Vesicular Stomatitis Virus Protein Synthesis Research Articles

P108 PMLIV inhibits VSV replication through the specific induction of IFN-beta and IFN-lambda

Promyelocytic Leukemia (PML) protein (also named TRIM19 for TRIpartite Motif protein 19), the organizer of small nuclear-matrix structures named nuclear bodies, has been implicated in the antiviral response toward diverse cytoplasmic replicating RNA viruses through different mechanisms. PML is covalently conjugated to the small ubiquitin-like modifier (SUMO). Splice variants transcribed from the PML gene yield several PML isoforms (PMLI to PMLVII) that share the same N-terminal region but differ in their C-termini. In consequence, each PML isoform is able to interact with specific partners and to display different functions. We have previously shown that PMLIII confers resistance to vesicular stomatitis virus (VSV) [1] . The antiviral activity of PML has been validated in vivo since knock out PML mice are more sensitive to VSV than wild-type mice [2] . The role of the other PML isoforms in VSV-infected cells is unknown. Therefore, we studied the implication of all PML isoforms during VSV infection. Viral proteins synthesis was determined in MEFs derived from knock out PML mice (MEFs PML-/-) and in human cells down-regulated for PML. U373MG cells stably expressing each PML isoform (PMLI to PMLVII) were infected with VSV at different MOIs, viral protein synthesis and VSV growth were determined. To investigate which viral step is targeted by PMLIV, we studied VSV entry, primary and secondary transcription. To determine whether PMLIV affects the expression of IFNs and/or pro-inflammatory cytokines, we quantified IFN-a, IFN-b, IFN-l, TNF-a and IL8 mRNAs in extracts from U373MG-EV and U373MG-PMLIV cells infected with VSV. We have shown that VSV protein synthesis were boosted in MEFs PML-/- or in human cells down-regulated for PML expression. Our comparative study performed with cells stably expressing each of PML isoform revealed that both PMLIII and PMLIV conferred resistance to VSV, whereas other nuclear isoforms (PMLI, II, V and VI) and cytoplasmic PMLVII failed to do so. The antiviral activity of PMLIV was higher than that of PMLIII. PMLIV did not alter VSV entry but inhibited viral mRNA and protein synthesis. PMLIV SUMOylation was required as the antiviral effect was not observed with PMLIV-3KR, in which lysines involved in conjugation to SUMO were mutated. The protective effect of PMLIII was independent of IFN. In contrast, PMLIV expression led to the induction of type I IFNs, particularly IFN-band IFN-lduring VSV infection. Here, we demonstrate for the first time that one particular isoform of PML (PMLIV) was implicated in the induction of type I IFNs, particularly IFN-band IFN-l, during VSV infection, suggesting a new role for PMLIV in the antiviral innate immunity.

Translation of mRNAs from Vesicular Stomatitis Virus and Vaccinia Virus Is Differentially Blocked in Cells with Depletion of eIF4GI and/or eIF4GII

Cytolytic viruses abrogate host protein synthesis to maximize the translation of their own mRNAs. In this study, we analyzed the eukaryotic initiation factor (eIF) 4G requirement for translation of vesicular stomatitis virus (VSV) and vaccinia virus (VV) mRNAs in HeLa cells using two different strategies: eIF4G depletion by small interfering RNAs or cleavage of eIF4G by expression of poliovirus 2A protease. Depletion of eIF4GI or eIF4GII moderately inhibits cellular protein synthesis, whereas silencing of both factors has only a slightly higher effect. Under these conditions, the extent of VSV protein synthesis is similar to that of nondepleted control cells, whereas VV expression is substantially reduced. Similar results were obtained when eIF4E was depleted. On the other hand, eIF4G cleavage by poliovirus 2A protease strongly inhibits translation of VV protein expression, whereas translation directed by VSV mRNAs is not abrogated, even though VSV mRNAs are capped. Therefore, the requirement for eIF4F activity is different for VV and VSV, suggesting that the molecular mechanism by which their mRNAs initiate their translation is also different. Consistent with these findings, eIF4GI does not colocalize with ribosomes in VSV-infected cells, while eIF2α locates at perinuclear sites coincident with ribosomes.

Different Effect of Proteasome Inhibition on Vesicular Stomatitis Virus and Poliovirus Replication

Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2α, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

The role of the proteasome–ubiquitin pathway in regulation of the IFN-γ mediated anti-VSV response in neurons

Pharmacologic inhibition of the proteasome resulted in increased NOS-1 protein levels and increased NO production by neuronal cells. This correlated with an increased antiviral effect of IFN-γ against the replication of vesicular stomatitis virus (VSV) replication in vitro. We also observed that a regulatory protein, Protein Inhibitor of NOS-1 (PIN) was down-regulated by IFN-γ treatment, and more ubiquitinated PIN accumulated in IFN-γ treated neurons. In cells of the reticuloendothelial system, IFN-γ treatment induces the expression of a set of low molecular weight MHC-encoded proteins (LMPs), which replace the β-subunit of the proteasome complex during the proteasome neosynthesis, resulting in a complex termed the immunoproteasome. LMP2, -7, and -10 were induced and the immunoproteasome was generated by IFN-γ treatment in neuronal cells. Importantly, we observed that IFN-γ induced inhibition of VSV protein synthesis was not dependent on ubiquitination.

Antiviral effect of the mammalian translation initiation factor 2α kinase GCN2 against RNA viruses

In mammals, four different protein kinases, heme-regulated inhibitor, double-stranded RNA-dependent protein kinase (PKR), general control non-derepressible-2 (GCN2) and PKR-like endoplasmic reticulum kinase, regulate protein synthesis in response to environmental stresses by phosphorylating the alpha-subunit of the initiation factor 2 (eIF2alpha). We now report that mammalian GCN2 is specifically activated in vitro upon binding of two nonadjacent regions of the Sindbis virus (SV) genomic RNA to its histidyl-tRNA synthetase-related domain. Moreover, endogenous GCN2 is activated in cells upon SV infection. Strikingly, fibroblasts derived from GCN2-/- mice possess an increased permissiveness to SV or vesicular stomatitis virus infection. We further show that mice lacking GCN2 are extremely susceptible to intranasal SV infection, demonstrating high virus titers in the brain compared to similarly infected control animals. The overexpression of wild-type GCN2, but not the catalytically inactive GCN2-K618R variant, in NIH 3T3 cells impaired the replication of a number of RNA viruses. We determined that GCN2 inhibits SV replication by blocking early viral translation of genomic SV RNA. These findings point to a hitherto unrecognized role of GCN2 as an early mediator in the cellular response to RNA viruses.

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.

Block of vesicular stomatitis virus endocytic and exocytic pathways by 1-cinnamoyl-3,11-dihydroxymeliacarpin, a tetranortriterpenoid of natural origin.

Previously, it has been shown that 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), a natural compound isolated from leaf extracts of Melia azedarach L., inhibits the vesicular stomatitis virus (VSV) multiplication cycle when added before or after infection. Here, we have established that the lack of VSV protein synthesis in CDM pre-treated Vero cells is ascribed to the inhibition of an initial step during virus multiplication, although indirect immunofluorescence (IFI) studies confirmed that the binding and uptake of [(35)S]methionine-labelled VSV was not affected by CDM pre-treatment. Instead, our findings revealed that this compound impedes the uncoating of VSV nucleocapsids in pre-treated Vero cells, since the antiviral action of CDM was partially reversed by inducing VSV direct fusion at the plasma membrane, and VSV M protein fluorescence was confined to the endosomes, even 2 h post-internalization. Furthermore, CDM induced cytoplasmic alkalinization, as shown by acridine orange staining, consistent with the inhibition of virus uncoating. Although VSV proteins are synthesized when CDM is added after infection, IFI studies revealed that G protein was absent from the surface of infected cells and co-localized with a Golgi marker. Therefore, CDM inhibits the transport of G protein to the plasma membrane. Taken together, these findings indicate that CDM exerts its antiviral action on the endocytic and exocytic pathways of VSV by pre- or post-treatment, respectively.

Resistance to virus infection conferred by the interferon-induced promyelocytic leukemia protein.

The interferon (IFN)-induced promyelocytic leukemia (PML) protein is specifically associated with nuclear bodies (NBs) whose functions are yet unknown. Two of the NB-associated proteins, PML and Sp100, are induced by IFN. Here we show that overexpression of PML and not Sp100 induces resistance to infections by vesicular stomatitis virus (VSV) (a rhabdovirus) and influenza A virus (an orthomyxovirus) but not by encephalomyocarditis virus (a picornavirus). Inhibition of viral multiplication was dependent on both the level of PML expression and the multiplicity of infection and reached 100-fold. PML was shown to interfere with VSV mRNA and protein synthesis. Compared to the IFN mediator MxA protein, PML had less powerful antiviral activity. While nuclear body localization of PML did not seem to be required for the antiviral effect, deletion of the PML coiled-coil domain completely abolished it. Taken together, these results suggest that PML can contribute to the antiviral state induced in IFN-treated cells.

Effect of combined αIFN and prostaglandin A 1 treatment on vesicular stomatitis virus replication and heat shock protein synthesis in epithelial cells

The antiviral activity of prostaglandin A (PGA) and interferons (IFNs) has been widely described. In the present report, we investigated the effect of combined αIFN and PGA 1 treatment on vesicular stomatitis virus (VSV) replication and on heat shock protein (HSP) induction in monkey epithelial cells. In uninfected cells, PGA 1 caused a dose-dependent induction of HSP70, HSP90 and HSP110, while αIFN did not affect HSP synthesis. Alpha-IFN suppressed VSV replication dose-dependently, even when cells were treated after virus infection. VSV protein synthesis was not affected by αIFN, indicating a block at the level of virus assembly or maturation. PGA 1 caused a dose-dependent inhibition of VSV replication, and suppressed VSV protein synthesis at concentrations which induced the synthesis of high levels of HSP70. The combined treatment with low doses of αIFN or PGA 1, which only moderately inhibited VSV replication when administered separately, was found to suppress VSV production by more than 95%, and resulted in a 3-fold increase of HSP70 synthesis as compared to PGA 1 alone. These results demonstrate a co-operative effect of PGA 1 and αIFN against VSV infection and suggest that αIFN can potentiate the cellular response to HSP induction in virus-infected cells.

Inhibition of Vesicular Stomatitis Virus Replication by Epidermal Growth Factor in Human Epidermoid A-431 Cells

Here we report that epidermal growth factor (EGF) inhibits the replication of vesicular stomatitis virus (VSV) in human epidermoid carcinoma A-431 cells, as measured by reduction in infectious viral particle production, cell protection assay, and inhibition of viral protein synthesis. The observed antiviral effects of EGF were not apparently due to inhibitory effects on the initial events in VSV infection as postinfection treatment of cells with EGF also resulted in significant inhibition in the levels of viral proteins. The EGF-mediated inhibition of VSV protein synthesis was specific in nature as prior treatment of cells with an excess of anti-EGF receptor monoclonal antibody, which blocks EGF binding, reduced the inhibition of VSV proteins elicited by EGF. In summary, the observed antiviral action of EGF was an early effect of EGF and was mediated via an EGF-responsive cellular pathway(s) that could impair VSV replication.

Effects of Psoralen-Derivatized Oligonucleoside Methylphosphonates on Vesicular Stomatitis Virus (VSV) Protein SynthesisIn Vitroand in VSV-Infected Cells

Abstract Oligodeoxyribonucleoside methylphosphonates (16-mers) targeted to VSV mRNAs were derivatized with 4′-{[N-(aminoethyl)amino]methyl}-4,5′8-trimethylpsoralen. These oligomers specifically inhibit translation of their targeted mRNAs in vitro following UV irradiation of the oligomer-mRNA complexes. Psoralen-derivatized oligonucleoside methylphosphonates are stable in cells and can inhibit VSV protein synthesis in culture following UV-irradiation of VSV-infected cells.

Monensin and nigericin prevent the inhibition of host translation by poliovirus, without affecting p220 cleavage.

Addition of monensin or nigericin after poliovirus entry into HeLa cells prevents the inhibition of host protein synthesis by poliovirus. The infected cells continue to synthesize cellular proteins at control levels for at least 8 h after infection in the presence of the ionophore. Cleavage of p220 (gamma subunit of eukaryotic initiation factor 4 [eIF-4 gamma]), a component of the translation initiation factor eIF-4F, occurs to the same extent in poliovirus-infected cells whether or not they are treated with monensin. Two hours after infection there is no detectable intact p220, but the cells continue to translate cellular mRNAs for several hours at levels similar to those in uninfected cells. Nigericin or monensin prevented the arrest of host translation at all the multiplicities of poliovirus infection tested. At high multiplicities of infection, an unprecedented situation was found: cells synthesized poliovirus and cellular proteins simultaneously. Superinfection of vesicular stomatitis virus-infected HeLa cells with poliovirus led to a profound inhibition of vesicular stomatitis virus protein synthesis, while nigericin partially prevented this blockade. Drastic inhibition of translation also took place in influenza virus-infected Vero cells treated with nigericin and infected with poliovirus. These findings suggest that the translation of newly synthesized mRNAs is dependent on the integrity of p220, while ongoing cellular protein synthesis does not require an intact p220. The target of ionophore action during the poliovirus life cycle was also investigated. Addition of nigericin at any time postinfection profoundly blocked the synthesis of virus RNA, whereas viral protein synthesis was not affected if nigericin was added at 4 h postinfection. These results agree well with previous findings indicating that inhibitors of phospholipid synthesis or vesicular traffic interfere with poliovirus genome replication. Therefore, the action of nigericin on the vesicular system may affect poliovirus RNA synthesis. In conclusion, monensin and nigericin are potent inhibitors of poliovirus genome replication that prevent the shutoff of host translation by poliovirus while still permitting cleavage of p220.

Properties of Exonuclease-Resistant, Psoralen-Conjugated Oligodeoxyribonucleotidesin Vitroand in Cell Culture

We have prepared oligodeoxyribonucleotides that are modified at the 3'-terminal with N4-(4-aminobutyl)deoxycytidine and derivatized at the 5'-end with a 4'-([N-(aminoethyl)amino]methyl)-4,5',8-trimethylpsoralen, (ae)AMT, and whose sequences are complementary to vesicular stomatitis virus (VSV), N-protein mRNA, (ae)AMT-II, or VSV M-protein mRNA, (ae)AMT-III. (ae)AMT-II cross-links exclusively to VSV N-mRNA when a mixture of the oligomer and poly(A+) RNA from VSV-infected cells is irradiated in vitro with long wavelength UV light at either 20 degrees or 37 degrees C. N4-(4-Aminobutyl)deoxycytidine at the 3'-end of (ae)AMT-II does not appear to affect the binding or cross-linking of the oligomer to its target RNA. Oligomer (ae)AMT-II is completely resistant to hydrolysis by the 3'-5'-exonuclease activity found in fetal calf serum whereas a similar oligomer, (ae)AMT-I, which contains a 3'-terminal deoxycytidine, is hydrolyzed within 30 min when incubated at 37 degrees C. Intact (ae)AMT-II was found in both the cell lysate and cell culture medium after 12 hr of incubation with mouse L-cells along with d-(ae)AMTpT, which appears to result from endonuclease degradation of the oligomer. In contrast no intact (ae)AMT-I was found in either the cell lysate or the culture medium after 1 hr incubation. Although 10 microM (ae)AMT-II had no effect on VSV-protein synthesis in either unirradiated or UV-irradiated VSV-infected mouse L-cells, 10 microM (ae)AMT-III inhibited VSV protein synthesis 30% in irradiated cells. These results show that introduction of a N4-(4-aminobutyl)deoxycytidine at the 3'-end of an oligodeoxyribonucleotide significantly increases the resistance of the oligomer to degradation by 3'-5'-exonucleases but does not interfere with its ability to bind selectively to complementary RNA. Further derivatization with psoralen creates an oligomer that can be triggered to cross-link with RNA in a sequence-specific manner, is taken up intact by mammalian cells in culture, and exhibits biological activity. In combination, these two modifications endow the oligodeoxyribonucleotide with novel properties that could be exploited in the design of antisense or antigene reagents for use in controlling gene expression in mammalian cells.

Studies on the Mechanism of the Interferon-mediated Antiviral State to Vesicular Stomatitis Virus in Resting Mouse Peritoneal Macrophages

We have analysed the expression of vesicular stomatitis virus (VSV) proteins in virus-infected freshly explanted mouse peritoneal macrophages (resistant to virus replication), macrophages aged in vitro (permissive for virus replication) and freshly explanted macrophages from mice treated with antibody to interferon (IFN) alpha/beta (permissive for VSV replication). Our data showed that some VSV proteins (i.e. N/NS and G) were synthesized in virus-infected (1 p.f.u/cell) freshly harvested macrophages at early times after infection (3 to 6 h); the expression of such viral proteins was subsequently inhibited at 18 h post-infection. In contrast, a progressive increase in the expression of VSV proteins was observed in the macrophages aged in vitro and infected with VSV at 1 p.f.u./cell. Infection with a higher m.o.i. (16 p.f.u./cell) resulted in similar viral protein electrophoresis patterns for both aged macrophages and freshly explanted macrophages. Even at low m.o.i. a marked and progressive expression of all VSV proteins was observed in freshly harvested macrophages from mice treated with antibody to mouse IFN-alpha/beta. Higher levels of oligo-2',5'-adenylate synthetase (2-5AS) were found in freshly harvested macrophages than in either aged macrophages or those from mice treated with antibody to IFN. No dsRNA-dependent 67K protein kinase was detected in freshly harvested macrophages or peritoneal cells from untreated mice or mice treated with poly(rI).poly(rC) or Newcastle disease virus. The following conclusions can be drawn from these results. Low levels of spontaneous IFN-alpha/beta are responsible for the time-dependent inhibition of VSV protein synthesis in virus-infected freshly harvested macrophages; high levels of 2-5AS (in the absence of detectable levels of 67K protein kinase) appear to correlate with the progressive inhibition of VSV proteins; this natural antiviral state is highly effective only at low m.o.i.

Vaccinia virus stimulates the growth of vesicular stomatitis virus at the level of protein synthesis in mouse L cells

Coinfection with vaccinia virus increases the growth of vesicular stomatitis virus (VSV) in mouse L cells by 10- to 20-fold. Although vaccinia has no significant effect on RNA synthesis by VSV, VSV protein synthesis is dramatically stimulated by double infection. The enhancement of VSV growth is correlated with the ability of vaccinia to inhibit the VSV-mediated damage to the host translational machinery. Coinfection with vaccinia fails to stimulate the growth of a VSV mutant which is deficient in its ability to shut off protein synthesis during infection.

Poliovirus protease 2A is required for interference with vesicular stomatitis virus-specified protein synthesis.

A subunit of eukaryotic initiation factor-4F (eIF-4F) which is a component of the protein complex which binds to the methylated cap structure at the 5' end of most cellular mRNAs, is proteolytically cleaved in poliovirus-infected cells resulting in the shutoff of cellular protein synthesis. Poliovirus mRNA is selectively translated in infected cells, in part, because translation of the uncapped viral mRNA does not require an intact cap binding protein complex. Wild-type poliovirus also inhibits the translation of vesicular stomatitis virus (VSV) mRNAs in coinfected cells, however, it has been unclear whether similar mechanisms are employed by poliovirus to interfere with cellular and VSV protein synthesis. Degradation of eIF-4F appears to be an indirect function of the poliovirus-encoded protease 2A. A poliovirus mutant in 2A failed to mediate eIF-4F cleavage and selectively terminate translation of capped cellular mRNAs. Unlike wild-type poliovirus, 2A-1 does not interfere with VSV-specified protein synthesis. These results indicate that the same viral protein, 2A protease, is required not only to effectively terminate host protein synthesis, but also to interfere with expression of a heterologous virus, VSV. In addition, 2A-1 specifies a function, heretofore undescribed for poliovirus, which interferes with VSV-induced shutoff of protein synthesis.

Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site.

Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression, but their efficient introduction within intact cells proved to be difficult to realize. As a step toward this goal, small (13- or 15-mer) synthetic oligodeoxyribonucleotides have been coupled at their 3' ends to epsilon-amino groups of lysine residues of poly(L-lysine) (Mr, 14,000). A 15-mer oligonucleotide-poly(L-lysine) conjugate complementary to the initiation region of vesicular stomatitis virus (VSV) N-protein mRNA specifically inhibits the synthesis of VSV proteins and exerts an antiviral activity against VSV when added in the cell culture medium at doses as low as 100 nM. Neither synthesis of cellular proteins nor multiplication of encephalomyocarditis virus was affected significantly by this oligonucleotide conjugate. The data suggest that oligonucleotide-poly(L-lysine) conjugates might become effective for studies on gene expression regulation and for antiviral chemotherapy.

Persistence of vesicular stomatitis virus in cloned interleukin-2-dependent natural killer cell lines.

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.

Inhibition of vesicular stomatitis virus protein synthesis and infection by sequence-specific oligodeoxyribonucleoside methylphosphonates.

Oligodeoxyribonucleoside methylphosphonates which have sequences complementary to the initiation codon regions of N, NS, and G vesicular stomatitis virus (VSV) mRNAs were tested for their ability to inhibit translation of VSV mRNA in a cell-free system and in VSV-infected mouse L cells. In a rabbit reticulocyte lysate cell-free system, the oligomers complementary to N (oligomer I) and NS (oligomer II) mRNAs inhibited translation of VSV N and NS mRNAs whereas oligomer III had only a slight inhibitory effect on N protein synthesis. At 100 and 150 microM, oligomer I specifically inhibited N protein synthesis in the lysate. In contrast, at 150 microM, oligomer II inhibited both N and NS protein synthesis. This reduced specificity of inhibition may be due to the formation of partial duplexes between oligomer II and VSV N mRNA. The oligomers had little or no inhibitory effects on the synthesis of globin mRNA in the same lysate system. Oligomers I-III specifically inhibited the synthesis of all five viral proteins in VSV-infected cells in a concentration-dependent manner. The oligomers had no effects on cellular protein synthesis in uninfected cells nor on cell growth. An oligothymidylate which forms only weak duplexes with poly(rA) had just a slight effect on VSV protein synthesis and yield of virus. Oligomers I-III have extensive partial complementarity with the coding regions of L mRNA. The nonspecific inhibition of viral protein synthesis in infected cells may reflect the role of N, NS, and/or L proteins in the replication and transcription of viral RNA or result from duplex formation between the oligomers and complementary, plus-strand viral RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of interferon action inhibition of vesicular stomatitis virus in human amnion U cells by cloned human leukocyte interferon

The effects of a subsaturating, long treatment (24 h) dose of a highly purified cloned subspecies of human leukocyte interferon (IFN-αA) on vesicular stomatitis virus (VSV) primary macromolecular synthesis in ts G41-infected human amnion U cells were examined. IFN-αA, under these conditions, was found to inhibit primary VSV protein synthesis ten-fold while producing no detectable effect on the amount or integrity of primary viral message transcripts. There was no selective reduction by IFN-αA of the VSV G or M proteins.