Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.     

Translation of capped viral mRNAs in poliovirus-infected HeLa cells.

HeLa cells doubly infected with Semliki Forest virus (SFV) and poliovirus synthesize either more poliovirus proteins or more SFV late proteins depending on the time of super-infection with poliovirus. Under some conditions, the infected cells translate uncapped poliovirus mRNA and capped 26S mRNA from SFV simultaneously, even though host protein synthesis has been shut down. Vesicular stomatitis virus (VSV) protein synthesis is depressed drastically when VSV-infected cells are super-infected with poliovirus. In cells doubly infected with VSV and encephalomyocarditis (EMC) virus or with VSV and SFV, dominance of one of the viruses depends on the time of addition of the challenge virus. The influence of external conditions on the relative translation of capped or uncapped viral mRNA in doubly infected cells has also been analysed.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected Cell Extracts

Crude cytoplasmic extracts from vesicular stomatitis virus (VSV)-infected HeLa cells incorporate radioactive amino acids into hot trichloroacetic acid-precipitable material linearly for 10 to 20 min. The material synthesized in vitro corresponds in molecular weight to four of the five VSV structural proteins. However, synthesis of the viral glycoprotein (G) is significantly reduced, whereas the relative amounts of viral structural proteins L and NS synthesized are increased compared with the ratio of the proteins found in the virion. Fractionation of a VSV-infected crude cytoplasmic extract into a cytoplasmic pellet (20,000 x g for 30 min) and a cytoplasmic supernatant results in a significant reduction in protein synthesizing activity of both fractions, although both contain polysomes. The products synthesized by a cytoplasmic supernatant-directed system included all the VSV structural proteins except the glycoprotein, whereas in an in vitro system directed by the cytoplasmic pellet there is a marked reduction in synthesis of the nucleoprotein (N) and also a small relative increase in synthesis of the glycoprotein. Addition of uninfected, preincubated HeLa or L-cell S10 or a HeLa ribosomal fraction to the VSV-infected cytoplasmic pellet results in a 30- to 60-fold stimulation of (35)S-methionine incorporation. However, these uninfected extracts do not stimulate (35)S-methionine incorporation by the infected crude cytoplasmic extract or the cytoplasmic supernatant. The products synthesized by the stimulated cytoplasmic pellet now include sizeable amounts of the glycoprotein in addition to the other VSV structural proteins.     

Cytolytic T lymphocytes from the BALB/c-H-2dm2 mouse recognize the vesicular stomatitis virus glycoprotein and are restricted by class II MHC antigens.

BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.     

Inhibition of protein synthesis in vesicular stomatitis virus infected Chinese hamster ovary cells: role of virus mRNA-ribonucleoprotein particle

Although host protein synthesis is preferentially inhibited, there is a steady decline in the ability of Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) to synthesize both host and viral proteins. We previously reported finding an mRNA-ribonucleoprotein particle (mRNP) that contained all five VSV mRNAs and viral N protein exclusively. This particle apparently regulates translation by sequestering a majority of the VSV mRNA made late in infection and thus rendering it unavailable for protein synthesis. In the present investigation the mRNP was also shown to inhibit in vitro protein synthesis in rabbit reticulocyte and wheat germ lysates programmed with mRNA isolated from VSV-infected cells. The synthesis of eIF-2 X GTP X Met-tRNA (ternary) complex, the first step in initiation of protein synthesis, was markedly inhibited by the mRNP. The inhibition was partially reversed by addition of purified eIF-2 to the inhibited lysate or ternary complex formation reaction. These results indicate a dual role of the mRNP in regulating protein synthesis during infection. Nucleocapsid also inhibited in vitro protein synthesis, although this inhibition was not reversed by eIF-2. Nucleocapsid did not inhibit ternary complex formation in vitro. Consequently, nucleocapsid may also regulate in vivo protein synthesis, but by a mechanism different from the mRNP.     

The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.     

Inhibition of host and viral translation during vesicular stomatitis virus infection. eIF2 is responsible for the inhibition of viral but not host translation

In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the alpha subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2alpha did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2alpha showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2alpha phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2alpha phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2alpha, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2alpha phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.     

Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus.

After infection of baby hamster kidney cells with vesicular stomatitis virus (VSV), processing and assembly of small nuclear ribonucleoproteins (snRNP) were rapidly inhibited. The U1 and U2 snRNAs accumulated as precursor species approximately 3 and 10 nucleotides longer, respectively, than the mature RNAs. Alteration in snRNP assembly was noted because the precursor snRNAs were not associated with the U-series RNA-core protein complex in infected cells. However, antibodies specific for the U2 RNA-binding protein, A', were able to precipitate pre-U2 RNAs from VSV-infected cells. These results indicated that precursors to U2 RNA were bound to A' and remained bound during virus infection. Analysis of the synthesis of proteins normally associated with U1 and U2 RNAs indicated that synthesis was unaffected at times when snRNP assembly with core proteins was blocked by the VSV. These findings suggested that the core proteins associate with one another in the absence of the snRNAs in VSV-infected cells. They further suggest a correlation between the inability of the core complex to bind the U-series snRNPs and the failure to process the 3' ends of U1 and U2 RNAs in VSV-infected cells. These effects of VSV on snRNP assembly may be related to the shutoff of host-cell macromolecular synthesis.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.     

Fewer CTL, not enhanced NK cells, are sufficient for viral clearance from the lungs of immunocompromised mice

Activation of the aryl hydrocarbon receptor (AhR) causes numerous defects in anti-viral immunity, including suppressed CTL generation and impaired host resistance. However, despite a reduced CTL response, mice that survive infection clear the virus. Therefore, we examined the contribution of NK cells and pro-inflammatory cytokines to viral clearance in influenza virus-infected mice exposed to TCDD, the most potent AhR agonist. Infection caused transient increases in pulmonary TNFalpha, IL-1, and IFNalpha/beta levels, but neither the kinetics nor magnitude of this response was affected by AhR activation. No IL-18 was detected at any time point examined. Exposure to TCDD enhanced NK cell numbers in the lung but did not affect their IFNgamma production. Furthermore, depletion of NK cells did not alter anti-viral cytolytic activity. In contrast, removal of CD8+ T cells ablated virus-specific cytolytic activity. These results demonstrate that the pulmonary CTL response to influenza virus is robust and few CTL are necessary for viral clearance.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.