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Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication

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Qin-Qin Song a,1 , Ming-Zhi Lu a,1 , Juan Song a,1 , Miao-Miao Chi a , Lin-Jun Sheng a , Jie Yu a , Xiao-Nuan Luo a , Lu Zhang a , Hai-Lan Yao b , Jun Han a,∗ a State Key Laboratory for Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases (Hangzhou), National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Beijing 102206, China b Molecular Immunology Laboratory, Capital Institute of Pediatrics, 2 YaBao Rd, Beijing 100020, China

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Article history: Received 1 April 2015 Received in revised form 23 May 2015 Accepted 25 May 2015 Available online xxx

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Keywords: Coxsackievirus B3 2A protease EMCV IRES Protein translation

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1. Introduction

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To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13–18 RVVNRH, aa65–70 KNKHYP, or aa88–93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133–147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases. © 2015 Published by Elsevier B.V.

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Cap-dependent and internal ribosome entry site (IRES) elements are two major mechanisms involved in the initiation of mRNA translation in eukaryotic cells. In eukaryotes, protein synthesis requires a modified nucleotide ‘cap’ on the mRNA and proteins that recruit and position the ribosome (Merrick, 2004; Pestova et al., 2001; Pisarev et al., 2005; Sonenberg and Hinnebusch, 2009). Many pathogenic viruses also use an alternative, capindependent mechanism (IRES) that substitutes RNA structure to translate proteins. Some IRESs are able to bind directly to the ribosome through a specific RNA structure (Doudna and Sarnow, 2007; Hellen and Sarnow, 2001; Jackson, 2005). In animals, major groups of viruses encoding mRNAs with IRES elements include picornaviruses, dicistrovirus, flaviviruses, pestiviruses, and retroviruses (Balvay et al., 2009; Bienz et al., 1982; Jang, 2006; Nakashima and Uchiumi, 2009). These viral IRESs have been classified into five major structural groups (Asnani et al., 2015). IRES type I is typical

∗ Corresponding author. Tel.: +86 10 58900680; fax: +86 10 58900680. E-mail address: hanjun [email protected] (J. Han). 1 These authors contributed equally to this work.

of entero/rhinoviruses with poliovirus as the prototype, while type II is present in cardio/aphthoviruses, the prototype being encephalomyocarditis virus (EMCV). Hepatitis A virus is Type III, and Hepatitis C virus and Classical swine fever virus (CSFV) is Type IV (Asnani et al., 2015), Type V IRESs were recently identified in members of the genera Kobuvirus, Salivirus, and Oscivirus (Sweeney et al., 2012; Yu et al., 2011). Up to five groups may be present in picornaviruses, although there are two that are more representative (Asnani et al., 2015; Belsham, 2009; Hellen and Wimmer, 1995). Footnote: internal ribosome entry site (IRES), encephalomyocarditis virus (EMCV), untranslated regions (UTR), cricket paralysis virus (CrPV), eukaryotic initiation factor 4G (eIF4G), classical swine fever virus (CSFV). The Picornaviridae family includes a large number of animal pathogens, which are the causative agents of important diseases found worldwide. Their genomes consist of similarly organized single sense-strand RNA molecules, where untranslated regions (UTR) flank a single open reading frame that encodes a large polyprotein. The 5 end of the viral RNA is covalently linked to a small viral polypeptide, VPg, while the 3 end is polyadenylated (MartinezSalas et al., 2008). However, each picornavirus genera is defined by its unique features found in the UTR and coding regions (Oberste

http://dx.doi.org/10.1016/j.virusres.2015.05.020 0168-1702/© 2015 Published by Elsevier B.V.

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et al., 2007). Genomes of picornaviruses as well as other positive strand RNA viruses possess distinct functions in the viral life cycle. These include viral protein synthesis, negative strand RNA replication, and viral packaging into virions. To perform these multiple roles, regulatory mechanisms have evolved to finish these functions during the different stages of the viral cycle. Many of these regulatory mechanisms rely on structural elements located in the non-coding regions of the genome, in which specialized motifs are recognized by RNA-binding proteins encoded by the host or the viral RNA. The genome of each genus also contains distinctive features found along the coding region. One of the differences among members of the Picornaviridae family involves the virus-encoded proteases. The encoded 2A protease by enteroviruses differs structurally and functionally from other 2A proteins by members of other genera. As an example, the 2A protein derived from cardioviruses lack sequence homology to the 2A protease of enterovirus and protease activity. The 2A protease of enterovirus and rhinovirus are cysteine proteinases which share significant sequence homology to the trypsin-like small serine proteases. Enterovirus 2A protease processes the viral polyprotein and various other host proteins. These include eukaryotic initiation factor 4G (eIF4G) I, eIF4GII, and poly(A)-binding protein. It has been shown that during enterovirus infection, the 2A protease inhibits host translation and modulates viral RNA replication, viral translation, and viral RNA stability (Morrison and Racaniello, 2009). There are unique structural and mechanistic classes of IRES, which remains undetermined to whether 2A protease facilitates other type IRES dependent translation and promotes other genera virus replication. Here, we report that the 2A protease of Coxsackievirus B3 (CVB3), which is an enterovirus of the picornavirus family with IRES type I, confers high efficiency EMCV IRES (Type II)-dependent protein translation, inhibits classical capdependent protein translation, and promote EMCV replication. Similar to poliovirus 2A protease, expression of CVB3 2A protease led to cleavage of eIF4G I, a key factor for host protein synthesis. Moreover, deletions of short motif (aa13–18 RVVNRH, aa65–70 KNKHYP, or aa88–93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal basic amino acid motif (aa133–147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decrease its activity to inhibit EMCV IRES-dependent translation. We have also observed that 2A protease localized in perinuclear membrane and cytoplasm, and deletions of any NSL-like sequences prevented its localization to perinucleus. Deletion of C-terminal aa133–147 of the protease 2A facilitated its nuclear localization. These results demonstrated CVB3 2A protease upregulated EMCV IRES-dependent viral genome translation over cap-dependent host mRNA translation and facilitated EMCV replication by cross genera. A previous study has shown that there were apparent coinfections with more than one picornavirus in a single patient stool specimens from acute flaccid paralysis (AFP) cases and healthy contacts (Nix et al., 2013). Our results may provide better understanding into more effective interventions and treatments for co-infection of two picornavirus.

2. Materials and methods 2.1. Cell lines and virus HeLa, 293T, and BHK-21 cells were cultured in double modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 5% CO2 at 37 ◦ C. CVB3 (Nancy strain) was propagated and titrated on HeLa cell monolayer. Cells were seeded on culture dishes a day before infection with CVB3 at 1× TCID50. Cells were then incubated at 37 ◦ C throughout the infection. To determine the effects of CVB3 2A protease on the replication of EMCV(BJC3),

5 × 104 BHK-21 cells were used to inoculate with 1×, or 10× TCID50 EMCV in 96-well plates. After 1 h adsorption, the cell layer was rinsed with 1× PBS and DMEM containing 2% FBS was added. For virus RNA copy numbers determination, BHK-21 cells per well were grown for 12 h in DMEM with 2% FBS. 2.2. Real-time reverse transcriptase-polymerase chain reaction To quantify EMCV RNA copy numbers, cells inoculated with virus were collected, and viral RNAs were isolated using a QIAamp viral RNA minikit (Qiagen, Germany). Real-time quantitative RT-PCR (RT-qPCR) was performed using the onestep reaction mixture (ABI) containing 100 ng of each primer (forward, 5 -GGTGAGAGCAAGCCTCGCAAAGACAG-3 and reverse, 5 -CCCTACCTCACGGAATGGGGCAAAG-3 ) and 0.8 ␮l of probe (5 -FAM-TCGGCCTGTCATGAACAGAGAGGCG-Tamara-3 ). Reaction conditions were 48 ◦ C for 30 min and 95 ◦ C for 2 min, followed by 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. All further analysis was done using Biorad Software (Biorad CFX96, USA). 2.3. Plasmid construction The cDNAs encoding CVB3 2A protease were obtained by reverse transcription PCR of total RNA from CVB3-infected HeLa cells. The DNA was cloned into EcoRI and Hind III restriction sites of the eukaryotic expression vector pcDNA3.1 formed vector p2A which produce Flag-tagged proteins. The three DNA fragments encoding CVB3 2A protease with the basic amino acid-rich region truncation of 13–18 (RVVNRH), 65–70 (KNKHYP), and 88–93 (PRRYQS) amino acids and the one fragment with acidic amino acid-rich truncation of 133–147 (DIRDLLWLEDDAMEQ) were also cloned, respectively, into pcDNA3.1 to form plasmids p2A13, p2A65, p2A88, and p2A133 with Flag-tag (Fig. 1). Plasmid pFluc–EMCV–IRES–Rluc was constructed by inserting the EMCV IRES and Fluc fragment into plasmid pRluc. Plasmid pEMCV–IRES–EGFP was inserted with EMCV IRES sequences following with GFP sequences. Plasmid pEGFP-N1 with CMV promoter and GFP sequences was purchased from Promega Corporation (USA). All of the expression plasmids were verified by DNA sequencing. 2.4. Immunofluorescence assay staining 293T and BHK-21 cells were seeded at a density of about 2.5 × 105 cells per 35 mm culture dish. After overnight incubation, cells were transfected with plasmids (0.5–2 ␮g) using Lipofectamine 2000 reagent according to the manufacturer’s instructions (Invitrogen, USA). After 48 h of transfection, GFP expressions in cells were assessed by Fluorescence microscopy (Nikon, Japan). Transfected cells were fixed with acetone for 10 min, washed with buffer (1% skim milk in PBS) twice for 5 min each, and then incubated with the anti-Flag antibody (1:200 dilution) at 37 ◦ C for 30 min. Cells were washed with PBS at room temperature for 5 min three times, and then incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:2000 dilution) at 37 ◦ C for 30 min. Cells were washed three more times with PBS, and then DAPI (Merck, Germany) was used to stain the nucleus. 2.5. Luciferase assay Approximately 4 × 105 BHK-21 cells were plated on a 96well tissue culture Plates 3 h before transfection. Cells were cotransfected with pFluc–EMCV–IRES–RLuc (0.05 ␮g/well) and p2A/pcDNA3.1 (0.2 ␮g/well). Luciferase activity was measured by the dual-luciferase assay system (Promega, USA) according to the

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Fig. 1. Schematics for constructing CVB3 2A and 2A deletion mutant plasmids. Full length of CVB3 2A and deletion mutation with three potential NLS-like regions with basic amino acid-rich region and one acidic amino acid-rich region are indicated, respectively. Predicted values of PI change of 2A protease is shown for each deletion mutant

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recommended protocol by the manufacturer at 24 h, 36 h, and 48 h post-transfection.

2.6. Western blot analysis 293T cells were respectively harvested at 48 h following transfection of plasmids p2A, p2A13, p2A65, p2A88, and p2A133. HeLa cells inoculated with CVB3 were harvested at 2 h, 4 h, 6 h, 8 h, and 10 h. Cells were prepared by washing with cold PBS and lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP-40) was added. Cell extracts were then mixed with an equal volume of 2× loading buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.2% bromophenol blue), separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Blots were incubated with mouse anti-eIF4GI antibody (1:2000 dilution; Biovision, USA). Blots were then incubated with secondary antibody of horseradish peroxidise (HRP)-conjugated goat anti-mouse (Sigma, USA). Proteins were detected using the Enhanced Chemiluminescence Western Blotting kit (GE, USA).

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3. Results

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3.1. CVB3 2A protease facilitated EMCV replication

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To determine the role of CVB3 2A on the replication of EMCV, BHK-21 cells were transfected for 12 h with plasmid p2A expressing CVB3 2A protease or control plasmid pcDNA3.1. After transfection, 5 × 104 BHK-21 cells were infected with 50 ␮l 1× TCID50 or 10× TCID50 EMCV virus. To prevent the residual effects of the virus after infection, cells were washed with PBS after absorption for 1 h. After 12 h infection, cells were collected for RNA extraction to determine the relative amounts of virus by RT-qPCR. The results showed that relative amounts of viral RNA did not increase in BHK21 cells after inoculation with two virus dilution for 12 h. Similar results were observed in control cells transfected with pcDNA3.1. However, relative higher amounts of viral RNA in cells treated with p2A were observed after infection by two different viral dilutions (Fig. 2a). Cytopathic effect (CPE) were also showed similar results after transfection with p2A following 12 h post infection (p.i.) with EMCV virus. CPE rapidly increase to over 75% with 10× TCID50 EMCV virus and over 25% with 1× TCID50 virus (Fig. 2b). However, no obvious CPE were observed in cells transfected with pcDNA3.1 vector within 12 h of infection with two virus (Fig. 2b). Altogether, these results reveal that CVB3 2A protease plays an important role in the replication of EMCV.

3.2. CVB3 2A protease promotes EMCV IRES-dependent protein translation and induces eIF4G cleavage and inhibits cap-dependent protein translation To determine whether CVB3 2A protease facilitates EMCV replication through EMCV IRES structure, both pIRES-GFP and p2A vector were co-transfected into 293T cells or BHK-21 cells. GFP expression was respectively observed under fluorescence microscopy 48 h post-transfection. The results showed that CVB3 2A protease strongly promoted EMCV IRES-dependent GFP expression within 48 h in both 293T and BHK-21 cells (Fig. 3a, row 3). CVB3 2A also promoted EMCV IRES-dependent GFP expression in both of these cell lines with longer transfection periods (data not shown). However, the control group had minimal GFP expression in 293T and BHK-21 cells when co-transfected with pcDNA3.1 and pIRES-GFP (Fig. 3a, row 2). Many enterovirus 2A protease have been reported to abrogate cap-dependent translation in host cells (Jurgens et al., 2006). To ascertain whether CVB3 2A protease itself imposed an inhibitory effect upon the translation of protein in this test, both GFP expression vector, pEGFP-N1 with GFP protein being translated in a cap-dependent manner, and p2A vector were co-transfected into 293T cells or BHK-21 cells. The results showed that CVB3 2A protease strongly inhibited GFP expression in both 293T and BHK-21 cells 48 h post-transfection (Fig. 3b, row 3). However, no differences were observed between in the control group co-transfecting with pcDNA3.1 and pEGFP-N1 (Fig. 3b, row 2) and cells transfected alone with pEGFP-N1 (Fig. 3b, row 1). Most cellular mRNAs are translated by a cap-dependent mechanism that requires the binding of the trimerical complex eIF4F, comprised of eIF4G, eIF4E, and eIF4A, to the 7-methyl GpppN cap structure at the 5 end of the mRNA. eIF4GI is a component of the eIF4F, which plays a pivotal role in the interaction between capped mRNA and the 40S ribosomal subunit. We sought to determine whether eIF4GI is also cleaved after CVB3 infection. HeLa cells were infected by CVB3 and extracts were prepared from the infected cells which were harvested at 2 h, 4 h, 6 h, 8 h, and 10 h p.i. By immunoblot analysis, we observed that eIF4GI was cleaved at 4 h, 6 h, 8 h, and 10 h p.i. (Fig. 3c, Lane 1–5), while the protein remained intact in mock-infected cells (Fig. 3c, Lane 6). We further investigated that 2A deletion mutant affect the eIF4GI cleavage activity of 2A protease. At 48 h post-transfection with p2A, p2A13, p2A65, p2A88, and p2A133, as well as control vector pcDNA3.1, immunoblot analysis revealed that the cleavage of eIF4GI was only induced by p2A (Fig. 3d, Lane 5) in 293T cells, but all p2A deletion mutants did not cleave eIF4GI (Fig. 3d, Lane 1–4). The intact form of eIF4GI was detected in mock-transfected cells (Fig. 3d, Lane 6) or cells transfected with the control plasmid

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Fig. 2. CVB3 2A protease promotes EMCV replication. After transfection with 150 ng of each of the plasmids p2A and pcDNA3.1 for at least 12 h, BHK-21 cells were used to inoculate with 1×, or 10× TCID50 EMCV in 96-well plates. After 12 h viral growth, cell were harvested, nucleic acids extracted, and RNA detected by RT-qPCR (a). Twelve hours after the infection, the CPE was observed under a microscope. Magnification, ×400 (b).

Fig. 3. CVB3 2A protease promotes EMCV IRES-GFP expression and inhibits cap-dependent GFP expression. (a) CVB3 2A protease increases EMCV IRES-GFP expression in both 293T and BHK-21 cells. (b) CVB3 2A protease inhibits cap-dependent GFP expression. Magnification, ×200. (c) CVB3 infection induces eIF4G cleavage of HeLa cells. Whole HeLa cell lysates prepared from mock-infected cells or cells that had been infected with CVB3 for the indicated length of time were analyzed by immunoblotting with eIF4G I antibody. Molecular mass markers are indicated in kilodaltons (kD). Lane 1–5: eIF4G of HeLa cells in the presence of 100× TCID50 CVB3 for 2 h, 4 h, 6 h, 8 h, and 10 h; Lane 6: Cell control. (d) eIF4G cleavage of 293T cells expressing CVB3 2A protease and deletion mutation of 2A protease. The whole 293T cells lysates prepared from mock-infected cells or cells that had been transfected with p2A, p2A13, p2A65, p2A88, and p2A133 respectively for 48 h were analyzed by immunoblotting with eIF4G I antibody. Lane 1–5: eIF4G of 293T cells tranfected respectively with plasmids p2A13, p2A65, p2A88, p2A133, and p2A, pcDNA3.1; Lane 6: Cell control. Molecular mass markers are indicated in kilodaltons (kD).

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(Fig. 3d, Lane 7). These results also indicate that protein structural integrity is a prerequisite for 2A protein to play the function of enzymes.

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3.3. Functional region analysis of CVB3 2A protease

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3.3.1. Cellular localization of CVB3 2A protease and deletion mutants Though no potential NLS was identified in CVB3 2A protease, its sequence is similar to that of polio virus and EMCV (as determined by http://tw.expasy.org/index.html). Previous reports demonstrated that 2A protease of poliovirus and EMCV localized in the nucleus (Aminev et al., 2003; Bienz et al., 1982). To assessed the functional regions of CVB3 2A protease which contains 147 amino acids, the following plasmids of 2A protease mutants were respectively constructed with deletions in three basic amino acidrich region: p2A13, p2A65, p2A88, and in one acidic amino acid-rich region: p2A133. These mutant plasmids contained truncation in CVB3 2A protease of amino acid 13–18 (RVVNRH), 65–70 (KNKHYP), 88–93 (PRRYQS), and 133–147 (DIRDLLWLEDDAMEQ), respectively (Fig. 1). The plasmids p2A, p2A13, p2A65, p2A88, and p2A133 were subsequently transfected into 293T cells to determine their effects on cellular localization of the CVB3 2A protein. The results demonstrated that the 2A protein localized to both cytoplasm and the nuclear membrane (Fig. 4, line 1, row 2, 3) but not the nucleus. The 2A protein with deletion of one of three basic amino acids-rich regions was found to localize fully in the cytoplasm (Fig. 4, line 2–4, row 2, 3). These results suggested that three basic amino acids-rich region may facilitate the localization of CVB3 2A to the nuclear membrane. However, it is notable that CVB3 2A protein fully localized to the nucleus after deletion of the acidic amino acid rich C-terminus (Fig. 4, line 5, row 3), which may contain a inhibition structure of CVB3 2A protease nuclear location.

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3.3.2. Deletion mutations of CVB3 2A protease inhibits its EMCV IRES-dependent and cap-dependent protein translation To determine what amino acid regions of CVB3 2A protease affect its IRES-dependent protein translation activity, we cotransfected BHK-21 cells with pIRES-GFP or pEGFP-N1 along with either p2A, p2A13, p2A65, p2A88, or p2A133. The results showed CVB3 2A protease promoted EMCV IRES-dependent GFP expression (Fig. 5a, line 1, row 2) and inhibit cap-dependent GFP protein translation (Fig. 5a, line 2, row 2). However, all deletions of CVB3 2A protease without acidic or basic amino acid-rich region did not promote both EMCV IRES-dependent (Fig. 5a, line 1, row 3–6) and cap-dependent GFP protein translation (Fig. 5a, line 2, row 3–6). To further study that the effects of CVB3 2A protease and subsequent deletion mutations on EMCV IRES-dependent protein translation, BHK-21 cells were co-transfected with deletion mutants (as described above) and pFluc–EMCV–IRES–RLuc, which contains a CMV promoter to study cap-dependent Fluc and EMCV IRES-directed Rluc translation. After transfection p2A for 24 h, 36 h, and 48 h, the Rluc/Fluc luciferase activities ratio results showed CVB3 2A protease rapidly increased up to 1.1, 6, 12.5 times relative to the activity ratio detected in control cells transfected with pcDNA3.1 (Fig. 5b). However, all deletion mutations of 2A protease did not significantly change the Rluc/Fluc luciferase activities ratio compared with control group (Fig. 5b). These results showed that the activity of CVB3 2A protease promoted IRES-dependent translation is required the whole protein structure.

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4. Discussion

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viral genomes. One strategy that a number of viruses utilize to compete for and hijack host ribosomes is to engage in active translation of cellular mRNAs to inhibit host translation (Walsh and Mohr, 2011). Another strategy used by viruses is to recruit ribosomes through alternative, cap-independent mechanisms (IRESs) binding with subset of canonical initiation factors. Among them, picornaviruses belong to an important group of animal pathogens that have evolved to use IRES structure to recruit ribosomes and promote translation (Doudna and Sarnow, 2007; MartinezSalas et al., 2001). Although there are dissimilarity in sequence between different viral families, covariation of IRESs are associated with conserved RNA secondary structures that play an important role in the function of IRES in the same virus genus. Conserved structural motifs, in addition to the factors required for optimal translation differentiating the viral IRESs, can assist in classifying the picornaviruses IRESs into distinct groups (Filbin and Kieft, 2009; Martinez-Salas et al., 2008). The success of the picornavirus replication cycle and the effectiveness of infection are dependent on intact and functional IRES regions. The IRES region is one of determinants of viral pathogenesis and virulence (Malnou et al., 2002; Pilipenko et al., 2001) and is therefore a target for antiviral drugs that aims to inactivate or inhibit IRES (Gutiérrez et al., 1994). To sustain viability, viruses exploit the translational machinery of host cells and have developed complex mechanisms over time to inhibit translation of the host while simultaneously producing their own viral proteins. In addition to cleaving viral polyproteins, picornavirus 2A protease is known to cleave eIF4G which inhibits cap-dependent translation of cellular mRNA without affecting the translation of viral RNA. Consistently, our results demonstrated that the cleavage of eIF4GI by CVB3 2A protease inhibited capdependent protein translation. Independent of the shutoff of host protein synthesis, several studies have reported that picornavirus 2A protease also stimulated the translation of virus RNA by eIF4G cleavage, which is mediated by the C-terminal region of the 2A protease. Therefore, 2A protease enhances viral protein synthesis in infected cells both by inhibiting host cell protein synthesis and by stimulating the translation of viral RNA (Hunt et al., 1999; Ziegler et al., 1995). Several genetic studies suggest that the poliovirus protease 2A is able to increase translation of genomic mRNAs containing IRES from poliovirus or EMCV, but not those of hepatitis C or Cricket paralysis virus (CrPV) (Sanz et al., 2010). Our results further demonstrated that CVB3 2A protease, which belongs to enterovirus genera, not only inhibited host cell protein synthesis, but also promoted EMCV IRES-dependent protein translation in cardiovirus genera. This suggested that the 2A protease could affect the viral interaction between two different genera. Our results showed that CVB3 2A protease played a direct role in EMCV virus progeny. In agreement that 2A protease participates in viral protein translation by IRES, the presence of 2A protein increased EMCV IRES-directed GFP/Fluc translation and suppressed cap-dependent GFP expression by cleavage of eIF4G. These results further demonstrate that CVB3 2A protease interacted with EMCV IRES to regulate EMCV viral replication. Thus the 2A protease may cleavage eIF4G, which is an essential scaffolding protein interacting between eIF4E (cap-binding protein), eIF4A (an RNA helicase), and eIF3 to recruit the small ribosomal subunit (Gingras et al., 1999) for the assembly of cap-dependent eIF4F complexes, to inhibit cap-dependent cellular protein translation and through binding to EMCV IRES to initiate viral protein translation and EMCV replication. Further studies need to be done to prove that piconavirus 2A protease facilitates virus replication by cross genera. Previous reports demonstrated that the 2A protease of poliovirus and EMCV is localized to the nucleus (Aminev et al., 2003; Bienz et al., 1982). Although no potential NLS was identified

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Fig. 4. Cellular localization of CVB3 2A protease and deletion mutations of 2A protease. The plasmids p2A, p2A13, p2A65, p2A88, and p2A133 were transfected

Q4 respectively into 293T cells. Transfected cells were fixed and then incubated with the anti-Flag antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Green color, Row 2). DAPI was used to stain the nucleus (Blue color, Row 1). Merged picture is shown in the row 3 (Blue white color). Line 1–6, cells were respectively transfected plasmids p2A, p2A13, p2A65, p2A88, and p2A133. Scale bar: 20 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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within the CVB3 2A protease, we found three potential NLS-like regions with basic amino acid-rich region and one acidic amino acid-rich region in CVB3 2A protease. In this study, we constructed four 2A protease mutant plasmids that contain deletion of acidic or basic amino acid rich regions. In these p2A deletion mutants, the isoelectric point (PI) of wild type CVB3 2A protease was altered from 4.87 to 4.58, 4.77, 4.77, and 6.16 (Fig. 1). We found that deletion of any NSL-like region comprising basic amino acids did not prevent cytoplasm accumulation of 2A protease to nucleus. In particular, deletion of acidic amino acids 133–147 in the C-terminus of

CVB3 2A protease allowed for nuclear localization. Thus, the acidic amino acid-rich region may affect nuclear localization of CVB3 2A protease, which is potentially mediated by three potential NLS containing rich basic amino acids regions. Our results showed that CVB3 2A protease was largely distributed in the cytoplasm, with some detection on the nuclear membrane, but not the nucleus (Fig. 4). Our results may provide further insight into the structural components that regulate cellular distribution of the 2A protein through modulation of nuclear pore complex (NPC) proteins (Watters and Palmenberg,

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Fig. 5. Effects of deletion mutation of CVB3 2A protease on protein translation. (a) Deletion mutation of CVB3 2A protease affect on EMCV IRES-GFP translation(Line 1) and capdependent GFP protein expression (Line 2) Row 1–6, cells were respectively transfected plasmids pcDNA3.1, p2A, p2A13, p2A65, p2A88, and p2A133. Magnification, ×200. (b) Deletion mutation of CVB3 2A protease affect on EMCV IRES-Rluc and cap-dependent Fluc. BHK-21 cells in 96-well plates were transfected with respectively the plasmids p2A and p2A13, p2A65, p2A88, and p2A133. At the indicated times of post-transfection, cell extracts were harvested and tested for luciferase activity and Rluc/Fluc ratio was calculated.

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2011). We suspect that localization of entrovirus 2A protease to the nuclear membrane may regulate both cellular and viral protein expression through either open or close of nuclear pore. Replication of picornavirus occurs in the cytoplasm, while mature EMCV 2A protease accumulates in nucleoli shortly after infection (Groppo et al., 2011). Thus, CVB3 2A has different roles from EMCV 2A protease, though both inhibit cellular capdependent translation. We speculated that EMCV 2A protein may regulate mRNA transcript whose mechanism remains unknown, while CVB3 2A protease still regulate both cellular and viral protein translation activity. Our study demonstrated that CVB3 2A proteases may repress cellular protein expression, while enhancing viral replication of EMCV through viral IRES regions. Further studies are needed to determine spatial structure of CVB3 2A proteases combining with EMCV IRES during viral replication. Determining the biological mechanisms, by which CVB3 2A protease promote EMCV replication, may help explain co-infection of two different cytoplasmic replicating picornavirus. Effective therapies for prevention and treatment of the diseases caused by picornavirus infection are not currently available (Lall et al., 2004). The 2A protease is an attractive target for the development of therapeutic antiviral agents. Identifying novel therapeutic antiviral agents by using in vivo screening to known 2A proteases

may provide effective interventions and treatments for co-infection of viral diseases. 5. Conclusions In summary, CVB3 2A protease promote EMCV replication through inhibition of cellular cap-dependent protein translation and enhance EMCV IRES associated protein translation. CVB3 2A protease is largely distributed in the cytoplasm and in the nuclear membrane, but not the nucleus. Deletion of the NLS-like and the acidic domain at the C-terminus of 2A break both its protease activity and IRES-dependent protein translation. Deletion of acidic domain in the C-terminus of CVB3 2A proteases allowed for nuclear localization of CVB3 2A protease from the cytoplasm. Acknowledgments We gratefully acknowledge Prof. Han-Chun Yang for the gifts of EMCV(BJC3) strain. This work was supported by the China Mega-Project for Infectious Disease (2011ZX10004-001, Q3 2012ZX10004215, and 2013ZX10004805002), National Natural Science Foundation of China Grants (31371397), Beijing Natural Science Foundation (7144197) and the SKLID Development Grant (2011SKLID104).

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Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication.

To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II I...
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