Influenza Virus mRNA Translation Revisited: Is the eIF4E Cap-Binding Factor Required for Viral mRNA Translation?

Influenza virus mRNAs bear a short capped oligonucleotide sequence at their 5' ends derived from the host cell pre-mRNAs by a "cap-snatching" mechanism, followed immediately by a common viral sequence. At their 3' ends, they contain a poly(A) tail. Although cellular and viral mRNAs are structurally similar, influenza virus promotes the selective translation of its mRNAs despite the inhibition of host cell protein synthesis. The viral polymerase performs the cap snatching and binds selectively to the 5' common viral sequence. As viral mRNAs are recognized by their own cap-binding complex, we tested whether viral mRNA translation occurs without the contribution of the eIF4E protein, the cellular factor required for cap-dependent translation. Here, we show that influenza virus infection proceeds normally in different situations of functional impairment of the eIF4E factor. In addition, influenza virus polymerase binds to translation preinitiation complexes, and furthermore, under conditions of decreased eIF4GI association to cap structures, an increase in eIF4GI binding to these structures was found upon influenza virus infection. This is the first report providing evidence that influenza virus mRNA translation proceeds independently of a fully active translation initiation factor (eIF4E). The data reported are in agreement with a role of viral polymerase as a substitute for the eIF4E factor for viral mRNA translation.     

Poliovirus switches to an eIF2-independent mode of translation during infection

Inhibition of translation is an integral component of the innate antiviral response and is largely accomplished via interferon-activated phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). To successfully infect a host, a virus must overcome this blockage by either controlling eIF2α phosphorylation or by utilizing a noncanonical mode of translation initiation. Here we show that enterovirus RNA is sensitive to translation inhibition resulting from eIF2α phosphorylation, but it becomes resistant as infection progresses. Further, we show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral internal ribosome entry site, plays a role in mediating viral translation under conditions that are nonpermissive for host cell translation. Together, these results provide a mechanism by which enteroviruses evade the antiviral response and provide insight into a noncanonical mechanism of translation initiation.     

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex

Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.     

La Crosse virus infection of mammalian cells induces mRNA instability.

La Crosse virus infection of BHK cells leads to a dramatic shutoff of not only host protein synthesis but also viral protein synthesis later in infection. This shutoff can be accounted for by the loss of the cytoplasmic cellular and viral mRNAs. The induction of mRNA instability requires extensive virus replication, since when cycloheximide is added early in infection the preexisting viral and cellular mRNAs do not decrease upon incubation of the cultures. Pretreatment of the cultures with actinomycin D does not affect the ability of La Crosse virus infection to induce mRNA instability, and examination of the rRNAs shows no evidence of specific degradation due to activation of the interferon-associated latent RNase. The induction of mRNA instability therefore does not appear to operate through an interferon pathway. Viral mRNA synthesis, on the other hand, is not turned off during infection, and the cap-dependent endonuclease involved in viral mRNA initiation may be responsible for the mRNA instability.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

Regulation of mRNA translation and cellular signaling by hepatitis C virus nonstructural protein NS5A

The NS5A nonstructural protein of hepatitis C virus (HCV) has been shown to inhibit the cellular interferon (IFN)-induced protein kinase R (PKR). PKR mediates the host IFN-induced antiviral response at least in part by inhibiting mRNA translation initiation through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). We thus examined the effect of NS5A inhibition of PKR on mRNA translation within the context of virus infection by using a recombinant vaccinia virus (VV)-based assay. The VV E3L protein is a potent inhibitor of PKR. Accordingly, infection of IFN-pretreated HeLa S3 cells with an E3L-deficient VV (VVDeltaE3L) resulted in increased phosphorylation levels of both PKR and eIF2alpha. IFN-pretreated cells infected with VV in which the E3L locus was replaced with the NS5A gene (VVNS5A) displayed diminished phosphorylation of PKR and eIF2alpha in a transient manner. We also observed an increase in activation of p38 mitogen-activated protein kinase in IFN-pretreated cells infected with VVDeltaE3L, consistent with reports that p38 lies downstream of the PKR pathway. Furthermore, these cells exhibited increased phosphorylation of the cap-binding initiation factor 4E (eIF4E), which is downstream of the p38 pathway. Importantly, these effects were reduced in cells infected with VVNS5A. NS5A was also found to inhibit activation of the p38-eIF4E pathway in epidermal growth factor-treated cells stably expressing NS5A. NS5A-induced inhibition of eIF2alpha and eIF4E phosphorylation may exert counteracting effects on mRNA translation. Indeed, IFN-pretreated cells infected with VVNS5A exhibited a partial and transient restoration of cellular and viral mRNA translation compared with IFN-pretreated cells infected with VVDeltaE3L. Taken together, these results support the role of NS5A as a PKR inhibitor and suggest a potential mechanism by which HCV might maintain global mRNA translation rate during early virus infection while favoring cap-independent translation of HCV mRNA during late infection.     

Inhibition of agonist-induced activation of phospholipase C following poxvirus infection.

Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.     

Replication and cytopathic effect of oncolytic vesicular stomatitis virus in hypoxic tumor cells in vitro and in vivo

Tumor hypoxia presents an obstacle to the effectiveness of most antitumor therapies, including treatment with oncolytic viruses. In particular, an oncolytic virus must be resistant to the inhibition of DNA, RNA, and protein synthesis that occurs during hypoxic stress. Here we show that vesicular stomatitis virus (VSV), an oncolytic RNA virus, is capable of replication under hypoxic conditions. In cells undergoing hypoxic stress, VSV infection produced larger amounts of mRNA than under normoxic conditions. However, translation of these mRNAs was reduced at earlier times postinfection in hypoxia-adapted cells than in normoxic cells. At later times postinfection, VSV overcame a hypoxia-associated increase in alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation and initial suppression of viral protein synthesis in hypoxic cells to produce large amounts of viral protein. VSV infection caused the dephosphorylation of the translation initiation factor eIF-4E and inhibited host translation similarly under both normoxic and hypoxic conditions. VSV produced progeny virus to similar levels in hypoxic and normoxic cells and showed the ability to expand from an initial infection of 1% of hypoxic cells to spread through an entire population. In all cases, virus infection induced classical cytopathic effects and apoptotic cell death. When VSV was used to treat tumors established in nude mice, we found VSV replication in hypoxic areas of these tumors. This occurred whether the virus was administered intratumorally or intravenously. These results show for the first time that VSV has an inherent capacity for infecting and killing hypoxic cancer cells. This ability could represent a critical advantage over existing therapies in treating established tumors.     

Inhibition of host protein synthesis and degradation of cellular mRNAs during infection by influenza and herpes simplex virus

Cloned DNA copies of two cellular genes were used to monitor, by blot hybridization, the stability of particular cell mRNAs after infection by influenza virus and herpesvirus. The results indicated that the inhibition of host cell protein synthesis that accompanied infection by each virus could be explained by a reduction in the amounts of cellular mRNAs in the cytoplasm, and they suggested that this decrease was due to virus-mediated mRNA degradation.     

Cell cycle dependency of murine cytomegalovirus replication in synchronized 3T3 cells.

Synchronized murine 3T3 cells have been used to investigate the possible dependency of murine cytomegalovirus replication upon the cell cycle. The normal latent period of 12 h characteristic of asynchronous 3T3 cells was protracted to more than 24 h after an early G1 infection in synchronous cells. In this case viral progeny were not detected until after the initiation of the host S-phase. Cells maintained in the G1 phase did not replicate virus. This failure could not be explained by a decrease in virus penetration but was apparently due to a requirement for an event associated with the host S-phase. Thymidine-induced inhibition of cell cycle traverse also blocked virus replication. Viral DNA synthesis did not initiate until after the initiation of host DNA. In contrast, herpes simplex virus type 1 replicated in 3T3 cells independently of the cell cycle.     

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors.     

Murine cytomegalovirus infection inhibits tumor necrosis factor alpha responses in primary macrophages

Despite robust host immune responses the betaherpesvirus murine cytomegalovirus (MCMV) is able to establish lifelong infection. This capacity is due at least in part to the virus utilizing multiple immune evasion mechanisms to blunt host responses. Macrophages are an important cell for MCMV infection, dissemination, and latency despite expression in the host of multiple cytokines, including tumor necrosis factor alpha (TNF-alpha), that can induce an antiviral state in macrophages or other cells. In this study, we found that MCMV infection of bone marrow-derived macrophages inhibited TNF-alpha-induced ICAM-1 surface expression and mRNA expression in infected cells via expression of immediate early and/or early viral genes. MCMV infection blocked TNF-alpha-induced nuclear translocation of NF-kappaB. This inhibition of TNF-alpha signaling was explained by a decrease in TNF receptor 1 (TNFR1) and TNFR2 that was due to decreased mRNA for the latter. These findings provide a mechanism by which MCMV can evade the effects of an important host cytokine in macrophages.     

Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.     

VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway

Interference with nucleocytoplasmic transport is a strategy employed by certain viruses to compromise host cellular function. While it has been shown that the matrix (M) protein of the vesicular stomatitis virus (VSV) inhibits nuclear export of host cell mRNAs, the underlying mechanism has not been fully established. Here we show that VSV M protein binds the mRNA export factor Rae1/mrnp41. A mutant of M protein defective in Rae1 binding is unable to inhibit mRNA nuclear export. We further show that increased expression of Rae1 fully reverts the inhibition of mRNA export induced by M protein or following virus infection. We found that Rae1 is induced by interferon-gamma, a cytokine that plays a critical role in the immune response to viruses, such as VSV. Thus, these results demonstrate that VSV M protein blocks mRNA export by disrupting Rae1 function, which can be reverted by induction of Rae1 expression.     

Cytoplasm from Poliovirus-infected HeLa Cells inhibits Cell-free Haemoglobin Synthesis

INFECTION of HeLa cells with poliovirus causes an inhibition of host cell protein synthesis with a concomitant conversion of polysomes to single ribosomes^{1, 2}. This process is thought to be mediated by a product of the viral genome, for infection of cells with virus particles which have been inactivated by ultraviolet irradiation does not affect the rate of protein synthesis¹. The inhibition acts directly on translation rather than on the production of mRNA^{1, 3}, yet neither chain elongation nor the release of polypeptide chains from ribosomes seems to be affected². Chain initiation, therefore, is the only possible target. Willems and Penman have suggested that cellular mRNA is inactivated after infection, preventing its reassociation with ribosomes³. To test these hypotheses, we assayed for the presence of inhibitors of initiation in poliovirus-infected HeLa cell cytoplasm, using a lysate of rabbit reticulocytes that initiates the synthesis of new polypeptide chains at a rate comparable with that in intact cells.     

Role for the phosphatidylinositol 3-kinase-Akt-TOR pathway during sindbis virus replication in arthropods

The efficient transmission of alphaviruses requires the establishment of a persistent infection in the arthropod vector; however, the nature of the virus-arthropod host interaction is not well understood. The phosphatidylinositol 3-kinase (PI3K)-Akt-TOR pathway is a signaling pathway with which viruses interact to manipulate cellular functions. The viral activation of this pathway can enhance translation and inhibit apoptosis, potentially promoting viral replication; conversely, repression can enhance cell death. Using a system to study Sindbis virus RNA replication in Drosophila melanogaster, we found that the overexpression of Akt enhanced Sindbis virus replication. In contrast, a decrease in viral replication was observed for flies hypomorphic for the Akt gene. Infection of cultured Drosophila cells led to the phosphorylation and activation of Akt. The chemical inhibition of PI3K, Akt, and TOR in mosquito cells reduced virus replication, suggesting that this pathway is proviral. Early after infection, there was an increase in the TOR-dependent phosphorylation of 4E-BP1 in mosquito cells and a consequent increase in the translation of a capped reporter mRNA. In contrast, no change in 4E-BP1 phosphorylation was seen in mammalian cells, and the level of translation of the reporter decreased following infection. Finally, we found that the increase in the phosphorylation of 4E-BP1 was stimulated by replicon RNA but not by UV-inactivated virus. Our data indicate that Sindbis virus replication complex formation in mosquito cells activates the PI3K-Akt-TOR pathway, causing the phosphorylation of 4E-BP1 and increasing the formation of eukaryotic initiation factor 4F (eIF4F), which promote cap-dependent translation. This virus-induced increase in cap-dependent translation allows the efficient translation of viral mRNA while minimizing the burden on the cell.