Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR

Indomethacin, a cyclooxygenase‐1 and ‐2 inhibitor widely used in the clinic for its potent anti‐inflammatory/analgesic properties, possesses antiviral activity against several viral pathogens; however, the mechanism of antiviral action remains elusive. We have recently shown that indomethacin activates the double‐stranded RNA (dsRNA)‐dependent protein kinase R (PKR) in human colon cancer cells. Because of the important role of PKR in the cellular defence response against viral infection, herein we investigated the effect of indomethacin on PKR activity during infection with the prototype rhabdovirus vesicular stomatitis virus. Indomethacin was found to activate PKR in an interferon‐ and dsRNA‐independent manner, causing rapid (< 5 min) phosphorylation of eukaryotic initiation factor‐2 α‐subunit (eIF2α). These events resulted in shutting off viral protein translation and blocking viral replication (IC₅₀ = 2 μM) while protecting host cells from virus‐induced damage. Indomethacin did not affect eIF2α kinases PKR‐like endoplasmic reticulum‐resident protein kinase (PERK) and general control non‐derepressible‐2 (GCN2) kinase, and was unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2‐aminopurine. In addition, small‐interfering RNA‐mediated PKR gene silencing dampened the antiviral effect in indomethacin‐treated cells. The results identify PKR as a critical target for the antiviral activity of indomethacin and indicate that eIF2α phosphorylation could be a key element in the broad spectrum antiviral activity of the drug.     

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.     

Phosphatidylserine is not the cell surface receptor for vesicular stomatitis virus

The envelope protein from vesicular stomatitis virus (VSV) has become an important tool for gene transfer and gene therapy. It is widely used mainly because of its ability to mediate virus entry into all cell types tested to date. Consistent with the broad tropism of the virus, the receptor for VSV is thought to be a ubiquitous membrane lipid, phosphatidylserine (PS). However, the evidence for this hypothesis is indirect and incomplete. Here, we have examined the potential interaction of VSV and PS at the plasma membrane in more detail. Measurements of cell surface levels of PS show a wide range across cell types from different organisms. We demonstrate that there is no correlation between the cell surface PS levels and VSV infection or binding. We also demonstrate that an excess of annexin V, which binds specifically and tightly to PS, does not inhibit infection or binding by VSV. While the addition of PS to cells does allow increased virus entry, we show that this effect is not specific to the VSV envelope. We conclude that PS is not the cell surface receptor for VSV, although it may be involved in a postbinding step of virus entry.     

Posttranslational modification of vesicular stomatitis virus glycoprotein, but not JNK inhibition, is the antiviral mechanism of SP600125

Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogen-activated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/β). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein.     

Persistent vesicular stomatitis virus infection mediates base substitutions in viral RNA termini.

We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.     

The eIF2&alpha; kinases inhibit vesicular stomatitis virus replication independently of eIF2 phosphorylation.

The eIF2alpha kinases have been involved in the inhibition of vesicular virus replication but the contribution of each kinase to this process has not been fully investigated. Using mouse embryonic fibroblasts (MEFs) from knock-out mice we show that PKR and HRI have no effects on VSV replication as opposed to PERK and GCN2, which exhibit strong inhibitory effects. When MEFs containing the serine 51 to alanine mutation of eIF2alpha were used, we found that VSV replication is independent of eIF2alpha phosphorylation. Nevertheless, the kinase domain of the eIF2alpha kinases is both necessary and sufficient to inhibit VSV replication in cultured cells. Induction of PI3K-Akt/PKB pathway by eIF2alpha kinase activation plays no role in the inhibition of VSV replication. Our data provide strong evidence that VSV replication is not affected by eIF2alpha phosphorylation or downstream effector pathways such as the PI3K-Akt/PKB pathway. Thus, the anti-viral properties of eIF2alpha kinases are not always related to their inhibitory effects on host protein synthesis as previously thought and are possibly mediated by phosphorylation of proteins other than eIF2alpha.     

Formalin inactivation of vesicular stomatitis virus impairs T-cell- but not T-help-independent B-cell responses.

The effects of formalin on the infectivity and immunogenicity of vesicular stomatitis virus (VSV) serotype Indiana were investigated. We found that formalin inactivation of VSV prevents infection of Vero cells in a concentration- and time-dependent manner, as shown by fluorometric cell analysis and inhibition of plaque formation. Inactivated VSV failed to induce significant cytotoxic T-lymphocyte responses in vivo or after restimulation in vitro. In contrast, the early immunoglobulin M (IgM) response, which is T help independent in the VSV system, was unaltered, suggesting normal antigenicity for and induction of B cells. However, no switch to IgG occurred, demonstrating failure of induction of T help. If cross-reactive T help was provided by previous infection with a second serotype of VSV (New Jersey), the IgG response was almost completely restored, confirming that the absence of IgG was due to lack of T help. A formalin-treated preparation of glycoprotein of VSV led to a delayed but otherwise normal IgG response, whereas treatment of VSV with UV light or beta-propiolactone reduced IgG titers to the same extent as did formalin. These results suggest that loss of infectivity and the ensuing lack of amplification of viral antigens of formaldehyde-inactivated VSV is the major factor impairing induction of specific T-helper cell responses.     

RNase III cleaves vesicular stomatitis virus genome-length RNAs but fails to cleave viral mRNA''s.

The procaryotic RNA processing enzyme RNase III (endoribonuclease III [EC 3.1.4.24]) was used to probe vesicular stomatitis virus (VSV) RNAs for specific sites that could be recognized and cleaved. The effect of the enzyme on the RNAs was monitored by measuring their subsequent migration in denaturing agarose-urea gels. VSV virion RNA (negative strand; Mr, 4 X 10(6)) was cleaved by the enzyme to yield a set of discrete fragments which ranged on size from 3.5 X 10(6) to 0.2 X 10(6) daltons. The cleavage was a function of enzyme concentration, salt concentration, and time. A maximum of 20 to 22 fragments was generated under conditions of low enzyme concentration or short times of incubation. VSV genome-length intracellular RNA of both + and - polarity was also cleaved by RNase III. In contrast to the findings with virion-length RNA, however, the migration rates of VSV mRNA's purified by chromatography on polyuridylic acid-Sepharose were unaffected by treatment with RNase III. These results show that specific sites in the virion RNA and its full-length complement can be recognized by RNase III. Sites of this type are not present in the polyadenylic acid-containing mRNA, however.     

DBC2 is essential for transporting vesicular stomatitis virus glycoprotein

DBC2 is a tumor suppressor gene linked to breast and lung cancers. Although DBC2 belongs to the RHO GTPase family, it has a unique structure that contains a Broad-Complex/Tramtrack/Bric a Brac (BTB) domain at the C terminus instead of a typical CAAX motif. A limited number of functional studies on DBC2 have indicated its participation in diverse cellular activities, such as ubiquitination, cell-cycle control, cytoskeleton organization and protein transport. In this study, the role of DBC2 in protein transport was analyzed using vesicular stomatitis virus glycoprotein (VSVG) fused with green fluorescent protein. We discovered that DBC2 knockdown hinders the VSVG transport system in 293 cells. Previous studies have demonstrated that VSVG is transported via the microtubule motor complex. We demonstrate that DBC2 mobility depends also on an intact microtubule network. We conclude that DBC2 plays an essential role in microtubule-mediated VSVG transport from the endoplasmic reticulum to the Golgi apparatus.     

Noninfectious vesicular stomatitis virus particles deficient in the viral nucleocapsid.

Several temperature-sensitive mutants of vesicular stomatitis virus in complementation group III produce, at nonpermissive temperature, noninfectious particles which contain the viral M (matrix) and G (glycoprotein) proteins but less than 10% of the normal proportion of N protein or RNA. Since group III mutants are thought to be defective in the structural gene for the virus M protein, these findings demonstrate that an interaction between M and the nucleocapsid is of importance in virus budding. Taken together with earlier results, they suggest that M is the key protein in bud formation.     

Translation of individual host mRNA''s in MPC-11 cells is differentially suppressed after infection by vesicular stomatitis virus.

Infection of MPC-11 mouse plasmacytoma cells by vesicular stomatitis virus results in 30 to 35% reduction in [35S]methionine incorporation into total proteins within 30 min postinfection. By 6 h postinfection, total protein synthesis is reduced by 80 to 90%. However, even by 30 min postinfection, a differential suppression of the synthesis of individual host protein is observed. The synthesis of the immunoglobin G (IgG) heavy chain (H), and, in particular, the synthesis of IgG light chain (L), is considerably more resistant to vesicular stomatitis virus-induced inhibition than is the synthesis of the non-IgG proteins as a whole; e.g., when the synthesis of non-IgG proteins was reduced by 41%, the synthesis of the H and L chains was reduced by 28 and 7%, respectively. Furthermore, these alterations in the relative synthesis of the L chain, H chain, and non-IgG are comparable to the alterations previously observed in uninfected MPC-11 cells when the overall rate of polypeptide chain initiation was selectively reduced (D.L. Nuss and G. Koch, 1976). These results are discussed in terms of the strategy of virus-directed suppression of host mRNA translation.     

The alpha subunit of eukaryotic initiation factor 2B (eIF2B) is required for eIF2-mediated translational suppression of vesicular stomatitis virus

Eukaryotic translation initiation factor 2B (eIF2B) is a heteropentameric guanine nucleotide exchange factor that converts protein synthesis initiation factor 2 (eIF2) from a GDP-bound form to the active eIF2-GTP complex. Cellular stress can repress translation initiation by activating kinases capable of phosphorylating the alpha subunit of eIF2 (eIF2α), which sequesters eIF2B to prevent exchange activity. Previously, we demonstrated that tumor cells are sensitive to viral replication, possibly due to the occurrence of defects in eIF2B that overcome the inhibitory effects of eIF2α phosphorylation. To extend this analysis, we have investigated the importance of eIF2Bα function and report that this subunit can functionally substitute for its counterpart, GCN3, in yeast. In addition, a variant of mammalian eIF2Bα harboring a point mutation (T41A) was able overcome translational inhibition invoked by amino acid depravation, which activates Saccharomyces cerevisiae GCN2 to phosphorylate the yeast eIF2α homolog SUI2. Significantly, we also demonstrate that the loss of eIF2Bα, or the expression of the T41A variant in mammalian cells, is sufficient to neutralize the consequences of eIF2α phosphorylation and render normal cells susceptible to virus infection. Our data emphasize the importance of eIF2Bα in mediating the eIF2 kinase translation-inhibitory activity and may provide insight into the complex nature of viral oncolysis.     

Requirements for eIF4A and eIF2 during translation of Sindbis virus subgenomic mRNA in vertebrate and invertebrate host cells

We have examined the requirements for the initiation factors (eIFs) eIF4A and eIF2 to translate Sindbis virus (SV) subgenomic mRNA (sgmRNA) in the natural hosts of SV: vertebrate and arthropod cells. Notably, this viral mRNA does not utilize eIF4A in SV‐infected mammalian cells. However, eIF4A is required to translate this mRNA in transfected cells. Therefore, SV sgmRNA exhibits a dual mechanism for translation with respect to the use of eIF4A. Interestingly, SV genomic mRNA requires eIF4A for translation during the early phase of infection. In sharp contrast to what is observed in mammalian cells, active eIF2 is necessary to translate SV sgmRNA in mosquito cells. However, eIF4A is not necessary for SV sgmRNA translation in this cell line. In the SV sgmRNA coding region, proximal to the initiation codon is a hairpin structure that confers eIF2 independence only in mammalian cells infected by SV. Strikingly, this structure does not provide independence for eIF4A neither in mammalian nor in mosquito cells. These findings provide the first evidence of different eIF requirements for translation of SV sgmRNA in vertebrate and invertebrate cells. These observations can help to understand the interaction of SV with its host cells.     

Pseudotypes of vesicular stomatitis virus with the mixed coat of reticuloendotheliosis virus and vesicular stomatitis virus.

Vesicular stomatitis virus (VSV) forms pseudotypes with envelope components of reticuloendotheliosis virus (REV). The VSV pseudotype possesses the limited host range and antigenic properties of REV. Approximately 70% of the VSV, Indiana serotype, and 45% of VSV, New Jersey serotype, produced from the REV strain T-transformed chicken bone marrow cells contain mixed envelope components of both VSV and REV. VSV pseudotypes with mixed envelope antigens can be neutralized with excess amounts of either anti-VSV antiserum or anti-REV antiserum.     

Ferritin heavy chain is the host factor responsible for HCV-induced inhibition of apoB-100 production and is required for efficient viral infection

Hepatic fat export occurs by apolipoprotein B-100-containing lipoprotein production, whereas impaired production leads to liver steatosis. Hepatitis C virus (HCV) infection is associated to dysregulation of apoB-100 secretion and steatosis; however, the molecular mechanism by which HCV affects the apoB-100 secretion is not understood. Here, combining quantitative proteomics and computational biology, we propose ferritin heavy chain (Fth) as being the cellular determinant of apoB-100 production inhibition. By means of molecular analyses, we found that HCV nonstructural proteins and NS5A appear to be sufficient for inducing Fth up-regulation. Fth in turn was found to inhibit apoB-100 secretion leading to increased intracellular degradation via proteasome. Notably, intracellular Fth down-regulation by siRNA restores apoB-100 secretion. The inverse correlation between ferritin and plasma apoB-100 concentrations was also found in JFH-1 HCV cell culture systems (HCVcc) and HCV-infected patients. Finally, Fth expression was found to be required for robust HCV infection. These observations provide a further molecular explanation for the onset of liver steatosis and allow for hypothesizing on new therapeutic and antiviral strategies.