblastoid cells infected with different viruses

Summary A semicontinuous infection system was used to test viral replication and interferon induction in lymphoblastoid cells: measles virus, Newcastle disease virus (NDV), Sendai virus, human parainfluenza virus (type II and III), Semliki forest virus (SFV) and Vesicular stomatitis virus (VSV). With the exception of Sendai virus, all viruses replicated in the Namalva cell line. Only measles virus was able to induce high levels of interferon. Three other cell lines, NC37, Raji (TK⁺-variant) and Raji (TK^–-variant) were tested using measles virus as inducer. The interferon yields from these cells were inferior to those obtained from Namalva cells.     

Prolonged infection of L cells with vesicular stomatitis virus. Defective interfering forms and temperature-sensitive mutants as factors in the infection.

When L_y cells were treated with 100 units per milliliter of mouse interferon and then infected with vesicular stomatitis virus (VSV) at a multiplicity of 10–60 PFU/cell, a prolonged infection of cultures ensued, lasting in one case longer than 60 days. After four passages in L_y cells at high multiplicities of VSV from a prolonged infection, there was no inhibition of virus growth, and virus from the prolonged infection had the same distribution of intracellular and extracellular RNA forms as was found in wild-type virus passed four times in L_y cells. Also, the intracellular and extracellular RNA forms of virus taken directly from a prolonged infection of L_y cells were indistinguishable from those of the wild-type virus. A morphological examination of VSV from a prolonged infection did not show a significant number of small defective viral forms after four passages in L_y cells at a high multiplicity of infection. There was, however, evidence for the emergence of temperature-sensitive mutants of VSV during the course of the prolonged infection. Changing the incubation temperature of the cultures from 37° to 32° resulted in an increase in virus production and virus-induced cytopathology. The virus produced during prolonged infections grew to higher titers at 32° than at 37° and its plaque size at 39° progressively decreased with the length of time that the infection persisted. Furthermore, interferon production also seemed to have a role in the persistence of infections such as the treatment of cultures with rabbit anti-mouse interferon globulin and resulted in a significant rise in virus production and viral cytopathology in the cell cultures.     

Nucleic acid synthesis during the focus formation by Shope fibroma virus on green monkey kidney cells

Summary Nucleic acid synthesis during the focus formation on Shope fibroma virus (SFV)-infected cells was studied. When African green monkey kidney (AGMK) cells were infected with SFV, the cell-focus was observed as a local piling up of cells at 3 to 5 days after infection. The number of foci increased in proportion to the size of the SFV inoculum. In the SFV-AGMK cell system, however, the growth rate of virus was comparatively low and no apparent cytopathic effect was demonstrated as a result of virus infection. The incorporation of³H-thymidine into the nuclear fraction of infected cells was suppressed early in the cycle of virus replication and before focus formation. On the other hand, the rate of DNA synthesis increased markedly in the infected cytoplasm. Cytoplasmic RNA synthesis also tended to increase gradually accompanying the induction of the viral DNA synthesis. In addition, the sedimentation properties of the newly synthesized DNA were investigated by sucrose density gradient centrifugation.     

The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the noncytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against noncytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-γ against vaccinia virus) and neutralizing antibodies (against VSV and SFV).     

Restriction of vesicular stomatitis virus in a nonpermissive rabbit cell line is at the level of protein synthesis

Replication of vesicular stomatitis virus (VSV) is restricted in a line of rabbit cornea (RC-60) cells; less than one infectious particle is produced per infected cell. We show that VSV is blocked at the level of viral-specific protein synthesis. VSV proteins are synthesized early in infection but the rate of VSV protein synthesis declines rapidly as infection proceeds. At times when synthesis of VSV proteins is barely detectable, VSV mRNA is produced and polyribosome structures are present. The VSV mRNA recovered from the polysome region directs the synthesis of VSV proteins in an in vitro reticulocyte translation system. This suggests that protein synthesis is blocked at some step beyond the level of initiation possibly at the level of elongation. Coinfection with vaccinia virus converts RC-60 cells to a permissive host. In contrast to the abortive infection with VSV alone, VSV proteins are synthesized throughout the replication cycle in doubly infected cells. Vaccinia supplies a product essential for sustained protein synthesis in the abortive system. We have confirmed that the replication of genome length 42 S RNA does not occur at late times in the abortive infection. This lack of 42 S RNA replication is explained by the shut-off of VSV protein synthesis, since continuous protein synthesis is required for the replication of VSV 42 S RNA.     

Biochemical characterization of vesicular stomatitis virus-infected Aedes albopictus cells deprived of methionine

Aedes albopictus cells infected with vesicular stomatitis virus (VSV) and maintained in medium lacking methionine produced 1000-fold less infectious virus than cultures maintained in complete medium. Analysis of viral macromolecular synthesis in cells maintained in the presence of varying concentrations of methionine showed that the reduction in virus yield was directly correlated with a reduction in viral RNA and protein synthesis. Of the viral mRNA which was made in methionine-starved cells the majority was not polysome associated. In contrast, virtually all of the virus mRNA in cells maintained in complete medium was polysome associated. In vitro translation of those mRNAs from methionine-starved cells, which were not polysome associated, indicated that they could be translated in vitro as efficiently as polysome-associated virus mRNA but only if the methylation inhibitor, S-adenosylhomocysteine, was not present. These results strongly suggest that methionine starvation of A. albopictus cells inhibits VSV replication by preventing cap methylation of the viral mRNAs, and thus reducing the efficiency with which they are translanted.     

Effect of ouabain on Sindbis virus replication in ouabain-sensitive and ouabain-resistant Aedes albopictus cells (Singh)

The effects of ouabain on Sindbis virus replication in cloned sublines of ouabain-sensitive (B₁C₄) and -resistant (Oua₅) Aedes albopictus cells have been studied. The addition of ouabain (0.1 mM) to B₁C₄ cells 1 hr after infection reduced the yield of infectious virus 500-fold. The same concentration had no effect on the yield from Oua.5 cells. Analysis of [³H]-uridine-labeled RNA by gel electrophoresis showed that ouabain markedly inhibited Sindbis virus-specific RNA synthesis in B₁C₄ cells but only partially inhibited host RNA synthesis. In Oua₅ cells, ouabain had little effect on either host or viral RNA synthesis. A close correlation was observed between the inhibition of host protein synthesis by ouabain and the inhibition of virus replication.     

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.     

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.     

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis.

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inhibitor of lipid synthesis. The presence of this agent from the beginning of infection blocks the appearance of viral proteins. However, when the antibiotic is added at later stages of infection it has no effect on protein synthesis, the cleavage of viral proteins and their acylation by palmitic acid. Cerulenin is a powerful inhibitor of viral RNA synthesis, as analyzed by [3H]uridine incorporation, incorporation of [32P]phosphate into viral replication complexes, or Northern blot analysis of viral RNAs hybridized with minus- or plus-stranded riboprobes. Finally, analysis of phospholipids made in SFV-infected cells indicates that viral infection clearly stimulates the synthesis of phosphatidyl choline and modifies the membrane formed as analyzed by sucrose gradient centrifugation. Cerulenin blocks the synthesis of phospholipids and inhibits the formation of new membranes. These results show that, when the synthesis of lipids is blocked by cerulenin, SFV RNA replication is hampered, suggesting that the synthesis of viral RNAs needs continuous lipid synthesis and membrane formation.     

Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus. II. Effect of UV-inactivated vaccinia and metabolic inhibitors

The rescue of vesicular stomatitis virus (VSV) from interferon-induced resistance in rabbit (RK-13) and mouse (L) cell cultures superinfected with vaccinia requires early (up to 2 hr) vaccinia DNA-dependent RNA synthesis. The inability of hydroxyurea to inhibit rescue of VSV in interferon-treated RK-13 and L cells, whether the drug is added at the time of or after infection, suggests that vaccinia DNA synthesis is not required for the rescue of VSV in cells superinfected with vaccinia.In both RK-13 and L cells pretreated with homologous interferon and then doubly infected with vaccinia and VSV, there was a significant increase (up to 8 hr) in the lag period before infective VSV progeny appeared. It appears that a product of vaccinia synthesis must accumulate before VSV replication can begin in cells pretreated with interferon. This product could be vaccinia-directed early RNA or a translation product of this RNA.In RK-13 cells pretreated with interferon, the ability of vaccinia to rescue VSV is much more resistant to UV-irradiation than the infectivity of the virus; in L cells there is a close correspondence in the inactivation rates of infectivity and the ability of vaccinia to rescue VSV. These results suggest a difference in the efficiency of uncoating of UV-irradiated vaccinia in RK-13 and L cells. In L cells it is possible that UV-irradiated vaccinia is not uncoated efficiently and the early vaccinia RNA product required for rescue of VSV is not synthesized.