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1 2 3 4 5 6 Heterogeneous nuclear ribonucleoprotein M facilitates enterovirus 7 infection 8 9 Julienne M Jagdeo1, Antoine Dufour2, Gabriel Fung3, Honglin Luo3, Christopher 10 M Overall1,2 and Eric Jan1* 11 1Department of Biochemistry and Molecular Biology, 2Department of Oral 12 Biological and Medical Sciences, 3Department of Pathology and Laboratory 13 Medicine, University of British Columbia, Vancouver BC V6T 1Z3 14 15 *corresponding author: ej@mail.ubc.ca 16 17 18 Keywords: proteinase, picornavirus, hnRNPM, translation, proteome, enterovirus19

JVI Accepted Manuscript Posted Online 29 April 2015J. Virol. doi:10.1128/JVI.02977-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 20 Picornavirus infection involves a dynamic interplay of host and viral protein 21 interactions that modulates cellular processes to facilitate virus infection and 22 evade host-antiviral defenses. Here, using a proteomics-based approach to 23 identify protease-generated neo-N-termini peptides, we identify a novel target of 24 the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M 25 (hnRNP M), which is a nucleo-cytoplasmic shuttling RNA-binding protein that is 26 primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by 27 poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in 28 poliovirus- and CVB3-infected HeLa cells and in hearts of CVB3-infected mice. 29 hnRNP M relocalizes from the nucleus to cytoplasm during poliovirus infection. 30 Finally, depletion of hnRNP M using siRNA knockdown approaches decreases 31 poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus IRES 32 translation and viral RNA stability. We propose that cleavage of and subverting 33 the function of hnRNP M is a general strategy utilized by picornaviruses to 34 facilitate viral infection. 35 36 37

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IMPORTANCE 38 Enteroviruses, a member of the picornavirus family, are RNA viruses that 39 cause a range of diseases including respiratory ailments, dilated cardiomyopathy 40 and paralysis. Although enteroviruses have been studied for several decades, 41 the molecular basis of infection and the pathogenic mechanisms leading to 42 disease are still poorly understood. Here, we have identified hnRNP M as a novel 43 target of a viral proteinase. We demonstrate that the virus subverts the function 44 of hnRNP M and redirects it to a step in the viral life cycle. We propose that 45 cleavage of hnRNP M is a general strategy that picornaviruses use to facilitate in 46 infection. 47

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INTRODUCTION 48 Proteases play fundamental roles in cells by ultimately changing the fate 49 and function of its substrates through proteolytic cleavage or degradation (1-9). 50 Many viruses have exploited these strategies to promote infection. For instance, 51 picornaviruses translate their RNA genome as a single polyprotein, which is then 52 processed by viral-encoded proteinases to produce the mature viral proteins. In 53 addition, viral proteinases target a subset of host proteins to modulate cellular 54 processes, inhibit antiviral responses and subvert host proteins to aid in specific 55 steps of the viral life cycle such as viral translation and replication. One of the 56 best characterized is the rapid host translational shutoff that occurs in poliovirus-57 infected cells, which is facilitated through cleavage of eukaryotic translation 58 initiation factors eIF4G and the poly(A) binding protein (PABP) by the viral 59 proteinases 2A and 3C (10-14). Overall inhibition of protein synthesis leads to 60 release of translation factors and ribosomes that are then diverted to poliovirus 61 protein synthesis. The identification of host protein substrates of viral proteinases 62 has provided important insights into the virus-host interaction strategies that 63 contribute to the viral life cycle. 64 Polyprotein processing by viral-encoded proteinases is a common strategy 65 among RNA viruses. Members of the picornavirus family, which include 66 poliovirus and coxsackievirus B3 (CVB3), are cytoplasmic RNA viruses that 67 possess a positive single-stranded RNA genome of approximately ~7500 bases 68 in length encoding a single open reading frame (15, 16). The 5' non-coding 69 region contains an internal ribosome entry site (IRES) that directs translation to 70

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produce a 220 kDa polyprotein, which is then processed by viral proteinases to 71 produce mature structural and non-structural viral proteins (17, 18). All 72 picornaviruses encode the 3C proteinase (3Cpro), a chymotrypsin-like protease 73 with an active-site cysteine nucleophile rather than a serine nucleophile. This 74 enzyme or its polypeptide precursor (3CD) performs the majority of the 75 polyprotein processing. A subset of picornaviruses encodes a second proteinase, 76 either the 2A proteinase (2Apro) or the leader proteinase that directs minor 77 cleavages of the polyprotein (19-21). The 3Cpro cleaves primarily between 78 glutamine and glycine (Q↓G) residues at eight distinct sites within the 79 polyprotein (22). The 2Apro has at least a single cleavage site within the 80 polyprotein, autocatalytically cleaving itself at its N-terminus between a tyrosine 81 and glycine (Y↓G) residue (20, 23). Mutagenesis of substrate peptides has 82 shown that P and P’ amino acids flanking the N- and C-terminus of the cleavage 83 site, respectively, are also important for substrate recognition and proteinase 84 activity (24-26). For example, poliovirus 3Cpro shows a strong preference for 85 proline and alanine at the P2 and P4 positions, respectively, whereas 2Apro 86 prefers isoleucine/leucine, threonine/serine, and proline at the P4, P2 and P2’ 87 positions, respectively (23-26). Furthermore, secondary structures adopted 88 during the folding process of the polyprotein are also required to mediate 89 substrate specificity and temporally regulate cleavage (27). Currently, there are 90 approximately 20 known host proteins that are targeted by picornavirus 2Apro and 91 3Cpro, and have been shown to disrupt several cellular processes, including 92 transcription, RNA metabolism, nucleo-cytoplasmic transport, cytoskeleton 93

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dynamics and stress granule formation (28-36). Despite the identification of many 94 host substrates, it is likely that the full repertoire of host substrates has yet to be 95 identified. 96 Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of 97 nucleo-cytoplasmic shuttling RNA-binding proteins that were originally identified 98 based on their association with pre-mRNAs (37). There are approximately 20 99 hnRNPs, named hnRNP A to U, that all contain at least one RNA-binding 100 domain, either an RNA recognition motif (RRM) or an hnRNP K-homologues 101 (KH) domain. Most hnRNPs are primarily involved in pre-mRNA splicing but they 102 also aid in diverse aspects of RNA metabolism, including translational control, 103 telomere biogenesis, mRNA stability and trafficking (37, 38). hnRNP activity also 104 contributes to different steps of the picornavirus life cycle. For example, hnRNPs 105 A1, I (more commonly known as the PTB) and K interact with the 5’UTR IRES of 106 several picornaviruses to facilitate viral translation and replication (2, 5, 6). 107 Moreover, viral proteinases target a subset of hnRNPs to regulate specific steps 108 of virus infection. Poly(rC)-binding protein 2 (PCBP2), also known as hnRNP E2, 109 binds to the poliovirus IRES to facilitate translation initiation, however, at late 110 times of infection, cleavage of PCBP2 by 3Cpro modifies its association with the 5’ 111 UTR to inhibit viral translation and thereby switches to viral replication (4, 7). 112 Thus, poliovirus has evolved a strategy to regulate PCBP2 function via cleavage 113 by 3Cpro in order to temporally regulate viral translation and replication. Not all 114 hnRNPs are pro-viral, as some hnRNPs have antiviral effects; hnRNP D, also 115 known as AU-rich biding factor (AUF1), binds directly to stem-loop IV of the 116

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poliovirus IRES to inhibit viral translation (3, 8, 9). However, in poliovirus- and 117 CVB3-infected cells, this antiviral activity is inhibited through 3Cpro-mediated 118 cleavage of AUF1 (8, 9). 119 In this study, we identified hnRNP M as a novel substrate of both 120 poliovirus and CVB3 3Cpro. hnRNP M is cleaved in both poliovirus- and CVB3 121 infected HeLa cells and mouse tissues, producing two cleavage products that 122 persist during infection. We demonstrate that endogenous hnRNP M relocalizes 123 from the nucleus to the cytoplasm during poliovirus infection and that hnRNP M 124 promotes poliovirus and CVB3 infection. Depletion of hnRNP M does not affect 125 IRES translation nor viral RNA stability. In summary, our data reveals a strategy 126 utilized by poliovirus and CVB3 to target hnRNP M by the 3Cpro to aid in virus 127 infection. 128

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MATERIALS AND METHODS 129 Cell Culture and Virus Stocks 130 HeLa cells were cultured in Dulbecco’s modified Eagle medium (DMEM) 131 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin 132 (P/S) at 37°C. Poliovirus (Mahoney type 1 strain) was generated from 133 transfection of in vitro transcribed RNA from a poliovirus infectious clone, 134 pT7pGemPolio (generously provided by Kurt Gustin, University of Arizona) into 135 HeLa cells. Poliovirus and CVB3 (Kandolf strain) were both propagated and 136 titred in HeLa cells. 137 138 Plasmids and Transfections 139 The full-length hnRNP M open reading frame (NM_005968) was PCR-140 amplified and cloned into the KpnI and NotI restriction sites of a p3XFlag-CMV-141 7.1 vector (Sigma) with a 3XHA-tag cloned downstream using XbaI and BamHI 142 sites. Full-length CVB3 3Cpro (M88483) and a CVB3 3Cpro C147A mutant were 143 PCR-amplified and cloned into the NotI and NdeI restriction sites of pET28b. 144 Constructs were verified by sequencing. 145 For DNA transfections, HeLa cells were transfected with 1-2 ug of plasmid 146 using Lipofectamine 2000 (Invitrogen) according to the manufacture’s protocol. 147 Cells were transfected in antibiotic-free media for 5 hours, then replaced with 148 complete media for 24-48 hours. For siRNA transfections, HeLa cells were 149 transfected (30-40% confluency) with either hnRNP M (Ambion; s9259, s9261, 150

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s9260) or scrambled siRNA (Ambion) using Lipofectamine RNAimax (Invitrogen). 151 Knockdown efficiency was validated by Western blot analysis. 152 The pIRES-poliovirus and pIRES-dEMCV bicistronic reporter constructs 153 (generously provided by Gabriele Fuchs and Peter Sarnow, Stanford University) 154 were transfected into HeLa cells for 1 hour and then infected with poliovirus. 155 Cells were harvested and luciferase activity was monitored using a dual 156 Luciferase reporter assay kit (Promega). Luminescence was measured using a 157 Centro LB 960 luminometer (Berthod Technologies). 158 159 Virus Infections 160 Virus was absorbed with HeLa cells at the indicated multiplicity of infection 161 (MOI) for 1 hour in serum-free DMEM at 37°C, then washed with phosphate 162 buffered saline (PBS) and replaced with complete media. For virus infections in 163 the presence of Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD-FMK, Calbiochem), 164 zVAD-FMK was added to serum-free DMEM containing virus at a final 165 concentration of 50 μM. 166 For pulse chase experiments, media was replaced with methionine- and 167 cysteine-free media containing 30 μCi of [35S]-EasyTag™ Express Protein 168 Labeling Mix (Perkin Elmer) for 30 minutes prior to harvesting. Cells were lysed 169 in RIPA buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% 170 Triton-X100, 0.02% Na-deoxycholate, 0.1% SDS) supplemented with protease 171 inhibitors (Roche), and protein concentrations were determined by Bradford 172

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assay. Proteins were resolved by SDS-PAGE and analyzed by phosphorimager 173 analysis. 174 For plaque assays, virus-infected cells were washed twice with PBS, 175 harvested in serum-free DMEM and lysed by 3 cycles of freeze thawing. Serial 176 dilutions of cell supernatants were incubated with HeLa cells for 1 hour at 37°C. 177 Cells were washed twice with PBS and overlaid with DMEM containing 2% FBS, 178 1% P/S and 1% methylcellulose. After 72 hours, cells were fixed with 50% 179 methanol and stained with 1% crystal violet. Plaques were counted and viral titer 180 was calculated as plaque forming units (PFU) per milliliter. 181 182 Western Blot Analysis 183 Equal amounts of protein were resolved by SDS-PAGE and transferred to 184 a PVDF membrane. Antibodies used in this study were as follows: 1:1000 hnRNP 185 M, 1:500 α-tubulin, 1:1000 actin (Santa Cruz Biotechnologies), 1:3000 G3BP1 186 (BD Transduction Science), 1:3000 VP1 (Dako), 1:1000 PABP (generously 187 provided by Dr. Richard Lloyd, Baylor College of Medicine), 1:1000 PARP 188 (Pharmingen), 1:1000 GAPDH (Abcam). 189 190 Northern Blot Analysis 191 Total RNA was isolated from cells using Trizol reagent (Invitrogen). RNA 192 was resolved on a denaturing agarose gel and transferred to Zeta-probe blotting 193 membrane (Biorad). Radiolabeled DNA hybridization probes were generated 194 using the Deca labeling kit (Fermentas). The amount of radiolabled probe 195

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hybridized to the blot was analyzed and quantified using a phosphorimager 196 (Typhoon, Amersham Biosciences). 197 198 Immunofluoresence 199 HeLa cells on coverslips were fixed with cold 100% methanol for 10 200 minutes, washed three times with PBS and then blocked with 5% bovine serum 201 albumin (BSA) in PBS for 1 hour, followed by 1 hour incubation with primary 202 antibody with 1% BSA in PBS at room temperature. The primary antibodies were 203 used as follows: 1:25 hnRNP M and 1:50 HA (Santa Cruz Biotechnologies), 204 1:400 double stranded RNA (English & Scientific Consulting Bt), 1:100 Flag 205 (Sigma). Coverslips were washed three times with PBS then incubated with 206 1:500 secondary antibody (goat anti-rabbit or goat anti-mouse Texas red, and 207 goat anti-mouse Alexa Fluor 488 (Life Technologies) with 1% BSA in PBS and 208 Hoescht to stain for nuclei. Following three washes, coverslips were mounted 209 onto slides using Prolong Gold Antifade Reagent (Life Technologies). Cells were 210 imaged and analyzed using a Nikon Eclipse Ti confocal microscope and pictures 211 were taken using the NIS-elements software. 212 213 Protein Purification 214 Wild type and catalytically inactive (C109A) CVB3 2Apro and His-tagged 215 wild type and catalytically inactive mutant (C147A) poliovirus 3Cpro were purified 216 using expression plasmids, pET-Cx2A, pET-Cx2A C109A, pET3Chc and 217 pET3Chc C147A (generously provided by Richard Lloyd, Baylor College of 218

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Medicine). Wild type CVB3 3Cpro and a C147A catalytically inactive mutant 219 proteinase were cloned into a pET28b expression vector containing an N-220 terminal His-tag. 2Apro proteinases were expressed in and purified from BL21 221 bacterial cells by ion exchange chromatography and size exclusion 222 chromatography as previously described (10, 39). 3Cpro was expressed in and 223 purified from BL21 bacterial cells by Nickle-nitrilotriacetic acid (Ni-NTA) chelating 224 resin affinity chromatography. Fractions containing purified 3Cpro

were then 225 pooled and dialyzed in 20 mM Hepes (pH 7.4), 100 mM NaCl, 7 mM β-226 mercaptoethanol, and 20% glycerol. Expression plasmid containing 3CD was 227 generously provided by Bert Semler (UC-Irvine). Recombinant 3CD was purified 228 as described (8). The integrity and purity of the purified protein were verified by 229 Coomassie R-250 staining using SDS-PAGE analysis. 230 231 In Vitro Cleavage Assay 232 HeLa cell lysates were prepared by harvesting and pelleting cells in cold 233 PBS and then resuspending in 2-3X pellet volumes of cleavage assay buffer (20 234 mM Hepes (pH 7.4), 150 mM KOAc and 1 mM DTT) supplemented with protease 235 inhibitors (Roche). Cells were incubated on ice for 10 minutes, and then lysed 236 with 25 strokes in a dounce homogenizer. Lysates were then clarified by 237 centrifugation at 13,000 rpm for 15 minutes at 4°C. 238 Purified hnRNP M (20 pg, Origene) or HeLa cell lysates were incubated 239 with purified wild type or catalytically inactive CVB3 2Apro (5 ng/μl), poliovirus 240 3Cpro (100 ng/μl) or CVB3 3Cpro (100 ng/μl) in cleavage assay buffer at 37°C for 241

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different periods of times as indicated. Reactions were resolved by SDS-PAGE 242 and proteins were assessed by Western blot analysis. 243 244 N-terminal TAILS proteomics 245 Equal amounts of HeLa cell lysates were incubated with either wild type or 246 catalytically inactive C147A mutant purified poliovirus 3Cpro in cleavage assay 247 buffer at 100:1 (w/w) ratio of protein to enzyme. TAILS was performed as 248 previously described (40). In brief, after the protein was denatured and reduced, 249 cysteines were alkylated and samples were isotopically labeled at the protein 250 level by reductive dimethylation of primary amines. Thus, any protein alpha 251 amine natural N-terminus or proteinase generated neo-N-terminus were labeled 252 and so could be identified after trypsin digestion. Heavy (wild type proteinase-253 treated) and light (C147A-proteinase treated) isotopically labeled samples were 254 combined, salts removed and the samples concentrated by methanol 255 precipitation. The sample was then subject to trypsin digestion, followed by 256 enrichment of labeled peptides by a negative selection step using a dendritic 257 polyglycerol aldehyde polymer purchased from Flintbox 258 (http://flintbox.com/public/project/1948) as described (40). Unbound labeled N-259 termini peptides were separated from the polymer-bound peptides by 260 centrifugation through a 10-kDa Microcon filter (Millipore). The flow through was 261 collected and fractionated by strong-cation exchange high-performance liquid 262 chromatography as previously described (40). Samples were then analyzed by 263 liquid chromatography-MS/MS on an Agilent G4240A ChipCube interfaced 264

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directly to an Agilent G6550A Q-TOF mass spectrometer (Agilent Technologies). 265 The full comprehensive list of protein targets will be provided in a follow-up 266 publication. 267 268 Mass Spectrometry Data Analysis 269 MS peaks were searched by MASCOT (version 2.2, Matrix Science, 270 London, UK) against a human database at a 1% false discovery rate. MASCOT 271 searches of MS data were performed separately for heavy- and light-labeled 272 peptides. Searches were performed using the following modifications: fixed 273 carbamidomethylation of cysteines (+57.021 Da (Cys)), fixed heavy lysine 274 (+34.0631 Da (Lys)), or light lysine (+28.0311 Da (Lys)); variable methionine 275 oxidation (+15.995 Da (Met)), and fixed and variable modifications of the N-276 termini with heavy formaldehylde (+34.0641 Da (N-termini)), light formaldehyde 277 (+28.0311 Da (N-termini)), and acetylation (+42.011 Da (N-termini)). The 278 additional search criteria used were as follows: semi-ArgC cleavage specificity 279 with up to three missed cleavages; a monoisotopic mass error window for the 280 parent ion of 0.4 to 0.6 Da; and peptide mass tolerance of 0.4 Da for MS/MS 281 fragment ions. Allowed peptide charge states were 1+, 2+, and 3+. Quantification 282 of the heavy to light isotopically labeled peptides was achieved by using 283 ProteoIQ. Statistically significant quantified peptides were determined by box plot 284 analysis. 285 286 Mouse Infection by CVB3 287

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A/J mice (Jackson Laboratory #000646) at approximately 5 weeks of age 288 were infected with 105 plaque-forming units by intraperitoneal injection. Mock 289 infections were performed using equal volumes of PBS. At 9 days post-infection, 290 mouse hearts were harvested, lysed and immunoblotted as indicated. 291 292 Statistical Analysis 293 All statistical analyses were performed using GraphPad Prism. All graphs 294 represent the mean ± standard deviation (s.d.). P values were determined using 295 a paired t-test and statistical significance was determined as p<0.05. 296 297

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RESULTS 298 hnRNP M is targeted by poliovirus 3C proteinase 299 Although several host protein targets of picornavirus 3Cpro are known, we 300 hypothesized that additional host proteins are targeted by 3Cpro under virus 301 infection. To identify novel host substrates of 3Cpro, we used a proteomics-based 302 approach called TAILS that simultaneously identifies protease substrates and 303 their cleavage sites by tandem mass spectrometry (MS/MS) (40, 41). TAILS uses 304 a negative selection to enrich for cleaved substrate neo-N-termini and natural N-305 termini after binding and removal of internal tryptic peptides to a polyaldehyde 306 polymer. Briefly, HeLa cell lysates were incubated with purified wild type or a 307 catalytically inactive (C147A) recombinant poliovirus 3Cpro, and then processed 308 by TAILS followed by tandem MS/MS to identify proteins from enriched neo-N-309 termini peptides. After isotopic ratio analysis of the identified peptides and 310 substrate winnowing of a list of high-confidence, high-ratio peptides (wild 311 type/mutant samples), we identified a spectra of a neo-N-termini peptide 312 390GGGGGGGSVPGIER from hnRNP M with a cleavage site at position 313 389Gln↓Gly (Fig. 1A, 1B). The 389Q and 390G at P1 and P1` positions, respectively, 314 is consistent with the consensus cleavage site of poliovirus 3Cpro (22). hnRNP M 315 is a nucleo-cytoplasmic shuttling protein primarily known for its role in pre-mRNA 316 splicing and alternative splicing (42-47). There are 4 alternatively spliced 317 isoforms of hnRNP M derived from a single pre-mRNA transcript, all of which 318 contain three RRMs (48). All four isoforms are highly similar in size and typically 319 migrate as a closely spaced doublet referred to as M1/2 and M3/4 (48). The M4 320

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isoform encodes the longest isoform of 730 amino acids, with a predicted 321 molecular weight of 77 kDa (Fig. 1A) (48). The M1 isoform encodes a 690 amino 322 acid variant of M4 of 74 kDa, containing a 39 amino acid deletion between RRM1 323 and RRM2 (48). Notably the same cleavage site sequence is found in all 324 isoforms of hnRNP M. The cleavage site falls between RRM2 and RRM3 at 325 amino acid 389 within the M4 isoform, which would result in two cleavage 326 products of approximately 41 and 36 kDa. 327 To confirm that hnRNP M is targeted by poliovirus 3Cpro, we used an in 328 vitro cleavage assay by incubating purified recombinant poliovirus 3Cpro with 329 HeLa cell lysates. Incubation of wild type but not mutant 3Cpro with lysates 330 resulted in the expected cleavage products of PABP, a known substrate of 3Cpro 331 (Fig. 1C) (10). Immunoblotting using the hnRNP M (1D8) antibody detected a 332 prominent band at approximately 77 kDa, which corresponds to the mass of the 333 M4 isoform. Addition of the wild type poliovirus 3Cpro resulted in the accumulation 334 of a cleavage product of approximately 36 kDa, which was detected as early as 5 335 minutes of incubation, whereas no cleavage was observed with the mutant 3Cpro 336 after incubating for 60 minutes (Fig. 1C). Detection of the 36 kDa cleavage 337 product is consistent with the predicted size of the C-terminal protein product 338 generated from cleavage at the site identified by TAILS and suggests that the 339 1D8 antibody recognizes the C-terminal half of hnRNP M. Addition of a 340 recombinant CVB3 2Apro to HeLa cell lysates resulted in cleavage of PABP but 341 not hnRNP M, suggesting that cleavage of hnRNP M is 3Cpro-specific (Fig. 1D). 342 To further assess whether hnRNP M is a direct substrate for 3Cpro, we incubated 343

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poliovirus 3Cpro with purified recombinant hnRNP M. Wild type 3Cpro, but not the 344 mutant, generated a 36 kDa cleavage product similar to that observed from the in 345 vitro cleavage assay (Fig. 1E). Finally, to confirm the cleavage site identified by 346 TAILS, we subcloned either the wild type or a Q389E/G390P mutant hnRNP M 347 into a pCMV mammalian expression vector fused in-frame with a 3X FLAG-tag 348 and 3X HA-tag at the N- and C-terminus, respectively (FLAG-hnRNP M-HA, Fig. 349 1F). The Q389E/G390P mutations are predicted to disrupt the consensus 350 cleavage site of 3Cpro and thus this mutant should be proteinase insensitive. The 351 expected molecular weight of FLAG-hnRNP M-HA is approximately 84 kDa. 352 Using the 1D8 hnRNP M antibody, immunoblotting analysis detected two proteins 353 in lysates of cells transfected with FLAG-hnRNP M-HA, the endogenous hnRNP 354 M at 77 kDa and the slower migrating tagged protein at approximately 84 kDa 355 (Fig. 1G). Moreover, expression of FLAG-hnRNP M-HA was also detected using 356 a FLAG antibody (Fig. 1H). After establishing expression of FLAG-hnRNP M-HA 357 in HeLa cells, we obtained lysates from HeLa cells expressing either the wild type 358 or a Q389E/G390P mutant FLAG-hnRNP M-HA and incubated the lysates in vitro 359 with either wild type or inactive 3Cpro. Wild type, but not the inactive 3Cpro 360 generated a 43 kDa cleavage product that was detected by anti-FLAG antibody 361 (Fig. 1H). As predicted, the Q389E/G390P FLAG-hnRNP M-HA mutant was 362 insensitive to 3Cpro cleavage (Fig 1H). Because 3Cpro is also expressed as 3CD 363 (49), we determined whether 3CD targets hnRNP M. As shown with 3Cpro, 364 recombinant 3CDpro targeted wild-type but not mutant Q389E/G390P FLAG-365 hnRNP M-HA in the in vitro cleavage assay (Fig. 1I). Taken together, these 366

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results demonstrate that hnRNP M is a bona fide substrate of poliovirus 3Cpro 367 and 3CDpro that is cleaved directly between amino acid pair 389Q↓G. 368 369 hnRNP M is cleaved in poliovirus-infected HeLa cells 370 To examine whether cleavage of hnRNP M occurs under virus infection, 371 HeLa cells were either mock- or poliovirus-infected, then harvested at different 372 times after infection. Cleavage of PABP was observed beginning at 3 hours post 373 infection, producing the expected cleavage products (Fig. 2A) (11). Similar to the 374 timing of PABP cleavage, the levels of full-length hnRNP M began to decrease at 375 3 hours post infection, which is concurrent with the appearance of two proteins at 376 36 and 39 kDa (Fig. 2A). By 7 hours post infection, the full-length hnRNP M was 377 completely degraded, whereas the two cleavage products remained detectable 378 throughout infection (Fig. 2A). The presence of two cleaved proteins suggests 379 that hnRNP M may be cleaved more than once during infection. 380 Apoptosis-induced activation of caspases can occur in picornavirus 381 infections (50, 51). To assess whether cleavage of hnRNP M is a result of 382 caspase activity, poliovirus-infected HeLa cells were incubated in the presence or 383 absence of the general caspase inhibitor, zVAD-FMK. Both hnRNP M cleavage 384 products were still observed in poliovirus-infected cells in the presence of zVAD-385 FMK, whereas cleavage of poly(ADP) ribose polymerase (PARP), a known 386 caspase-3 substrate, was inhibited (Fig. 2B). Thus, we demonstrate that hnRNP 387 M is cleaved to completion under poliovirus infection in a caspase-independent 388 manner and that the cleavage products persist during infection. 389

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390 Subcellular relocalization of hnRNP M in poliovirus-infected cells 391 hnRNP M is a predominantly nuclear localized protein (52). Thus, it is of 392 interest to determine how hnRNP M is targeted by a cytoplasmic RNA virus. To 393 address this, we monitored the localization of hnRNP M in mock- and poliovirus-394 infected cells by immunofluorescence confocal microscopy. In mock-infected 395 cells, hnRNP M was predominantly localized to the nucleus, in agreement with its 396 role as a nuclear protein involved pre-mRNA splicing (Fig. 3, mock-infected). 397 However, upon infection, hnRNP M underwent a dramatic relocalization to the 398 cytoplasm beginning at 3 hours post infection to near completion at 5 and 7 hours 399 post infection (Fig. 3, poliovirus-infected). This subcellular redistribution from the 400 nucleus to the cytoplasm is similar to that observed with other hnRNPs such as 401 hnRNP K and A1 during poliovirus infection (53). Given that hnRNP M is cleaved 402 nearly to completion and that the cleaved fragments of hnRNP M persist in 403 poliovirus-infected cells at 5 and 7 hours post infection (Fig. 2A), the 404 immunoflourescence signal detected in the cytoplasm most likely represents the 405 cleaved forms of hnRNP M. 406 We next assessed the subcellular location of the N- and C-terminal 407 hnRNP M cleavage products during infection. We expressed FLAG-hnRNP M-HA 408 in HeLa cells and monitored the fate of N- and C-terminal cleavage products by 409 FLAG and HA antibodies during infection. As previously shown (Fig. 1G), a 410 predominant 84 kDa protein was detected by FLAG and HA antibodies in 411 transfected cells, indicative of FLAG-hnRNP M-HA expression (Fig. 4B, mock-412

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infected). Expression of FLAG-hnRNP M-HA had a reproducible moderate effect 413 on cell viability in HeLa cells, suggesting that overexpression of hnRNP M is 414 somewhat toxic (Fig. 4A). When probed with the hnRNP M antibody, both the 415 endogenous hnRNP M and the C-terminal HA-tagged fragment of hnRNP M 416 were detected (Fig. 4B, long exposure). We then subjected HeLa cells 417 expressing FLAG-hnRNP M-HA to poliovirus infection. Immunoblotting for FLAG 418 detected two N-terminal cleavage products of approximately 44 and 47 kDa at 5 419 and 7 hours post infection, which is slightly delayed compared to when 420 endogenous hnRNP M is cleaved (Fig. 4B). It is probable that the overexpression 421 of the tagged hnRNP M delays infection. Interestingly, the HA antibody detected 422 only a single protein at approximately 42 kDa (Fig. 4B). It is noted that the full-423 length endogenous hnRNP M and FLAG-hnRNP M-HA were not cleaved to 424 completion as observed in Figure 2. It is likely that overexpression of FLAG-425 hnRNP M-HA may affect the extent of protein processing of endogenous hnRNP 426 M by the virus during infection. Similar to that of the endogenous hnRNP M, the 427 tagged hnRNP M is cleaved and the N- and C-terminal cleavage products persist 428 during poliovirus infection. However, it is noted that the C-terminal tagged HA-429 hnRNP M is less stable at 7 h.p.i. which is similar to that observed using the 430 hnRNP M antibody for detection. 431 We then monitored expression of FLAG-hnRNP M-HA in poliovirus-432 infected HeLa cells by immunofluoresence, to assess the N- and C-terminal 433 cellular localization of hnRNP M. A dsRNA antibody was used to monitor the 434 accumulation of viral replication intermediates. In mock-infected cells transfected 435

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with FLAG-hnRNP M-HA, HA and FLAG signals were detected primarily in the 436 nucleus, similar to nuclear localization of the endogenous protein (Fig. 5A and B, 437 mock-infected). Beginning at 3 h.p.i., which is the time prior to cleavage of FLAG-438 hnRNP M-HA, both FLAG and HA antibodies showed a diffuse cytoplasmic 439 staining (Fig. 5A and B). FLAG and HA signals accumulated in the cytoplasm at 5 440 and 7 h.p.i. (Fig. 5A and B). As expected, dsRNA antibody staining was only 441 detected in the cytoplasm of poliovirus-infected cells (Fig. 5). Interestingly, no 442 colocalization was observed between either FLAG or HA and dsRNA signals, 443 which would suggest that hnRNP M does not have a direct effect on viral 444 replication. In summary, FLAG-hnRNP M-HA recapitulates the subcellular 445 localization and cleavage pattern of endogenous hnRNP M. 446 447 Expression of mutant hnRNP M Q389E/G390P in cells 448 Our results indicated that mutant Q389E/G390P hnRNP M is resistant to 449 cleavage by poliovirus 3Cpro (Fig. 1H). To determine whether this mutant is 450 resistant to cleavage in poliovirus-infected Hela cells, we transfected the FLAG-451 hnRNP M-HA expression construct that contains the Q389E/G390P mutations 452 and followed the fate of the protein by immunoblot analysis. Surprisingly, despite 453 being resistant to 3Cpro and 3CDpro in the in vitro cleavage assay, the 454 Q389E/G390P FLAG-hnRNP M-HA was still cleaved at roughly the same time 455 and extent as the wild-type version in poliovirus-infected cells (Fig. 6A). 456 Furthermore, the cleavage products of mutant and wild-type hnRNP M in infected 457 cells migrated similarly by immunoblot analysis. This result suggests that hnRNP 458

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M is cleaved at a distinct site(s), likely close to the 3Cpro-sensitive Q389/G390 459 site. One possibility is that 3Cpro may cleave at multiple sites on hnRNP M. 460 Surveying for putative 3C proteinase sites nearby, we found two sites at QE336-7 461 and QE349-50 that if cleaved by 3Cpro would result in cleavage products similar 462 in mass as that observed during infection. However, expression of mutant FLAG-463 hnRNP M-HA containing mutations at these sites resulted in cleavage products 464 during poliovirus infection (Fig. 6B). Nevertheless, we next determined whether 465 the Q389E/G390P FLAG-hnRNP M-HA localized to the same cellular 466 compartments as the wild-type version during polivirus infection. As observed 467 with endogenous hnRNP M and the wild-type FLAG-hnRNP M-HA, the FLAG 468 and HA signals were predominantly nuclear localized in mock-infected cells and 469 was localized to the cytoplasm in poliovirus-infected cells to the same extent and 470 time as the wild-type protein (data not shown). In summary, these results indicate 471 that although 3Cpro cleaves between amino acid pair 389Q↓G in vitro, hnRNP M 472 is likely cleaved at another site nearby during poliovirus-infected cells. Currently, 473 it is unclear whether the secondary site(s) is cleaved by 3Cpro or by another 474 protease. 475 476 hnRNP M facilitates poliovirus infection 477 We next explored the significance of hnRNP M during poliovirus infection 478 using a siRNA knockdown approach. Transfection of hnRNP M-specific siRNAs 479 but not scrambled-siRNAs in HeLa cells resulted in loss of hnRNP M protein 480 expression (Fig. 7A) and did not significantly affect cell viability (Fig. 7B). Cells 481

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transfected with scrambled or hnRNP M siRNAs for 72 hours were then mock- or 482 poliovirus-infected, and virus production was monitored by immunoblot and 483 Northern blot analysis. In scrambled-siRNA treated cells, cleavage of hnRNP M 484 was detected in poliovirus-infected cells at 5 and 7 hours post infection (Fig. 2 485 and 7C). As expected, hnRNP M was not detected in infected cells treated with 486 hnRNP M siRNAs (Fig. 7C). Interestingly, the viral structural protein VP1 was 487 significantly reduced in poliovirus-infected cells treated with hnRNP M siRNAs 488 compared to the scrambled control at 5 and 7 hours post infection (Fig. 7C). By 9 489 and 11 hours post infection, VP1 expression in hnRNP siRNA-treated cells 490 accumulated to similar levels as in scrambled treated cells (Fig. 7C). These 491 results suggest that loss of hnRNP M inhibits and delays poliovirus infection. 492 Furthermore, knockdown of hnRNP M decreased poliovirus genomic RNA at 5 493 and 7 h.p.i. (Fig. 7D) and resulted in a 5 to 6-fold decrease in viral titre of both 494 intracellular (5 h.p.i.) and extracellular viral yield (7 h.p.i.) compared to the 495 scrambled control (Fig. 7E). These results collectively demonstrate that hnRNP 496 M facilitates poliovirus infection in HeLa cells. 497 498 Role of hnRNP M in poliovirus IRES translation 499 We have shown that hnRNP M promotes poliovirus infection. Given that 500 hnRNP M does not colocalize with replication complexes under infection, we next 501 investigated a role for hnRNP M in viral translation. To examine this further, we 502 investigated whether hnRNP M affects host translation and viral protein synthesis 503 during infection. Mock- or poliovirus-infected cells that were pre-treated with 504

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hnRNP M siRNAs for 72 hours were pulse-labeled with [35S]-methionine/cysteine 505 for 30 minutes prior to harvesting at each time point. Knockdown of hnRNP M did 506 not significantly affect (94% ± 11%) overall protein synthesis as compared to 507 cells treated with scrambled siRNAs, which is in agreement with our observation 508 that knockdown of hnRNP M does not affect cell viability (Fig. 8A, mock infected 509 lanes). Host translational shutoff was observed beginning at 3-5 h.p.i. in 510 poliovirus-infected scrambled siRNA-treated cells at an MOI of 5, which is 511 concomitant with viral protein synthesis (Fig. 8A). Synthesis of viral proteins such 512 as P1, VP0, VP3, VP1 and 2BC was clearly observed in infected cells at an MOI 513 of 1 and 5 at 5 h.p.i. (Fig. 8A). By contrast, in cells depleted of hnRNP M, host 514 translational shutoff was delayed at 5 and 7 h.p.i. in infected cells at an MOI of 5 515 (Fig. 8A). Moreover, viral protein synthesis was reduced in these cells, which is 516 consistent with the observation that VP1 expression is decreased in hnRNP M 517 siRNA-treated, virus-infected cells (Fig. 8A and Fig. 7C). This result is most 518 evident in infected cells at an MOI of 1 where viral protein synthesis is barely 519 detected at 5 and 7 hours post infection (Fig. 8A). Thus, depletion of hnRNP M 520 results in a decrease in either viral protein synthesis or replication in infected 521 HeLa cells. 522 To examine more closely whether hnRNP M has a role in viral translation, 523 we monitored poliovirus IRES translation directly by using an IRES-containing 524 reporter construct (Fig. 8B). The bicistronic reporter construct contains the 525 poliovirus IRES within the intergenic region between the Renilla and firefly 526 luciferase genes, which monitor cap-dependent and poliovirus IRES-mediated 527

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translation, respectively (Fig. 8B). Because hnRNP M redistributes to the 528 cytoplasm during poliovirus infection, we monitored poliovirus IRES translation by 529 transfecting the IRES-containing reporter construct in poliovirus-infected cells. 530 Briefly, cells treated with scrambled or hnRNP M siRNAs for 72 hours were 531 transfected with the bicistronic construct for 1 hour, followed by mock or 532 poliovirus infection (Fig. 8B, flowchart). Cells were then harvested 5 hours later 533 and luciferase activities were measured. 534 In mock-infected cells, hnRNP M siRNA treatment decreased Renilla 535 luciferase activity by approximately 12% as compared to scrambled siRNA 536 treatment, indicating that depletion of hnRNP M had a slight effect on cap-537 dependent translation using this transfection reporter approach (Fig. 8C). In 538 contrast, firefly luciferase activity was detected at similar levels in both the 539 scrambled and hnRNP M siRNA treatments, suggesting that hnRNP M does not 540 have a role in IRES-dependent translation under basal conditions (Fig. 8C). In 541 poliovirus-infected cells, Renilla luciferase activity was inhibited to the same 542 extent in both the scrambled and hnRNP M siRNA treatments, which is a 543 reflection of shutoff of host translation during infection (Fig. 8C). In contrast, firefly 544 luciferase activity was still detected at similar levels in the hnRNP M siRNA 545 treated cells compared to the scrambled controls (Fig. 8C), suggesting that 546 hnRNP M is not required for poliovirus IRES translation. A bicistronic construct 547 containing an inactive IRES did not result in firefly luciferase expression, 548 indicating that IRES activity is being measured (Fig. 8D). In summary, the results 549

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suggest that hnRNP M is not required for poliovirus IRES translation during 550 infection, and likely participates in another step of the viral life cycle. 551 552 hnRNP M is not required for poliovirus genomic RNA stability 553 The decrease in viral RNA in poliovirus infected cells that are depleted of 554 hnRNP M may be due to an effect on viral replication or viral RNA stability. To 555 address whether hnRNP M is involved in viral RNA stability, we monitored the 556 fate of viral RNA in scrambled or hnRNP M siRNA-treated poliovirus-infected 557 cells after treating the cells at 4 h.p.i. with 2 mM guanidine hydrochloride 558 (GuHCL), a known inhibitor of poliovirus RNA synthesis (54). No significant 559 difference was observed between the stabilities of viral RNA between the 560 scrambled and hnRNP M siRNA-treated cells following the addition of guanidine 561 hydrochloride (Fig. 9). Thus, hnRNP M is not required for maintaining poliovirus 562 RNA stability during infection. 563 564 Role of hnRNP M in CVB3 infection 565 Our results have established a novel role of hnRNP M in poliovirus 566 infection. We next determined whether the requirement of hnRNP M is specific to 567 poliovirus infection. To address this, we asked whether hnRNP M facilitates 568 infection of another picornavirus, CVB3. Similar to that observed with poliovirus 569 3Cpro, immunoblot analysis detected the accumulation a cleavage product of 570 approximately 34 kDa in HeLa lysates incubated with purified CVB3 3Cpro but not 571 a catalytically inactive mutant CVB3 3Cpro (Fig. 10A). The cleavage product of 572

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hnRNP M by CVB3 3Cpro migrated slightly ahead than the cleavage product 573 produced by poliovirus 3Cpro, suggesting the CVB3 3Cpro may target another site 574 within hnRNP M (Fig. 10A). When monitored during CVB3 infection, a single 575 cleavage 55 kDa protein was observed at 5 hours post infection followed by 576 multiple cleavage products observed at 7 and 9 hours post infection, 577 demonstrating the hnRNP M is cleaved at multiple sites under CVB3 infection 578 (Fig. 10B). Cleavage of hnRNP M was still observed during CVB3 infection in the 579 presence of zVAD-FMK, demonstrating that targeting of hnRNP M is not due to 580 caspase activity (data not shown). The significance of hnRNP M in CVB3 581 infection was also explored by measuring viral titres following siRNA-mediated 582 knockdown of hnRNP M. We observed an approximately seven-fold decrease in 583 CVB3 titre in hnRNP M siRNA-treated cells (Fig 10C). 584 CVB3 is a prevalent contributor to dilated cardiomyopathy among young 585 children by targeting and ultimately destroying cardiomyocytes (55). To assess 586 whether cleavage of hnRNP M occurs under more physiologically relevant 587 conditions, we monitored hnRNP M in cardiomyocytes from mice infected with 588 CVB3. Cleavage products of hnRNP M were detected in the CVB3 treated mice 589 but not in the mock treated mice (Fig. 10D). Altogether, this data indicates that 590 cleavage of hnRNP M and the requirement of this protein for infection may be a 591 conserved strategy among picornaviruses to facilitate virus infection. 592

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DISCUSSION 593 We have identified hnRNP M as a direct substrate of poliovirus 3Cpro and 594 3CDpro in vitro, and that hnRNP M is cleaved in both poliovirus and CVB3-595 infected cells. We also demonstrate that hnRNP M promotes both poliovirus and 596 CVB3 infection. Several members of the hnRNP family, including hnRNP A1, 597 PCBP1/2, AUF1, and PTB, are important host factors for picornavirus infection 598 (2-9). A subset of these hnRNPs are modified through cleavage by picornavirus 599 proteinases in infected-cells and as a result, picornaviruses either can inhibit or 600 alter the function of these proteins, or exploit the function of their cleavage 601 products. Our work suggests that cleavage of hnRNP M is common strategy of 602 picornavirus infections and that picornaviruses hijack hnRNP M to facilitate 603 infection. 604 We demonstrate conclusively that hnRNP M is cleaved by poliovirus 3Cpro 605 and 3CDpro between 389Q↓G390 (M4 isoform numbering) to produce a 36 kDa 606 cleavage protein that is detected by the 1D8 hnRNP M antibody (Fig. 1). The 1D8 607 antibody recognizes an epitope within the C-terminal fragment of hnRNP M, 608 which is based on the observation that both 1D8 and HA antibodies detect the 609 same C-terminal cleavage product of the tagged FLAG-hnRNP M-HA in 610 poliovirus-infected cells (Fig. 4B). We posit that all variants of hnRNP M are 611 targeted as all isoforms harbor the region containing the identified cleavage site 612 and thus cleavage by 3Cpro would produce the same C-terminal end of hnRNP M. 613 Furthermore, hnRNP M is completely cleaved by 7 hours post infection in 614

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poliovirus-infected HeLa cells, which suggests that all four isoforms are being 615 targeted during infection (Fig. 2A). 616 We detected a single 36 kDa cleavage product of hnRNP M in vitro (Fig. 617 1B), however, two cleavage products of approximately 36 and 39 kDa were 618 consistently detected in poliovirus-infected cells (Fig. 2A). Accumulation of a 619 second cleavage product under infection may occur through more than one 620 mechanism. First, hnRNP M may be cleaved twice at a second site close in 621 proximity to the 389Q↓G390 cleavage site identified in vitro, through either 3Cpro or 622 another protease. We have determined that the viral 2Apro is unlikely involved 623 and ruled out the possibility of caspase-induced cleavage (Fig. 2B). Furthermore, 624 while mutating Q389E/G390P prevented direct cleavage by 3Cpro in vitro, we still 625 observed cleavage of this mutant under poliovirus infection (Fig. 1H, 6A). These 626 results suggest that hnRNP M is cleaved at one or more sites in close proximity 627 to the 3Cpro 389Q↓G390 in vitro cleavage site. Several candidate proteinase 628 cleavage sites were tested but all failed to prevent cleavage (Fig. 6B; data not 629 shown). Further mapping of the cleavage sites will provide insights into the 630 proteases that target hnRNP M during poliovirus infection. Interestingly, 631 expression of FLAG-hnRNP M-HA generates two cleavage products detected by 632 the FLAG epitope under poliovirus infection and only one cleavage product 633 detected by the HA epitope. This may indicate that a second cleavage event is 634 occuring on a cleavage product following destabilization of the hnRNP M 635 structure following the initial cleavage. Alternatively, the two cleavage products 636 may be due to two distinct isoforms that are cleaved during infection, or that the 637

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cleaved fragment is subject to post-translational modification. Further investigtion 638 is required to determine the cleavage activity of hnRNP M under infection. 639 During poliovirus and CVB3 infections, hnRNP M is relocalized to the 640 cytoplasm and is cleaved by the viral 3Cpro (Fig. 3, data not shown). The 641 subcellular relocalization of hnRNP M is similar to that observed of other hnRNP 642 proteins during poliovirus infection, thus suggesting a general strategy of 643 picornaviruses to redistribute RNA-binding proteins (53). Poliovirus and CVB3 644 infections lead to remodeling of the nuclear pore complex by viral proteinases, 645 which contributes to the inhibition of nuclear import of specific proteins (53, 56-646 58). Interestingly, it has previously been demonstrated that 3C in its precurser 647 form as 3CD is capable of entering the nucleus of virus-infected cells through a 648 3D nuclear localization signal (59). While we demonstrate that hnRNP M can also 649 serve as a substrate of 3CDpro (Fig. 1I), we observed that FLAG-hnRNP M-HA 650 begins to redistribute from the nucleus to the cytoplasm at 3 hours post infection 651 prior to being cleaved (Fig. 4B, 5). It is likely that hnRNP M relocalizes to the 652 cytoplasm due to blockage of nuclear import mediated by the 2A proteinase, and 653 is then targeted by the 3C proteinase. 654 Like other hnRNPs, hnRNP M is a RNA-binding protein that associates 655 with G-U rich regions of pre-mRNA (48). hnRNP M is part of pre-spliceosome 656 assembly complexes and functions in splice-site recognition and alternative 657 splicing (43-45, 52, 60, 61). In addition hnRNP M has also been implicated in 658 transcriptional controls, heat shock stress responses and cell signaling (62-64). 659 Our work shows for the first time that the function of hnRNP M is subverted 660

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during poliovirus infection and diverted towards a step in the viral life cycle. An 661 indirect effect of relocalization of hnRNP M to the cytoplasm is that splicing will 662 cease or be altered in the nucleus. Previous reports have shown that inhibition of 663 splicing may be a strategy utilized by picornaviruses to subvert host antiviral 664 responses (28, 65, 66). Thus, redistribution of hnRNP M contributes to this effect. 665 Although hnRNP M is cleaved during infection, our study shows that 666 hnRNP M is required for optimal picornavirus infection (Fig. 7E, 10C), suggesting 667 that hnRNP M and/or its cleavage products contribute to a specific step of the 668 viral life cycle. Several hnRNPs are exploited by picornaviruses to aid in viral 669 translation, replication or stability of the viral genome. For example, inhibition of 670 nucleo-cytoplasmic transport of protein during picornavirus infection leads to 671 relocalization of nuclear proteins, such as PTB and PCBP2, to the cytoplasm that 672 then aid in viral translation (33, 53, 57). While several hnRNP proteins have 673 specifically been identified as mediators of poliovirus translation through direct 674 interaction with the IRES (2, 4-7), our translation assays in hnRNP M-depleted 675 cells do not show an effect on poliovirus IRES-mediated translation (Fig. 8B). 676

Given that viral RNA levels are dampened in hnRNP M siRNA treated 677 cells, the simplest hypotheses are that the defect is at the step of RNA 678 metabolism replication or viral RNA stability (Fig. 7D). We showed that hnRNP M 679 does not have role in maintaining viral RNA stability in infected HeLa cells treated 680 with GuHCL (Fig. 9). Previous studies have implicated a role for hnRNP M in 681 replication of influenza A virus and Semiliki Forest virus (SFV) (67, 68). 682 Moreover, depletion of hnRNP M enhances SFV gene expression and 683

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replication, suggesting that hnRNP M may be anti-viral. Both the N- and C-684 terminal cleavage proteins of hnRNP M contain at least one RRM and thus 685 presumably have the ability to bind RNA. Moreover, because both the N- and C-686 terminal fragment persists at least until 5 h.p.i. (Figs. 2 and 4), we hypothesized 687 that the cleavage products of hnRNP M act in viral replication. However, 688 quantitation of the confocal images showed no co-localization of the tagged 689 hnRNP M and dsRNA antibody signals, which mark sites of replication (Fig. 5; 690 data not shown)(69). Moreover, viral RNA was not detected in hnRNP M 691 immunoprecipitation experiments (data not shown). Thus, hnRNP M does not 692 have a direct role in poliovirus replication. It is still possible that hnRNP M has an 693 indirect role in replication, possibly by interacting with and affecting the function 694 of a specific protein or an mRNA that encodes a protein involved in viral 695 replication. Alternatively, hnRNP M may interact with a protein or mRNA that 696 encodes a protein that have a role in the innate immune response or in stress 697 granules or P body formation, host processes that could affect poliovirus RNA 698 accumulation during infection (36, 70, 71). Further experiments to identify the 699 proteins and/or mRNAs that interact with the cleavage products of hnRNP M in 700 infected cells will undoubtedly shed light into the functions of hnRNP M in 701 infected cells. 702 Our findings are in line with the general theme that the RNA-binding family 703 of hnRNPs is targeted by picornavirus infections. However, not all hnRNPs 704 function similarly in infected cells. Depletion of a subset of hnRNPs does not 705 have an effect on virus infection whereas others do, thus highlighting that each 706

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hnRNP has specific roles in picornavirus-host interactions (2-9). Our work 707 demonstrates that hnRNP M plays in important role in poliovirus and CVB3 708 infections. It will be important to determine how the cleavage products contribute 709 to a specific step of the viral life cycle. Lastly, we identified hnRNP M through an 710 unbiased proteomics approach using TAILS N-terminome enrichment to identify 711 substrates of viral proteinases. This proof-in-principle study provides added 712 confidence that TAILS has great potential to reveal other proteinase substrates 713 that are important for picornavirus infection. 714 715

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ACKNOWLEDGEMENTS 716 We thank Kurt Gustin (University of Arizona) for insightful discussions and the 717 pT7GEMpolio poliovirus infection clone and helpful discussions. We thank 718 Richard Lloyd (Baylor College of Medicine) for the pET3Chc and pET-Cx2A 719 plasmids, as well as the PABP primary antibody. We thank Gaby Fuchs and 720 Peter Sarnow (Stanford) for the pIRES-PV bicistronic reporter construct. This 721 study was supported by CIHR operating grants to E.J. (MOP-81244) and C.M.O. 722 (MOP-37937) and by the British Columbia Proteomics Network training grant 723 (J.J.). A.D. is a CIHR Post Doctoral Fellow. E.J. is a CIHR New Investigator 724 scholar and a MSFHR scholar. CMO is a Canada Research Chair in Proteinase 725 Proteomics and Systems Biology. 726 727

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FIGURE LEGENDS 728 Figure 1. hnRNP M is cleaved by poliovirus 3C proteinase in vitro. (A) The 729 hnRNP M peptide identified by TAILS is shown, including the four amino acids 730 located directly upstream (P4-P1). Schematic of hnRNP M protein isoforms are 731 shown. RRM - RNA recognition motifs. Arrow denotes the cleavage site of 732 poliovirus 3Cpro. (B) Fragmented spectra of the doubly charged 733 GGGGGGGSVPGIER peptide identified following N-terminal enrichment by 734 TAILS. HeLa cell lysates were incubated with purified (C) wild type or mutant 735 (C147A) poliovirus 3Cpro (100 ng/μl) or (D) wild type or mutant (C109A) CVB3 736 2Apro (100 ng/μl) for the indicated times. hnRNP M, PABP, and α-tubulin were 737 detected by immunblot analysis. (E) Cleavage of recombinant hnRNP M by 738 purified poliovirus 3Cpro. Proteins were loaded on a SDS-PAGE and 739 immunobloted for hnRNP M. (F) Schematic of CMV-promoter driven mammalian 740 expression construct containing 3X FLAG and HA fused in frame with the full-741 length hnRNP M (FLAG-hnRNP M-HA). (G) Expression of FLAG-hnRNP M-HA in 742 HeLa cells. Cells were harvested at the times indicated and lysates were 743 immunobloted with hnRNP M and Actin antibodies. (H and I) Lysates from cells 744 expressing the wild type or mutant (Q389E/G390P) tagged hnRNP M (FLAG-745 hnRNP M-HA) were incubated with (H) wild type or mutant poliovirus 3Cpro or (I) 746 with poliovirus 3CDpro and immunoblotted for FLAG and PABP. cp - cleavage 747 protein. 748 749

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Figure 2. Cleavage of hnRNP M in poliovirus-infected HeLa cells. (A) HeLa cells 750 were mock or poliovirus-infected (MOI 10) for the indicated times. (B) Cleavage 751 of hnRNP M is insensitive to zVAD-FMK in poliovirus-infected cells. HeLa cells 752 were infected with poliovirus at an MOI of 10 in the presence or absence of 50 753 uM zVAD-FMK (7 hours post infection, h.p.i.). hnRNP M, poliovirus structural 754 protein VP1, PARP and α-tubulin were assessed by Western blot analysis. cp- 755 cleavage proteins. Shown are representative gels from at least two independent 756 experiments. 757 758 Figure 3. Subcellular Localization of hnRNP M in PV-infected HeLa cells. HeLa 759 cells were mock- or PV-infected (MOI 10) for the indicated times. Cells were 760 permeabilized, fixed and co-stained for hnRNP M (red) and DNA (Hoescht). 761 Representative confocal images are shown from at least three independent 762 experiments. 763 764 Figure 4. Cleavage of hnRNP M in PV-infected cells. (A) Cell viability of cells 765 transfected with FLAG-hnRNP M-HA for 48 hours. Cell viability was assessed by 766 the percentage of cells that are not stained with trypan blue. Averages ± s.d. are 767 shown, *p<0.05. (B) HeLa cells transfected with FLAG-hnRNP M-HA were either 768 mock- or poliovirus-infected (MOI 10) for the indicated times. Lysates were 769 immunoblotted for FLAG, HA, hnRNP M and α-tubulin. 770 771

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Figure 5. Subcellular localization of N- and C-terminal cleavage products of 772 hnRNP M in poliovirus-infected HeLa cells. (A) Subcellular localization of N-773 terminal and C-terminal cleavage products of hnRNP M. HeLa cells transfected 774 with FLAG-hnRNP M-HA for 48 hours, followed by either mock or poliovirus 775 infection (MOI 10) for the times indicated. Cells were fixed and co-stained for 776 FLAG (red in A) or HA (red in B), dsRNA (green) for detection of virus, and 777 Hoescht (blue). Shown are representative images from at least three 778 independent experiments. 779 780 Figure 6. Expression of mutant FLAG-hnRNP M-HA in poliovirus infected cells. 781 HeLa cells were transfected with either wild-type or mutant Q389E/G390P (A), or 782 E337K, E350K FLAG-hnRNP M-HA expression plasmids for 48 hours (B), 783 followed by mock- or poliovirus-infection (MOI 1) for the indicated times. Lysates 784 were immunoblotted with anti-FLAG. 785 786 Figure 7. Poliovirus infection is inhibited in HeLa cells lacking hnRNP M. (A) 787 hnRNP M knockdown by transfection of siRNA in HeLa cells. hnRNP M and α-788 tubulin were assessed by immunoblot. (B) Cell viability of cells treated with 789 siRNAs for 72 hours was calculated by the percentage of cells that are not 790 stained with trypan blue. Averages ± s.d. are shown. N.D., no statistical 791 difference, p<0.05. (C) HeLa cells were transfected with either scrambled 792 (siSCX) or hnRNP M (sihnRNP M) siRNA for 72 hours, followed by poliovirus 793 infection (MOI 1) for the indicated times. Immunoblots of hnRNP M, poliovirus 794

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structural protein VP1 and α-tubulin are shown. (D) Northern blot analysis of 795 poliovirus genomic RNA in poliovirus-infected HeLa cells (MOI 1) treated with 796 siSCX or sihnRNP M. (E) Viral titres of intracellular (5 h.p.i.) and extracellular (7 797 h.p.i.) virus from poliovirus-infected cells (MOI 0.1) pre-treated with siSCX or 798 sihnRNP M. Titres were calculated as plaque forming units (p.f.u./ml ± s.d., 799 *p<0.05) from three independent experiments. 800 801 Figure 8. Role of hnRNP M in poliovirus IRES translation. (A) Pulse-labeling 802 using [35S]-methionine/cysteine at the indicated times after poliovirus infection 803 (MOI 1 and 5) in cells treated with siSCX or sihnRNP M for 72 hours prior to 804 infection. The cells were pulse-labeled for 30 minutes prior to harvesting at each 805 time point. A representative gel is shown from at least two independent 806 experiments. (B) Flowchart of the transfection protocol to monitor poliovirus IRES 807 translation. A schematic of the bicistronic reporter construct containing the 808 poliovirus IRES within the intergenic region is shown below. (C) and (D) Cap-809 dependent Renilla and IRES-mediated firefly luciferase activities of the (C) PV 810 IRES bicistronic reporter construct and (D) mutant EMCV IRES bicistronic 811 reporter construct. Relative luminescence was calculated as a mean ± s.d. of 812 three independent experiments. N.D., no statistical difference, *p<0.05. 813 814 Figure 9. Stability of viral genomic RNA in PV-infected cells. Hela cells 815 pretreated with siSCX or sihnRNP M for 24 hours were infected with poliovirus 816 (MOI 5) and at 4 hours post infection, treated with 2mM guanidine hydrochloride 817

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(GuHCL). Cells were harvested at the indicated times and the viral RNA and 818 GAPDH mRNA assayed by Northern blot analysis. The ratio of viral RNA to 819 GAPDH at 4 hours post infection was set as 100%. Shown are the averages of 820 three independent experiments ± s.d. 821 822 Figure 10. hnRNP M in CVB3-infected cells. (A) Cleavage of hnRNP M in CVB3-823 infected cells. (A) Immunoblots of HeLa lysates incubated with purified wild type 824 or mutant (C147A) CVB3 3C proteinase. (B) Immunoblots of lysates from mock- 825 or CVB3-infected HeLa cells (MOI 10) or (D) hearts of CVB3-infected mice (two 826 independent experiments are shown). (C) Viral titers of CVB3-infected (MOI 1, 16 827 h.p.i.) HeLa cells that were pre-treated with siSCX or sihnRNP M (n=3, mean 828 ± s.d, *p<0.05). 829

41

REFERENCES 830 1. Turk B, Turk D, Turk V. 2012. Protease signalling: the cutting edge. EMBO J 831 31:1630-1643. 832 2. Back SH, Kim YK, Kim WJ, Cho S, Oh HR, Kim JE, Jang SK. 2002. 833 Translation of polioviral mRNA is inhibited by cleavage of polypyrimidine 834 tract-binding proteins executed by polioviral 3C(pro). Journal of virology 835 76:2529-2542. 836 3. Cathcart AL, Semler BL. 2014. Differential restriction patterns of mRNA 837 decay factor AUF1 during picornavirus infections. J Gen Virol 95:1488-1492. 838 4. Chase AJ, Daijogo S, Semler BL. 2014. Inhibition of poliovirus-induced 839 cleavage of cellular protein PCBP2 reduces the levels of viral RNA replication. 840 Journal of virology 88:3192-3201. 841 5. Lin JY, Li ML, Huang PN, Chien KY, Horng JT, Shih SR. 2008. 842 Heterogeneous nuclear ribonuclear protein K interacts with the enterovirus 843 71 5' untranslated region and participates in virus replication. J Gen Virol 844 89:2540-2549. 845 6. Lin JY, Shih SR, Pan M, Li C, Lue CF, Stollar V, Li ML. 2009. hnRNP A1 846 interacts with the 5' untranslated regions of enterovirus 71 and Sindbis virus 847 RNA and is required for viral replication. Journal of virology 83:6106-6114. 848 7. Perera R, Daijogo S, Walter BL, Nguyen JH, Semler BL. 2007. Cellular 849 protein modification by poliovirus: the two faces of poly(rC)-binding protein. 850 Journal of virology 81:8919-8932. 851 8. Rozovics JM, Chase AJ, Cathcart AL, Chou W, Gershon PD, Palusa S, 852 Wilusz J, Semler BL. 2012. Picornavirus modification of a host mRNA decay 853 protein. mBio 3:e00431-00412. 854 9. Wong J, Si X, Angeles A, Zhang J, Shi J, Fung G, Jagdeo J, Wang T, Zhong Z, 855 Jan E, Luo H. 2013. Cytoplasmic redistribution and cleavage of AUF1 during 856 coxsackievirus infection enhance the stability of its viral genome. FASEB J 857 27:2777-2787. 858 10. Joachims M, Van Breugel PC, Lloyd RE. 1999. Cleavage of poly(A)-binding 859 protein by enterovirus proteases concurrent with inhibition of translation in 860 vitro. Journal of virology 73:718-727. 861 11. Kuyumcu-Martinez NM, Joachims M, Lloyd RE. 2002. Efficient cleavage of 862 ribosome-associated poly(A)-binding protein by enterovirus 3C protease. 863 Journal of virology 76:2062-2074. 864 12. Etchison D, Milburn SC, Edery I, Sonenberg N, Hershey JW. 1982. 865 Inhibition of HeLa cell protein synthesis following poliovirus infection 866 correlates with the proteolysis of a 220,000-dalton polypeptide associated 867 with eucaryotic initiation factor 3 and a cap binding protein complex. J Biol 868 Chem 257:14806-14810. 869 13. Lamphear BJ, Yan R, Yang F, Waters D, Liebig HD, Klump H, Kuechler E, 870 Skern T, Rhoads RE. 1993. Mapping the cleavage site in protein synthesis 871 initiation factor eIF-4 gamma of the 2A proteases from human Coxsackievirus 872 and rhinovirus. J Biol Chem 268:19200-19203. 873

42

14. Kerekatte V, Keiper BD, Badorff C, Cai A, Knowlton KU, Rhoads RE. 1999. 874 Cleavage of Poly(A)-binding protein by coxsackievirus 2A protease in vitro 875 and in vivo: another mechanism for host protein synthesis shutoff? Journal of 876 virology 73:709-717. 877 15. Baltimore D, Huang A, Manly KF, Rekosh D, Stampfer M. 1971. The 878 synthesis of protein by mammalian RNA viruses. In: strategy of the viral 879 genome. Ciba Found Symp:101-110. 880 16. Jacobson MF, Baltimore D. 1968. Polypeptide cleavages in the formation of 881 poliovirus proteins. Proc Natl Acad Sci U S A 61:77-84. 882 17. Pelletier J, Sonenberg N. 1988. Internal initiation of translation of 883 eukaryotic mRNA directed by a sequence derived from poliovirus RNA. 884 Nature 334:320-325. 885 18. Jang SK, Krausslich HG, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer 886 E. 1988. A segment of the 5' nontranslated region of encephalomyocarditis 887 virus RNA directs internal entry of ribosomes during in vitro translation. J 888 Virol 62:2636-2643. 889 19. Kitamura N, Semler BL, Rothberg PG, Larsen GR, Adler CJ, Dorner AJ, 890 Emini EA, Hanecak R, Lee JJ, van der Werf S, Anderson CW, Wimmer E. 891 1981. Primary structure, gene organization and polypeptide expression of 892 poliovirus RNA. Nature 291:547-553. 893 20. Toyoda H, Nicklin MJ, Murray MG, Anderson CW, Dunn JJ, Studier FW, 894 Wimmer E. 1986. A second virus-encoded proteinase involved in proteolytic 895 processing of poliovirus polyprotein. Cell 45:761-770. 896 21. Nicklin MJ, Krausslich HG, Toyoda H, Dunn JJ, Wimmer E. 1987. 897 Poliovirus polypeptide precursors: expression in vitro and processing by 898 exogenous 3C and 2A proteinases. Proc Natl Acad Sci U S A 84:4002-4006. 899 22. Hanecak R, Semler BL, Anderson CW, Wimmer E. 1982. Proteolytic 900 processing of poliovirus polypeptides: antibodies to polypeptide P3-7c 901 inhibit cleavage at glutamine-glycine pairs. Proc Natl Acad Sci U S A 79:3973-902 3977. 903 23. Hellen CU, Lee CK, Wimmer E. 1992. Determinants of substrate recognition 904 by poliovirus 2A proteinase. Journal of virology 66:3330-3338. 905 24. Pallai PV, Burkhardt F, Skoog M, Schreiner K, Bax P, Cohen KA, Hansen 906 G, Palladino DE, Harris KS, Nicklin MJ, et al. 1989. Cleavage of synthetic 907 peptides by purified poliovirus 3C proteinase. The Journal of biological 908 chemistry 264:9738-9741. 909 25. Ventoso I, Barco A, Carrasco L. 1999. Genetic selection of poliovirus 2Apro-910 binding peptides. J Virol 73:814-818. 911 26. Weidner JR, Dunn BM. 1991. Development of synthetic peptide substrates 912 for the poliovirus 3C proteinase. Arch Biochem Biophys 286:402-408. 913 27. Ypma-Wong MF, Filman DJ, Hogle JM, Semler BL. 1988. Structural 914 domains of the poliovirus polyprotein are major determinants for proteolytic 915 cleavage at Gln-Gly pairs. J Biol Chem 263:17846-17856. 916 28. Almstead LL, Sarnow P. 2007. Inhibition of U snRNP assembly by a virus-917 encoded proteinase. Genes Dev 21:1086-1097. 918

43

29. Clark ME, Lieberman PM, Berk AJ, Dasgupta A. 1993. Direct cleavage of 919 human TATA-binding protein by poliovirus protease 3C in vivo and in vitro. 920 Mol Cell Biol 13:1232-1237. 921 30. de Breyne S, Bonderoff JM, Chumakov KM, Lloyd RE, Hellen CU. 2008. 922 Cleavage of eukaryotic initiation factor eIF5B by enterovirus 3C proteases. 923 Virology 378:118-122. 924 31. Graham KL, Gustin KE, Rivera C, Kuyumcu-Martinez NM, Choe SS, Lloyd 925 RE, Sarnow P, Utz PJ. 2004. Proteolytic cleavage of the catalytic subunit of 926 DNA-dependent protein kinase during poliovirus infection. Journal of 927 virology 78:6313-6321. 928 32. Neznanov N, Chumakov KM, Neznanova L, Almasan A, Banerjee AK, 929 Gudkov AV. 2005. Proteolytic cleavage of the p65-RelA subunit of NF-930 kappaB during poliovirus infection. J Biol Chem 280:24153-24158. 931 33. Shiroki K, Isoyama T, Kuge S, Ishii T, Ohmi S, Hata S, Suzuki K, Takasaki 932 Y, Nomoto A. 1999. Intracellular redistribution of truncated La protein 933 produced by poliovirus 3Cpro-mediated cleavage. Journal of virology 934 73:2193-2200. 935 34. Wong J, Zhang J, Yanagawa B, Luo Z, Yang X, Chang J, McManus B, Luo H. 936 2012. Cleavage of serum response factor mediated by enteroviral protease 937 2A contributes to impaired cardiac function. Cell Res 22:360-371. 938 35. Zaragoza C, Saura M, Padalko EY, Lopez-Rivera E, Lizarbe TR, Lamas S, 939 Lowenstein CJ. 2006. Viral protease cleavage of inhibitor of kappaBalpha 940 triggers host cell apoptosis. Proc Natl Acad Sci U S A 103:19051-19056. 941 36. White JP, Cardenas AM, Marissen WE, Lloyd RE. 2007. Inhibition of 942 cytoplasmic mRNA stress granule formation by a viral proteinase. Cell Host 943 Microbe 2:295-305. 944 37. Martinez-Contreras R, Cloutier P, Shkreta L, Fisette JF, Revil T, Chabot B. 945 2007. hnRNP proteins and splicing control. Adv Exp Med Biol 623:123-147. 946 38. Han SP, Tang YH, Smith R. 2010. Functional diversity of the hnRNPs: past, 947 present and perspectives. Biochem J 430:379-392. 948 39. Liebig HD, Ziegler E, Yan R, Hartmuth K, Klump H, Kowalski H, Blaas D, 949 Sommergruber W, Frasel L, Lamphear B, et al. 1993. Purification of two 950 picornaviral 2A proteinases: interaction with eIF-4 gamma and influence on 951 in vitro translation. Biochemistry 32:7581-7588. 952 40. Kleifeld O, Doucet A, auf dem Keller U, Prudova A, Schilling O, Kainthan 953 RK, Starr AE, Foster LJ, Kizhakkedathu JN, Overall CM. 2010. Isotopic 954 labeling of terminal amines in complex samples identifies protein N-termini 955 and protease cleavage products. Nat Biotechnol 28:281-288. 956 41. Kleifeld O, Doucet A, Prudova A, auf dem Keller U, Gioia M, 957 Kizhakkedathu JN, Overall CM. 2011. Identifying and quantifying 958 proteolytic events and the natural N terminome by terminal amine isotopic 959 labeling of substrates. Nature protocols 6:1578-1611. 960 42. Dery KJ, Gaur S, Gencheva M, Yen Y, Shively JE, Gaur RK. 2011. 961 Mechanistic control of carcinoembryonic antigen-related cell adhesion 962 molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear 963

44

ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M. J Biol Chem 964 286:16039-16051. 965 43. Hovhannisyan RH, Carstens RP. 2007. Heterogeneous ribonucleoprotein m 966 is a splicing regulatory protein that can enhance or silence splicing of 967 alternatively spliced exons. J Biol Chem 282:36265-36274. 968 44. Kafasla P, Patrinou-Georgoula M, Guialis A. 2000. The 72/74-kDa 969 polypeptides of the 70-110 S large heterogeneous nuclear ribonucleoprotein 970 complex (LH-nRNP) represent a discrete subset of the hnRNP M protein 971 family. Biochem J 350 Pt 2:495-503. 972 45. Kafasla P, Patrinou-Georgoula M, Lewis JD, Guialis A. 2002. Association of 973 the 72/74-kDa proteins, members of the heterogeneous nuclear 974 ribonucleoprotein M group, with the pre-mRNA at early stages of 975 spliceosome assembly. Biochem J 363:793-799. 976 46. Lleres D, Denegri M, Biggiogera M, Ajuh P, Lamond AI. 2010. Direct 977 interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative 978 splice site choice. EMBO Rep 11:445-451. 979 47. Park E, Iaccarino C, Lee J, Kwon I, Baik SM, Kim M, Seong JY, Son GH, 980 Borrelli E, Kim K. 2011. Regulatory roles of heterogeneous nuclear 981 ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine 982 D2 receptor pre-mRNA. J Biol Chem 286:25301-25308. 983 48. Datar KV, Dreyfuss G, Swanson MS. 1993. The human hnRNP M proteins: 984 identification of a methionine/arginine-rich repeat motif in 985 ribonucleoproteins. Nucleic Acids Res 21:439-446. 986 49. Ypma-Wong MF, Dewalt PG, Johnson VH, Lamb JG, Semler BL. 1988. 987 Protein 3CD is the major poliovirus proteinase responsible for cleavage of the 988 P1 capsid precursor. Virology 166:265-270. 989 50. Tolskaya EA, Romanova LI, Kolesnikova MS, Ivannikova TA, Smirnova 990 EA, Raikhlin NT, Agol VI. 1995. Apoptosis-inducing and apoptosis-991 preventing functions of poliovirus. Journal of virology 69:1181-1189. 992 51. Blondel B, Autret A, Brisac C, Martin-Latil S, Mousson L, Pelletier I, 993 Estaquier J, Colbere-Garapin F. 2009. Apoptotic signaling cascades 994 operating in poliovirus-infected cells. Front Biosci (Landmark Ed) 14:2181-995 2192. 996 52. Aidinis V, Sekeris CE, Guialis A. 1995. Two immunologically related 997 polypeptides of 72/74 kDa specify a novel 70-100S heterogeneous nuclear 998 RNP. Nucleic Acids Res 23:2742-2753. 999 53. Gustin KE, Sarnow P. 2001. Effects of poliovirus infection on nucleo-1000 cytoplasmic trafficking and nuclear pore complex composition. The EMBO 1001 journal 20:240-249. 1002 54. Simoes EA, Sarnow P. 1991. An RNA hairpin at the extreme 5' end of the 1003 poliovirus RNA genome modulates viral translation in human cells. Journal of 1004 virology 65:913-921. 1005 55. Marchant D, Si X, Luo H, McManus B, Yang D. 2008. The impact of CVB3 1006 infection on host cell biology. Curr Top Microbiol Immunol 323:177-198. 1007

45

56. Castello A, Izquierdo JM, Welnowska E, Carrasco L. 2009. RNA nuclear 1008 export is blocked by poliovirus 2A protease and is concomitant with 1009 nucleoporin cleavage. J Cell Sci 122:3799-3809. 1010 57. Park N, Katikaneni P, Skern T, Gustin KE. 2008. Differential targeting of 1011 nuclear pore complex proteins in poliovirus-infected cells. Journal of virology 1012 82:1647-1655. 1013 58. Park N, Skern T, Gustin KE. 2010. Specific cleavage of the nuclear pore 1014 complex protein Nup62 by a viral protease. J Biol Chem 285:28796-28805. 1015 59. Sharma R, Raychaudhuri S, Dasgupta A. 2004. Nuclear entry of poliovirus 1016 protease-polymerase precursor 3CD: implications for host cell transcription 1017 shut-off. Virology 320:195-205. 1018 60. Kiesler E, Hase ME, Brodin D, Visa N. 2005. Hrp59, an hnRNP M protein in 1019 Chironomus and Drosophila, binds to exonic splicing enhancers and is 1020 required for expression of a subset of mRNAs. The Journal of cell biology 1021 168:1013-1025. 1022 61. Cho S, Moon H, Loh TJ, Oh HK, Choy HE, Song WK, Chun JS, Zheng X, Shen 1023 H. 2014. hnRNP M facilitates exon 7 inclusion of SMN2 pre-mRNA in spinal 1024 muscular atrophy by targeting an enhancer on exon 7. Biochim Biophys Acta 1025 1839:306-315. 1026 62. Marko M, Leichter M, Patrinou-Georgoula M, Guialis A. 2014. Selective 1027 interactions of hnRNP M isoforms with the TET proteins TAF15 and 1028 TLS/FUS. Mol Biol Rep 41:2687-2695. 1029 63. Bajenova OV, Zimmer R, Stolper E, Salisbury-Rowswell J, Nanji A, 1030 Thomas P. 2001. Heterogeneous RNA-binding protein M4 is a receptor for 1031 carcinoembryonic antigen in Kupffer cells. The Journal of biological 1032 chemistry 276:31067-31073. 1033 64. Gattoni R, Mahe D, Mahl P, Fischer N, Mattei MG, Stevenin J, Fuchs JP. 1034 1996. The human hnRNP-M proteins: structure and relation with early heat 1035 shock-induced splicing arrest and chromosome mapping. Nucleic Acids Res 1036 24:2535-2542. 1037 65. Alvarez E, Castello A, Carrasco L, Izquierdo JM. 2011. Alternative splicing, 1038 a new target to block cellular gene expression by poliovirus 2A protease. 1039 Biochem Biophys Res Commun 414:142-147. 1040 66. Liu YC, Kuo RL, Lin JY, Huang PN, Huang Y, Liu H, Arnold JJ, Chen SJ, 1041 Wang RY, Cameron CE, Shih SR. 2014. Cytoplasmic viral RNA-dependent 1042 RNA polymerase disrupts the intracellular splicing machinery by entering the 1043 nucleus and interfering with Prp8. PLoS Pathog 10:e1004199. 1044 67. Jorba N, Juarez S, Torreira E, Gastaminza P, Zamarreno N, Albar JP, Ortin 1045 J. 2008. Analysis of the interaction of influenza virus polymerase complex 1046 with human cell factors. Proteomics 8:2077-2088. 1047 68. Varjak M, Saul S, Arike L, Lulla A, Peil L, Merits A. 2013. Magnetic 1048 fractionation and proteomic dissection of cellular organelles occupied by the 1049 late replication complexes of Semliki Forest virus. Journal of virology 1050 87:10295-10312. 1051

46

69. Richards AL, Soares-Martins JA, Riddell GT, Jackson WT. 2014. 1052 Generation of unique poliovirus RNA replication organelles. mBio 5:e00833-1053 00813. 1054 70. Dougherty JD, White JP, Lloyd RE. 2011. Poliovirus-mediated disruption of 1055 cytoplasmic processing bodies. Journal of virology 85:64-75. 1056 71. Feng Q, Langereis MA, Lork M, Nguyen M, Hato SV, Lanke K, Emdad L, 1057 Bhoopathi P, Fisher PB, Lloyd RE, van Kuppeveld FJ. 2014. Enterovirus 1058 2Apro targets MDA5 and MAVS in infected cells. Journal of virology 88:3369-1059 3378. 1060 1061

1062

1206

RRM RRM RRM

IAKQ GGGG

38965414772 279 730

A

B

D E

Jagdeo2015_Figure 1

αTUB

αTUB

PABP

hnRNP MhnRNP M

PABP

cp

cp

cp

cp

0 5 15 30 60 60

WT C147A

PV 3Cpro

CVB3 2Apro

WT C57A

kDa

kDa

7055

35

70

55

55

70

35

7055

55

Protein

Heterogenous nuclear ribonucleoprotein M (hnRNP M)

P4-P1 Peptide identified by TAILS

390GGGGGGGSVPGIER386IAKQ

70

35

WT C147A

PV 3Cpro

1167

RRM RRM RRM

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H

3X FLAG hnRNP M 3X HA

CMV

anti-

hnRNP M

untr 24 48 hrs

40 Actin

70

100FLAG-hnRNP M-HAhnRNP M

kDa

705540

WT C147A WT C147A

WT QG389EP hnRNP M

PV 3Cpro

100

kDa

anti-FLAG

70

55

PABP

cp

cp

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cp

anti-PABP

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0.4

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nsity (

counts

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1.8

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y13

b3

b4

y9

y6

y5

y2

y1

b8

b9

b13

0

y5+

I

kDa

70

55

40

WT QG389EP hnRNP M

PV 3CDpro

100

anti-FLAG

70

55

PABP

cp

cp

hnRNP M

cp

anti-PABP

PV 3CDpro

+ -

A

Jagdeo2015_Figure 2

kDa

70

35

70

35

75

63

48

35

55

VP1

hnRNP M

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PV mock

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PARP

cp

VP1

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mock PV

DMSO zVAD DMSO zVAD

70

55

100

35

130

100

35

55

kDa

Jagdeo2015_Figure 3

hnRNP M

Hoescht

merge

poliovirus mock

1 3 5 7 7 h.p.i.

Jagdeo2015_Figure 4

A B

70

100

55

40

70

100

55

40

kDa

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cp

cp

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anti-hnRNP M

Short Exposure

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Long Exposure

FLAG-hnRNP M-HA

cpcp

hnRNP M

FLAG-hnRNP M-HA

cpcp

hnRNP M

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70

100

35

40

70

100

35

40

55

anti-HA

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ell

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ty

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50

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*

Jagdeo2015_Figure 5

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kDa

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55

40

WTQ389A WTQ389A

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WT WT

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anti-FLAG

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70

55

40

FLAG-hnRNP M-HA

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hnRNP M

cp

Jagdeo2015_Figure 6

A

B

Jagdeo2015_Figure 7

A

E

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55

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++

++

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poliovirus infection mock

1 3 5 7 7 h.p.i.

h.p.i.

C

kDa

35

55

70

35

55

hnRNP M

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++

++

++

++

poliovirus infection mock

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2

3

4

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1

2

3

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55

70

35

55

hnRNP M

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poliovirus infection mock

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VP1

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Jagdeo2015_Figure 8

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++

++

poliovirus (MOI 1) mock

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1.5

2.0

2.5

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0

2

3

4

5

Ren

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Luc

Ren

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FF

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sihnRNP M

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++

++

++

++

poliovirus (MOI 5) mock

1 3 5 7 7 hrs

VP3

VP02BC

P170

100

5543

34

17

26 VP3

VP02BC

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5543

34

17

26

VP1VP1

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Jagdeo2015_Figure 9

1 2

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iral R

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50

75

100

Hours After GuHCl Addtion

siSCXsihnRNP M

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A

cp

55

D

10070

55

35

kDa

cp

B

70

55

35

cp

G3BP1

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Jagdeo2015_Figure 10

aTUB

aTUB

VP1

CVB3 mock

1 3 5 7 9 9kDa

70

55

35

70

55

35

60 60301550 min

CVB3 3C

WT C147A

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hnRNP M

PABP

hnRNP M

hnRNP M

cpcp

mock mock CVB3

63

48

kDa CVB3

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siSCX sihnRNP M

1

0

2

3

4

5

p.f.u

. (X

105 )

Viral Titre6

*

cp

exp

#1

exp

#2

exp

#1

exp

#2