Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.     

Cell-free RNA replication systems based on a human cell extracts-derived in vitro translation system with the encephalomyocarditisvirus RNA

To study the relationship between translation and replication of encephalomyocarditisvirus (EMCV) RNA, we established a cell-free RNA replication system by employing a human cell extracts-based in vitro translation system. In this system, a cis-EMCV RNA replicon encoding the Renilla luciferase (R-luc) or GFP and the viral regulatory proteins efficiently replicated with simultaneous translation of the encoded protein. To examine how translation of the replicon RNA, but not the translated products, affected replication, a trans-EMCV RNA replicon encoding R-luc and the RNA replication elements was next constructed. The trans-replicon RNA replicated only in the presence of the regulatory proteins pre-expressed in trans. Incubation with cycloheximide, puromycin or a dominant-negative eukaryotic translation initiation factor 4A following expression of the regulatory proteins almost completely inhibited not only translation of the trans-replicon RNA but also replication of the RNA, suggesting that EMCV RNA translation promotes replication of the RNA. In conclusion, the cell-free RNA replication systems should become useful tools for the study of the viral RNA replication.     

Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube

Virus particles are used in vaccination, drug delivery, and material sciences. Here we devised a system where the RNA virus encephalomyocarditis virus (EMCV) is synthesized from DNA templates in vitro. When a plasmid or a PCR product harboring the full-length cDNA of EMCV in the T7 promoter/terminator unit was incubated in a HeLa cell extract supplemented with T7 RNA polymerase, EMCV was produced within 4 h at an efficiency of over 10-fold compared with the system programmed with the EMCV RNA. The EMCV RNA transcribed by the virally encoded RNA-dependent RNA polymerase was predominantly incorporated into the EMCV particle even in the presence of a larger amount of the EMCV RNA transcribed by T7 RNA polymerase from the plasmid.     

High-level expression of soluble viral structural protein in Escherichia coli

Pharmaceutically relevant virus-like particles (VLPs) can potentially be manufactured cheaply and efficiently through in vitro assembly of viral structural protein in cell-free reactors, but a bottleneck for this processing route is the currently low-level expression of soluble viral protein in efficient cell factories such as Escherichia coli (E. coli). Here, we report expression levels of up to 180 mg L(-1) that are achievable from low-cell-density E. coli cultures using a simple and low cost strategy. We investigated effects of host strain, plasmid, inducer concentration, pre-induction temperature and cell density at induction with design of experiment (DOE). The statistical approach successfully identified significant effects and their interactions, and provided insights into the role of codon-usage effects in expression of viral structural protein. In particular, our results support the notion that full codon optimization may be unnecessary to improve expression of viral genes rich in E. coli rare codons; using a strategically modified host cell could provide a simpler and cheaper alternative.     

Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location

The internal ribosomal entry site (IRES)from encephalomyocarditis virus (EMCV) is a popular RNA element used widely in experimental and pharmaceutical applications to express proteins in eukaryotic cells or cell-free extracts. Inclusion of the wild-type element in monocistronic or bicistronic messenger RNAs (mRNAs) confers a high level of cap-independent translation activity to appropriately configured cistrons. The history of this element and the experimental consequences of sequence derivations inherent to commercial IRES vectors are less well known. Compared head-to-head with dual-luciferase reporter constructs, a native EMCV IRES in a bicistronic configuration directed 8- to 10-fold more protein than a similarly configured pIRES vector. It also produced nearly twice as much protein as pCITE-1, an early monocistronic iteration, harboring a suboptimal A7 sequence in a crucial structural motif The results indicate that investigators should be aware of and carefully report the sequence of their IRES in any comparative study. The preferred IRES (viral bases 273-845) and the minimum IRES (viral bases 400-836) for optimum activity are illustrated.     

PKR-dependent mechanisms of gene expression from a subgenomic hepatitis C virus clone

Studies on hepatitis C virus (HCV) replication have been greatly advanced by the development of cell culture models for HCV known as replicon systems. The prototype replicon consists of a subgenomic HCV RNA in which the HCV structural region is replaced by the neomycin phosphotransferase II (NPTII) gene, and translation of the HCV proteins NS3 to NS5 is directed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). The interferon (IFN)-inducible protein kinase PKR plays an important role in cell defense against virus infection by impairing protein synthesis as a result of eIF-2alpha phosphorylation. Here, we show that expression of the viral nonstructural (NS) and PKR proteins and eIF-2alpha phosphorylation are all variably regulated in proliferating replicon Huh7 cells. In proliferating cells, induction of PKR protein by IFN-alpha is inversely proportional to viral RNA replication and NS protein expression, whereas eIF-2alpha phosphorylation is induced by IFN-alpha in proliferating but not in serum-starved replicon cells. The role of PKR and eIF-2alpha phosphorylation was further addressed in transient-expression assays in Huh7 cells. These experiments demonstrated that activation of PKR results in the inhibition of EMCV IRES-driven NS protein synthesis from the subgenomic viral clone through mechanisms that are independent of eIF-2alpha phosphorylation. Unlike NS proteins, HCV IRES-driven NPTII protein synthesis from the subgenomic clone was resistant to PKR activation. Interestingly, activation of PKR could induce HCV IRES-dependent mRNA translation from dicistronic constructs, but this stimulatory effect was mitigated by the presence of the viral 3' untranslated region. Thus, PKR may assume multiple roles in modulating HCV replication and protein synthesis, and tight control of PKR activity may play an important role in maintaining virus replication and allowing infection to evade the host's IFN system.     

Mutational analysis of the J-K stem-loop region of the encephalomyocarditis virus IRES.

Cap-independent translation of encephalomyocarditis virus (EMCV) RNA is controlled by a segment of the 5' untranslated region termed the internal ribosomal entry site, or IRES. The IRES contains a series of stem-loop structural elements. The J and K stems (EMCV bases 682 to 795), near the center of the IRES, are well conserved among all cardio-, aphtho-, and hepatoviruses. We have examined the biological roles of these elements by constructing mutations within the J-K sequences of EMCV and testing the mutations for activity in translation, translation competition, UV cross-linking, and viral infectivity assays. Mutations near the helical junction of J and K proved severely detrimental to both cellular translation and cell-free translation of downstream cistrons. The same mutations reduced the ability of the IRES to compete for cellular factors in competition assays and reduced the infectivity of viral genomes carrying these lesions. A mutation in the terminal loop of J gave similar results. In contrast, mutations within the terminal loop of K had minimal impact on in vitro translation activity and IRES competitive ability. However, in vivo analysis of the K-loop mutations revealed deficiencies during cellular translation and further showed markedly reduced infectivity in HeLa cells. UV cross-linking experiments identified a 49-kDa protein which interacts strongly with the J-K region, but the identity of this protein and its contribution to IRES activity are unclear.     

Encephalomyocarditis virus 2A protein is required for viral pathogenesis and inhibition of apoptosis

The encephalomyocarditis virus (EMCV), a Picornaviridae virus, has a wide host spectrum and can cause various diseases. EMCV virulence factors, however, are as yet ill defined. Here, we demonstrate that the EMCV 2A protein is essential for the pathogenesis of EMCV. Infection of mice with the B279/95 strain of EMCV resulted in acute fatal disease, while the clone C9, derived by serial in vitro passage of the B279/95 strain, was avirulent. C9 harbored a large deletion in the gene encoding the 2A protein. This deletion was incorporated into the cDNA of a pathogenic EMCV1.26 strain. The new virus, EMCV1.26Δ2A, was capable of replicating in vitro, albeit more slowly than EMCV1.26. Only mice inoculated with EMCV1.26 triggered death within a few days. Mice infected with EMCV1.26Δ2A did not exhibit clinical signs, and histopathological analyses showed no damage in the central nervous system, unlike EMCV1.26-infected mice. In vitro, EMCV1.26Δ2A presented a defect in viral particle release correlating with prolonged cell viability. Unlike EMCV1.26, which induced cytopathic cell death, EMCV1.26Δ2A induced apoptosis via caspase 3 activation. This strongly suggests that the 2A protein is required for inhibition of apoptosis during EMCV infection. All together, our data indicate that the EMCV 2A protein is important for the virus in counteracting host defenses, since Δ2A viruses were no longer pathogenic and were unable to inhibit apoptosis in vitro.     

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.     

Leader-Induced Phosphorylation of Nucleoporins Correlates with Nuclear Trafficking Inhibition by Cardioviruses

Picornaviruses disrupt nucleocytoplasmic trafficking pathways during infection. Poliovirus and rhinovirus inhibit nuclear protein import/export through a series of 2A protease-dependent cleavages within nuclear pore proteins (nucleoporins [Nups]), including Nup62, Nup98, and Nup153. Cardioviruses lack the same protease and instead affect trafficking inhibition through an activity mapped to their leader (L) protein, a 67- to 76-amino acid (aa) polypeptide with no known enzymatic activity. We have shown that L from encephalomyocarditis virus (EMCV) binds and inhibits the activity of Ran-GTPase, a key regulator of nucleocytoplasmic transport. We now report that recombinant EMCV L triggers the unregulated efflux of protein cargo from preloaded HeLa cell nuclei in cell-free reactions dependent upon Xenopus egg cytosol or HeLa cell-derived cytosol. Recombinant L was the only viral protein necessary for this activity or for nuclear protein import inhibition. Mutational disruption of the L protein zinc finger domain (C₁₉A) abrogated the inhibitory activity for both import and efflux in cell extracts, but mutations in the C-terminal acidic domain of L (aa 37 to 61) did not. Notably, HeLa cell nuclei treated with L, or those from EMCV-infected cells, showed reproducibly altered patterns of nucleoporin phosphorylation. Nup62, Nup153, and Nup214 each became hyperphosphorylated in an L-dependent manner. Staurosporine, a broad-spectrum kinase inhibitor, blocked this phosphorylation and rescued nuclear import/export activity from L-dependent inhibition. Therefore, cardioviruses target the same group of nucleoporins as enteroviruses, but the effector mechanism triggered by L (or L-Ran complexes) involves a unique cytosol-dependent phosphorylation cascade rather than proteolysis.     

Encephalomyocarditis virus induces PKR-independent mitogen-activated protein kinase activation in macrophages

In this study, we provide evidence that the double-stranded RNA-dependent protein kinase (PKR) is not required for virus-induced expression of inducible nitric oxide synthase (iNOS) or the activation of specific signaling pathways in macrophages. The infection of RAW264.7 cells with encephalomyocarditis virus (EMCV) induces iNOS expression and nitric oxide production, which are unaffected by a dominant-negative mutant of PKR. EMCV infection also activates the mitogen-activated protein kinase, cyclic AMP response element binding protein, and nuclear factor kappaB (NF-kappaB) signaling cascades at 15 to 30 min postinfection in PKR+/+ and PKR-/- macrophages. Activation of these signaling cascades does not temporally correlate with PKR activity or the accumulation of EMCV RNA, suggesting that an interaction between a structural component of the virion and the cell surface may activate macrophages. Consistent with this hypothesis, empty EMCV capsids induced comparable levels of iNOS expression, nitrite production, and activation of these signaling cascades to those induced by intact virions. These findings support the hypothesis that virion-host cell interactions are primary mediators of the PKR-independent activation of signaling pathways that participate in the macrophage antiviral response of inflammatory gene expression.     

Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.     

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.     

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.     

Rapamycin and Wortmannin Enhance Replication of a Defective Encephalomyocarditis Virus

Inhibitors of the phosphatidylinositol 3-kinase (PI3 kinase)–FKBP-rapamycin-associated protein (FRAP) pathway, such as rapamycin and wortmannin, induce dephosphorylation and activation of the suppressor of cap-dependent translation, 4E-BP1. Encephalomyocarditis virus (EMCV) infection leads to activation of 4E-BP1 at the time of host translation shutoff. Consistent with these data, rapamycin mildly enhances the synthesis of viral proteins and the shutoff of host cell protein synthesis after EMCV infection. In this study, two defective EMCV strains were generated by deleting portions of the 2A coding region of an infectious cDNA clone. These deletions dramatically decreased the efficiency of viral protein synthesis and abolished the virus-induced shutoff of host translation after infection of BHK-21 cells. Both translation and processing of the P1-2A capsid precursor polypeptide are impaired by the deletions in 2A. The translation and yield of mutant viruses were increased significantly by the presence of rapamycin and wortmannin during infection. Thus, inhibition of the PI3 kinase-FRAP signaling pathway partly complements mutations in 2A protein and reverses a slow-virus phenotype.