Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection

ABSTRACT

Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoprotein M (hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNP M is cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNP M relocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNP M using small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNP M is a general strategy utilized by picornaviruses to facilitate viral infection.

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

INTRODUCTION

Proteases play fundamental roles in cells by ultimately changing the fate and function of its substrates through proteolytic cleavage or degradation (1–9, 72). Many viruses have exploited these strategies to promote infection. For instance, picornaviruses translate their RNA genome as a single polyprotein, which is then processed by virus-encoded proteinases to produce the mature viral proteins. In addition, viral proteinases target a subset of host proteins to modulate cellular processes, inhibit antiviral responses, and subvert host proteins to aid in specific steps of the viral life cycle, such as viral translation and replication. One of the best characterized is the rapid host translational shutoff that occurs in poliovirus-infected cells, which is facilitated through cleavage of eukaryotic translation initiation factors eIF4G and the poly(A) binding protein (PABP) by the viral proteinases 2A and 3C (10–14). Overall inhibition of protein synthesis leads to release of translation factors and ribosomes that are then diverted to poliovirus protein synthesis. The identification of host protein substrates of viral proteinases has provided important insights into the virus-host interaction strategies that contribute to the viral life cycle.

Polyprotein processing by virus-encoded proteinases is a common strategy among RNA viruses. Members of the picornavirus family, which include poliovirus and coxsackievirus B3 (CVB3), are cytoplasmic RNA viruses that possess a positive single-stranded RNA genome of ∼7,500 bases in length encoding a single open reading frame (15, 16). The 5′ noncoding region contains an internal ribosome entry site (IRES) that directs translation to produce a 220-kDa polyprotein, which is then processed by viral proteinases to produce mature structural and nonstructural viral proteins (17, 18). All picornaviruses encode the 3C proteinase (3C^pro), a chymotrypsin-like protease with an active-site cysteine nucleophile rather than a serine nucleophile. This enzyme and its polypeptide precursor (3CD) perform the majority of the polyprotein processing. A subset of picornaviruses encodes a second proteinase, either the 2A proteinase (2A^pro) or the leader proteinase that directs minor cleavages of the polyprotein (19–21). The 3C^pro cleaves primarily between glutamine and glycine (Q↓G) residues at eight distinct sites within the polyprotein (22). The 2A^pro has at least a single cleavage site within the polyprotein, autocatalytically cleaving itself at its N terminus between a tyrosine and glycine (Y↓G) residue (20, 23). Mutagenesis of substrate peptides has shown that P and P′ amino acids flanking the N and C termini of the cleavage site, respectively, are also important for substrate recognition and proteinase activity (24–26). For example, poliovirus 3C^pro shows a strong preference for proline and alanine at the P2 and P4 positions, respectively, whereas 2A^pro prefers isoleucine/leucine, threonine/serine, and proline at the P4, P2, and P2′ positions, respectively (23–26). Furthermore, secondary structures adopted during the folding process of the polyprotein are also required to mediate substrate specificity and temporally regulate cleavage (27). Currently, there are approximately 20 known host proteins that are targeted by picornavirus 2A^pro and 3C^pro and have been shown to disrupt several cellular processes, including transcription, RNA metabolism, nucleocytoplasmic transport, cytoskeleton dynamics, and stress granule formation (28–36). Despite the identification of many host substrates, it is likely that the full repertoire of host substrates has yet to be identified.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of nucleocytoplasmic shuttling RNA-binding proteins that were originally identified based on their association with pre-mRNAs (37). There are approximately 20 hnRNPs, named hnRNP A to U, that all contain at least one RNA-binding domain, either an RNA recognition motif (RRM) or an hnRNP K homologue (KH) domain. Most hnRNPs are primarily involved in pre-mRNA splicing, but they also aid in diverse aspects of RNA metabolism, including translational control, telomere biogenesis, mRNA stability, and trafficking (37, 38). hnRNP activity also contributes to different steps of the picornavirus life cycle. For example, hnRNPs A1, I (more commonly known as the PTB), and K interact with the 5′ untranslated region (UTR) IRES of several picornaviruses to facilitate viral translation and replication (2, 5, 6). Moreover, viral proteinases target a subset of hnRNPs to regulate specific steps of virus infection. Poly(rC)-binding protein 2 (PCBP2), also known as hnRNP E2, binds to the poliovirus IRES to facilitate translation initiation, however, at late times of infection, cleavage of PCBP2 by 3C^pro modifies its association with the 5′ UTR to inhibit viral translation and thereby switches to viral replication (4, 7). Thus, poliovirus has evolved a strategy to regulate PCBP2 function via cleavage by 3C^pro in order to temporally regulate viral translation and replication. Not all hnRNPs are proviral, as some hnRNPs have antiviral effects; hnRNP D, also known as AU-rich biding factor (AUF1), binds directly to stem-loop IV of the poliovirus IRES to inhibit viral translation (3, 8, 9). However, in poliovirus- and CVB3-infected cells, this antiviral activity is inhibited through 3C^pro-mediated cleavage of AUF1 (8, 9).

In the present study, we identified hnRNP M as a novel substrate of both poliovirus and CVB3 3C^pro. hnRNP M is cleaved in both poliovirus- and CVB3-infected HeLa cells and mouse tissues, producing two cleavage products that persist during infection. We demonstrate that endogenous hnRNP M relocalizes from the nucleus to the cytoplasm during poliovirus infection and that hnRNP M promotes poliovirus and CVB3 infection. Depletion of hnRNP M does not affect IRES translation or viral RNA stability. In summary, our data reveal a strategy utilized by poliovirus and CVB3 to target hnRNP M by the 3C^pro to aid in virus infection.

MATERIALS AND METHODS

Cell culture and virus stocks.

HeLa cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C. Poliovirus (Mahoney type 1 strain) was generated from transfection of in vitro-transcribed RNA from a poliovirus infectious clone, pT7pGemPolio (generously provided by Kurt Gustin, University of Arizona) into HeLa cells. Poliovirus and CVB3 (Kandolf strain) were both propagated and titered in HeLa cells.

Plasmids and transfections.

The full-length hnRNP M open reading frame (NM_005968) was PCR amplified and cloned into the KpnI and NotI restriction sites of a p3×Flag-CMV-7.1 vector (Sigma) with a 3×HA tag cloned downstream using XbaI and BamHI sites. Full-length CVB3 3C^pro (M88483) and a CVB3 3C^pro C147A mutant were PCR amplified and cloned into the NotI and NdeI restriction sites of pET28b. Constructs were verified by sequencing.

For DNA transfections, HeLa cells were transfected with 1 to 2 μg of plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Cells were transfected in antibiotic-free medium for 5 h and then replaced with complete medium for 24 to 48 h. For siRNA transfections, HeLa cells were transfected (30 to 40% confluence) with either hnRNP M (s9259, s9261, and s9260 [Ambion]) or scrambled siRNA (Ambion) using Lipofectamine RNAimax (Invitrogen). The knockdown efficiency was validated by Western blotting.

The pIRES-poliovirus and pIRES-dEMCV bicistronic reporter constructs (generously provided by Gabriele Fuchs and Peter Sarnow, Stanford University) were transfected into HeLa cells for 1 h and then infected with poliovirus. The cells were harvested, and the luciferase activity was monitored by using a dual Luciferase reporter assay kit (Promega). Luminescence was measured using a Centro LB 960 luminometer (Berthold Technologies).

Virus infections.

Virus was absorbed with HeLa cells at the indicated multiplicity of infection (MOI) for 1 h in serum-free DMEM at 37°C, followed by washing with phosphate-buffered saline (PBS), and then the medium was replaced with complete medium. For virus infections in the presence of Z-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-FMK; Calbiochem), zVAD-FMK was added to serum-free DMEM containing virus at a final concentration of 50 μM.

For pulse-chase experiments, the medium was replaced with methionine- and cysteine-free medium containing 30 μCi of [³⁵S]EasyTag Express protein labeling mix (Perkin-Elmer) for 30 min prior to harvesting. Cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris [pH 8], 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% Triton X-100, 0.02% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented with protease inhibitors (Roche), and the protein concentrations were determined by Bradford assay. Proteins were resolved by SDS-PAGE and analyzed by phosphorimager analysis.

For plaque assays, virus-infected cells were washed twice with PBS, harvested in serum-free DMEM, and lysed by three cycles of freeze-thawing. Serial dilutions of cell supernatants were incubated with HeLa cells for 1 h at 37°C. The cells were washed twice with PBS and then overlaid with DMEM containing 2% FBS, 1% penicillin-streptomycin, and 1% methylcellulose. After 72 h, the cells were fixed with 50% methanol and stained with 1% crystal violet. The plaques were counted, and the virus titer was calculated as PFU/ml.

Western blot analysis.

Equal amounts of protein were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The dilutions and antibodies used were as follows: 1:1,000 hnRNP M, 1:500 α-tubulin, 1:1,000 actin (Santa Cruz Biotechnologies), 1:3,000 G3BP1 (BD Transduction Science), 1:3,000 VP1 (Dako), 1:1,000 PABP (generously provided by Richard Lloyd, Baylor College of Medicine), 1:1,000 PARP (Pharmingen), and 1:1,000 GAPDH (Abcam).

Northern blot analysis.

Total RNA was isolated from cells using TRIzol reagent (Invitrogen). RNA was resolved on a denaturing agarose gel and transferred to Zeta-probe blotting membrane (Bio-Rad). Radiolabeled DNA hybridization probes were generated using a Deca labeling kit (Fermentas). The amount of radiolabeled probe hybridized to the blot was analyzed and quantified using a phosphorimager (Typhoon; Amersham Biosciences).

Immunofluorescence.

HeLa cells on coverslips were fixed with cold 100% methanol for 10 min, washed three times with PBS, and then blocked with 5% bovine serum albumin (BSA) in PBS for 1 h, followed by a 1-h incubation with primary antibody with 1% BSA in PBS at room temperature. The primary antibodies and the dilutions used were as follows: 1:25 hnRNP M and 1:50 hemagglutinin (HA; Santa Cruz Biotechnologies), 1:400 double-stranded RNA (dsRNA; English & Scientific Consulting Bt), and 1:100 Flag (Sigma). Coverslips were washed three times with PBS and then incubated with 1:500 secondary antibody (goat anti-rabbit antibody or goat anti-mouse antibody conjugated to Texas Red and goat anti-mouse antibody conjugated to Alexa Fluor 488 [Life Technologies]) with 1% BSA in PBS and Hoechst to stain for nuclei. After three washes, coverslips were mounted onto slides using Prolong Gold antifade reagent (Life Technologies). The cells were imaged and analyzed using a Nikon Eclipse Ti confocal microscope, and pictures were taken using NIS-Elements software.

Protein purification.

Wild-type and catalytically inactive (C109A) CVB3 2A^pro and His-tagged wild-type and catalytically inactive mutant (C147A) poliovirus 3C^pro were purified by using the expression plasmids pET-Cx2A, pET-Cx2A C109A, pET3Chc, and pET3Chc C147A (generously provided by Richard Lloyd, Baylor College of Medicine). Wild-type CVB3 3C^pro and a C147A catalytically inactive mutant proteinase were cloned into a pET28b expression vector containing an N-terminal His tag. 2A^pro proteinases were expressed in and purified from BL21 bacterial cells by ion-exchange chromatography and size-exclusion chromatography as previously described (10, 39). 3C^pro was expressed in and purified from BL21 bacterial cells by nickel-nitrilotriacetic acid (Ni-NTA) chelating resin affinity chromatography. Fractions containing purified 3C^pro were then pooled and dialyzed in 20 mM HEPES (pH 7.4), 100 mM NaCl, 7 mM β-mercaptoethanol, and 20% glycerol. Expression plasmid containing 3CD was generously provided by Bert Semler (UC–Irvine). Recombinant 3CD was purified as described previously (8). The integrity and purity of the purified protein were verified by Coomassie R-250 staining using SDS-PAGE analysis.

In vitro cleavage assay.

HeLa cell lysates were prepared by harvesting and pelleting cells in cold PBS and then resuspending them in 2× to 3× pellet volumes of cleavage assay buffer (20 mM HEPES [pH 7.4], 150 mM potassium acetate, and 1 mM dithiothreitol) supplemented with protease inhibitors (Roche). The cells were incubated on ice for 10 min and then lysed with 25 strokes in a Dounce homogenizer. The lysates were then clarified by centrifugation at 13,000 rpm for 15 min at 4°C.

Purified hnRNP M (20 pg; Origene) or HeLa cell lysates were incubated with purified wild-type or catalytically inactive CVB3 2A^pro (5 ng/μl), poliovirus 3C^pro (100 ng/μl), or CVB3 3C^pro (100 ng/μl) in cleavage assay buffer at 37°C for different periods of time, as indicated. The reaction mixtures were resolved by SDS-PAGE, and proteins were assessed by Western blotting.

N-terminal TAILS proteomics.

Equal amounts of HeLa cell lysates were incubated with either wild-type or catalytically inactive C147A mutant purified poliovirus 3C^pro in cleavage assay buffer at a 100:1 (wt/wt) ratio of protein to enzyme. Terminal amine isotopic labeling of substrates (TAILS) was performed as previously described (40). In brief, after the protein was denatured and reduced, the cysteines were alkylated, and the samples were isotopically labeled at the protein level by reductive dimethylation of primary amines. Thus, any protein alpha amine natural N terminus or proteinase-generated neo-N terminus were labeled and so could be identified after trypsin digestion. Heavy (wild-type proteinase-treated) and light (C147A-proteinase-treated) isotopically labeled samples were combined, the salts were removed, and the samples were concentrated by methanol precipitation. The sample was then subjected to trypsin digestion, followed by enrichment of labeled peptides by a negative selection step using a dendritic polyglycerol aldehyde polymer purchased from Flintbox (http://flintbox.com/public/project/1948) as described previously (40). Unbound labeled N termini peptides were separated from the polymer-bound peptides by centrifugation through a 10-kDa Microcon filter (Millipore). The flowthrough was collected and fractionated by strong cation-exchange high-performance liquid chromatography as previously described (40). The samples were then analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) on an Agilent G4240A ChipCube interfaced directly to a G6550A Q-TOF mass spectrometer (Agilent Technologies). A full comprehensive list of protein targets will be provided in a follow-up publication.

MS data analysis.

MS peaks were searched by using MASCOT (version 2.2; Matrix Science, London, United Kingdom) against a human database at a 1% false discovery rate. MASCOT searches of MS data were performed separately for heavy- and light-labeled peptides. Searches were performed using the following modifications: fixed carbamidomethylation of cysteines (+57.021 Da [Cys]), fixed heavy lysine (+34.0631 Da [Lys]) or light lysine (+28.0311 Da [Lys]); variable methionine oxidation (+15.995 Da [Met]), and fixed and variable modifications of the N termini with heavy formaldehyde (+34.0641 Da [N termini]), light formaldehyde (+28.0311 Da [N termini]), and acetylation (+42.011 Da [N termini]). The additional search criteria used were as follows: semi-ArgC cleavage specificity with up to three missed cleavages, a monoisotopic mass error window for the parent ion of 0.4 to 0.6 Da, and peptide mass tolerance of 0.4 Da for MS/MS fragment ions. The allowed peptide charge states were 1+, 2+, and 3+. Quantification of the heavy to light isotopically labeled peptides was achieved by using ProteoIQ. Statistically significant quantified peptides were determined by box plot analysis.

Mouse infection by CVB3.

A/J mice (Jackson Laboratory, catalog no. 000646) at ∼5 weeks of age were infected with 10⁵ PFU by intraperitoneal injection. Mock infections were performed using equal volumes of PBS. At 9 days postinfection (p.i.), mouse hearts were harvested, lysed, and immunoblotted as indicated.

Statistical analysis.

All statistical analyses were performed using GraphPad Prism. All graphs represent means ± the standard deviations (SD). P values were determined using a paired Student t test, and statistical significance was determined as P < 0.05.

RESULTS

hnRNP M is targeted by poliovirus 3C proteinase.

Although several host protein targets of picornavirus 3C^pro are known, we hypothesized that additional host proteins are targeted by 3C^pro under virus infection. To identify novel host substrates of 3C^pro, we used a proteomics-based approach called TAILS that simultaneously identifies protease substrates and their cleavage sites by MS/MS (40, 41). TAILS uses a negative selection to enrich for cleaved substrate neo-N termini and natural N termini after binding and removal of internal tryptic peptides to a polyaldehyde polymer. Briefly, HeLa cell lysates were incubated with purified wild-type or a catalytically inactive (C147A) recombinant poliovirus 3C^pro and then processed by TAILS, followed by tandem MS/MS, to identify proteins from enriched neo-N-terminus peptides. After isotopic ratio analysis of the identified peptides and substrate winnowing of a list of high-confidence, high-ratio peptides (wild-type/mutant samples), we identified a spectra of a neo-N-terminus peptide ³⁹⁰GGGGGGGSVPGIER from hnRNP M with a cleavage site at position ³⁸⁹Gln↓Gly (Fig. 1A and B). The ³⁸⁹Q and ³⁹⁰G at P1 and P1′ positions, respectively, are consistent with the consensus cleavage site of poliovirus 3C^pro (22). hnRNP M is a nucleocytoplasmic shuttling protein primarily known for its role in pre-mRNA splicing and alternative splicing (42–47). There are four alternatively spliced isoforms of hnRNP M derived from a single pre-mRNA transcript, all of which contain three RRMs (48). All four isoforms are highly similar in size and typically migrate as a closely spaced doublet referred to as M1/2 and M3/4 (48). The M4 isoform encodes the longest isoform of 730 amino acids, with a predicted molecular mass of 77 kDa (Fig. 1A) (48). The M1 isoform encodes a 690-amino-acid variant of M4 of 74 kDa containing a 39-amino-acid deletion between RRM1 and RRM2 (48). Notably, the same cleavage site sequence is found in all isoforms of hnRNP M. The cleavage site falls between RRM2 and RRM3 at amino acid 389 within the M4 isoform, which would result in two cleavage products of approximately 41 and 36 kDa.

FIG 1.

hnRNP M is cleaved by poliovirus 3C proteinase in vitro. (A) The hnRNP M peptide identified by TAILS is shown, including the four amino acids located directly upstream (P4 to P1). Schematic of hnRNP M protein isoforms are shown. RRM-RNA recognition motifs. Arrow denotes the cleavage site of poliovirus 3C^pro. (B) Fragmented spectra of the doubly charged GGGGGGGSVPGIER peptide identified following N-terminal enrichment by TAILS. (C and D) HeLa cell lysates were incubated with purified wild-type or mutant (C147A) poliovirus 3C^pro (100 ng/μl) (C) or wild type or mutant (C109A) CVB3 2A^pro (100 ng/μl) (D) for the indicated times. hnRNP M, PABP, and α-tubulin were detected by immunoblot analysis. (E) Cleavage of recombinant hnRNP M by purified poliovirus 3C^pro. Proteins were loaded on an SDS-PAGE and immunoblotted for hnRNP M. (F) Schematic of cytomegalovirus (CMV) promoter-driven mammalian expression construct containing 3× FLAG and HA fused in frame with the full-length hnRNP M (FLAG-hnRNP M-HA). (G) Expression of FLAG-hnRNP M-HA in HeLa cells. Cells were harvested at the times indicated, and lysates were immunoblotted with hnRNP M and actin antibodies. (H and I) Lysates from cells expressing the wild-type or mutant (Q389E/G390P) tagged hnRNP M (FLAG-hnRNP M-HA) were incubated with wild type or mutant poliovirus 3C^pro (H) or with poliovirus 3CD^pro (I) and immunoblotted for FLAG and PABP. cp, cleavage protein.

To confirm that hnRNP M is targeted by poliovirus 3C^pro, we used an in vitro cleavage assay by incubating purified recombinant poliovirus 3C^pro with HeLa cell lysates. Incubation of wild-type but not mutant 3C^pro with lysates resulted in the expected cleavage products of PABP, a known substrate of 3C^pro (Fig. 1C) (10). Immunoblotting with the hnRNP M (1D8) antibody detected a prominent band at ∼77 kDa, which corresponds to the mass of the M4 isoform. Addition of the wild-type poliovirus 3C^pro resulted in the accumulation of a cleavage product of ∼36 kDa, which was detected as early as 5 min of incubation, whereas no cleavage was observed with the mutant 3C^pro after incubation for 60 min (Fig. 1C). Detection of the 36-kDa cleavage product is consistent with the predicted size of the C-terminal protein product generated from cleavage at the site identified by TAILS and suggests that the 1D8 antibody recognizes the C-terminal half of hnRNP M. Addition of a recombinant CVB3 2A^pro to HeLa cell lysates resulted in cleavage of PABP but not hnRNP M, suggesting that the cleavage of hnRNP M is 3C^pro specific (Fig. 1D). To further assess whether hnRNP M is a direct substrate for 3C^pro, we incubated poliovirus 3C^pro with purified recombinant hnRNP M. Wild-type 3C^pro, but not the mutant, generated a 36-kDa cleavage product similar to that observed from the in vitro cleavage assay (Fig. 1E). Finally, to confirm the cleavage site identified by TAILS, we subcloned either the wild type or a Q389E/G390P mutant hnRNP M into a pCMV mammalian expression vector fused in-frame with a 3× FLAG tag and 3× HA tag at the N and C termini, respectively (FLAG-hnRNP M-HA, Fig. 1F). The Q389E/G390P mutations are predicted to disrupt the consensus cleavage site of 3C^pro, and thus this mutant should be proteinase insensitive. The expected molecular weight of FLAG-hnRNP M-HA is ∼84 kDa. Using the 1D8 hnRNP M antibody, immunoblotting analysis detected two proteins in the lysates of cells transfected with FLAG-hnRNP M-HA: the endogenous hnRNP M at 77 kDa and the slower-migrating tagged protein at ∼84 kDa (Fig. 1G). Moreover, the expression of FLAG-hnRNP M-HA was also detected with a FLAG antibody (Fig. 1H). After establishing the expression of FLAG-hnRNP M-HA in HeLa cells, we obtained lysates from HeLa cells expressing either the wild-type or a Q389E/G390P mutant FLAG-hnRNP M-HA and incubated the lysates in vitro with either wild-type or inactive 3C^pro. Wild-type, but not the inactive 3C^pro generated a 43-kDa cleavage product that was detected by anti-FLAG antibody (Fig. 1H). As predicted, the Q389E/G390P FLAG-hnRNP M-HA mutant was insensitive to 3C^pro cleavage (Fig. 1H). Because 3C^pro is also expressed as 3CD (49), we determined whether 3CD targets hnRNP M. As shown with 3C^pro, recombinant 3CD^pro targeted wild-type but not mutant Q389E/G390P FLAG-hnRNP M-HA in the in vitro cleavage assay (Fig. 1I). Taken together, these results demonstrate that hnRNP M is a bona fide substrate of poliovirus 3C^pro and 3CD^pro that is cleaved directly between amino acid pair ³⁸⁹Q↓G.

hnRNP M is cleaved in poliovirus-infected HeLa cells.

To examine whether cleavage of hnRNP M occurs during virus infection, HeLa cells were either mock or poliovirus infected and then harvested at different times after infection. Cleavage of PABP was observed beginning at 3 h p.i., producing the expected cleavage products (Fig. 2A) (11). Similar to the timing of PABP cleavage, the levels of full-length hnRNP M began to decrease at 3 h p.i., which is concurrent with the appearance of two proteins at 36 and 39 kDa (Fig. 2A). By 7 h p.i., the full-length hnRNP M was completely degraded, whereas the two cleavage products remained detectable throughout infection (Fig. 2A). The presence of two cleaved proteins suggests that hnRNP M may be cleaved more than once during infection.

FIG 2.

Cleavage of hnRNP M in poliovirus-infected HeLa cells. (A) HeLa cells were mock or poliovirus infected (MOI of 10) for the indicated times. (B) Cleavage of hnRNP M is insensitive to zVAD-FMK in poliovirus-infected cells. HeLa cells were infected with poliovirus at an MOI of 10 in the presence or absence of 50 μM zVAD-FMK (7 h p.i.). hnRNP M, poliovirus structural protein VP1, PARP, and α-tubulin were assessed by Western blotting. cp, cleavage proteins. Shown are representative gels from at least two independent experiments.

Apoptosis-induced activation of caspases can occur in picornavirus infections (50, 51). To assess whether cleavage of hnRNP M is a result of caspase activity, poliovirus-infected HeLa cells were incubated in the presence or absence of the general caspase inhibitor, zVAD-FMK. Both hnRNP M cleavage products were still observed in poliovirus-infected cells in the presence of zVAD-FMK, whereas cleavage of poly(ADP) ribose polymerase (PARP), a known caspase-3 substrate, was inhibited (Fig. 2B). Thus, we demonstrate that hnRNP M is cleaved to completion under poliovirus infection in a caspase-independent manner and that the cleavage products persist during infection.

Subcellular relocalization of hnRNP M in poliovirus-infected cells.

hnRNP M is a predominantly nuclear localized protein (52). Thus, it is of interest to determine how hnRNP M is targeted by a cytoplasmic RNA virus. To address this, we monitored the localization of hnRNP M in mock- and poliovirus-infected cells by immunofluorescence confocal microscopy. In mock-infected cells, hnRNP M was predominantly localized to the nucleus, in agreement with its role as a nuclear protein involved pre-mRNA splicing (Fig. 3, mock infected). However, upon infection, hnRNP M underwent a dramatic relocalization to the cytoplasm beginning at 3 h p.i. to near completion at 5 and 7 h p.i. (Fig. 3, poliovirus infected). This subcellular redistribution from the nucleus to the cytoplasm is similar to that observed with other hnRNPs such as hnRNP K and A1 during poliovirus infection (53). Given that hnRNP M is cleaved nearly to completion and that the cleaved fragments of hnRNP M persist in poliovirus-infected cells at 5 and 7 h p.i. (Fig. 2A), the immunofluorescence signal detected in the cytoplasm most likely represents the cleaved forms of hnRNP M.

FIG 3.

Subcellular localization of hnRNP M in poliovirus-infected HeLa cells. HeLa cells were mock or poliovirus infected (MOI of 10) for the indicated times. Cells were permeabilized, fixed, and costained for hnRNP M (red) and DNA (blue [Hoechst]). Representative confocal images are shown from at least three independent experiments.

We next assessed the subcellular location of the N- and C-terminal hnRNP M cleavage products during infection. We expressed FLAG-hnRNP M-HA in HeLa cells and monitored the fate of N- and C-terminal cleavage products by FLAG and HA antibodies during infection. As previously shown (Fig. 1G), a predominant 84 kDa protein was detected by FLAG and HA antibodies in transfected cells, indicative of FLAG-hnRNP M-HA expression (Fig. 4B, mock infected). The expression of FLAG-hnRNP M-HA had a reproducible moderate effect on cell viability in HeLa cells, suggesting that overexpression of hnRNP M is somewhat toxic (Fig. 4A). When probed with the hnRNP M antibody, both the endogenous hnRNP M and the C-terminal HA-tagged fragment of hnRNP M were detected (Fig. 4B, long exposure). We then subjected HeLa cells expressing FLAG-hnRNP M-HA to poliovirus infection. Immunoblotting for FLAG detected two N-terminal cleavage products of approximately 44 and 47 kDa at 5 and 7 h p.i., which is slightly delayed compared to when endogenous hnRNP M is cleaved (Fig. 4B). It is probable that the overexpression of the tagged hnRNP M delays infection. Interestingly, the HA antibody detected only a single protein at ∼42 kDa (Fig. 4B). It is noted that the full-length endogenous hnRNP M and FLAG-hnRNP M-HA were not cleaved to completion as observed in Fig. 2. It is likely that overexpression of FLAG-hnRNP M-HA may affect the extent of protein processing of endogenous hnRNP M by the virus during infection. Similar to that of the endogenous hnRNP M, the tagged hnRNP M is cleaved and the N- and C-terminal cleavage products persist during poliovirus infection. However, it is noted that the C-terminal tagged HA-hnRNP M is less stable at 7 h p.i. which is similar to that observed using the hnRNP M antibody for detection.

FIG 4.

Cleavage of hnRNP M in poliovirus-infected cells. (A) Cell viability of cells transfected with FLAG-hnRNP M-HA for 48 h. Cell viability was assessed by determining the percentage of cells not stained with trypan blue. Averages ± the SD are shown. *, P < 0.05. (B) HeLa cells transfected with FLAG-hnRNP M-HA were either mock or poliovirus infected (MOI of 10) for the indicated times. Lysates were immunoblotted for FLAG, HA, hnRNP M, and α-tubulin.

We then monitored expression of FLAG-hnRNP M-HA in poliovirus-infected HeLa cells by immunofluorescence to assess the N- and C-terminal cellular localization of hnRNP M. A dsRNA antibody was used to monitor the accumulation of viral replication intermediates. In mock-infected cells transfected with FLAG-hnRNP M-HA, HA and FLAG signals were detected primarily in the nucleus, similar to nuclear localization of the endogenous protein (Fig. 5, mock infected). Beginning at 3 h p.i., which is the time prior to cleavage of FLAG-hnRNP M-HA, both FLAG and HA antibodies showed a diffuse cytoplasmic staining (Fig. 5). FLAG and HA signals accumulated in the cytoplasm at 5 and 7 h p.i. (Fig. 5). As expected, dsRNA antibody staining was only detected in the cytoplasm of poliovirus-infected cells (Fig. 5). Interestingly, no colocalization was observed between either FLAG or HA and dsRNA signals, which would suggest that hnRNP M does not have a direct effect on viral replication. In summary, FLAG-hnRNP M-HA recapitulates the subcellular localization and cleavage pattern of endogenous hnRNP M.

FIG 5.

Subcellular localization of N- and C-terminal cleavage products of hnRNP M in poliovirus-infected HeLa cells. (A) Subcellular localization of N-terminal and C-terminal cleavage products of hnRNP M. HeLa cells transfected with FLAG-hnRNP M-HA for 48 h, followed by either mock or poliovirus infection (MOI of 10) for the times indicated. The cells were fixed and costained for FLAG (red in panel A) or HA (red in panel B), and dsRNA (green) for the detection of virus and Hoechst (blue). Shown are representative images from at least three independent experiments.

Expression of mutant hnRNP M Q389E/G390P in cells.

Our results indicated that mutant Q389E/G390P hnRNP M is resistant to cleavage by poliovirus 3C^pro (Fig. 1H). To determine whether this mutant is resistant to cleavage in poliovirus-infected HeLa cells, we transfected the FLAG-hnRNP M-HA expression construct that contains the Q389E/G390P mutations and followed the fate of the protein by immunoblot analysis. Surprisingly, despite being resistant to 3C^pro and 3CD^pro in the in vitro cleavage assay, the Q389E/G390P FLAG-hnRNP M-HA was still cleaved at roughly the same time and extent as the wild-type version in poliovirus-infected cells (Fig. 6A). Furthermore, the cleavage products of mutant and wild-type hnRNP M in infected cells migrated similarly by immunoblot analysis. This result suggests that hnRNP M is cleaved at a distinct site(s), likely close to the 3C^pro-sensitive Q389/G390 site. One possibility is that 3C^pro may cleave at multiple sites on hnRNP M. Surveying for putative 3C proteinase sites nearby, we found two sites at QE336-7 and QE349-50 that if cleaved by 3C^pro would result in cleavage products similar in mass as that observed during infection. However, expression of mutant FLAG-hnRNP M-HA containing mutations at these sites resulted in cleavage products during poliovirus infection (Fig. 6B). Nevertheless, we next determined whether the Q389E/G390P FLAG-hnRNP M-HA localized to the same cellular compartments as the wild-type version during poliovirus infection. As observed with endogenous hnRNP M and the wild-type FLAG-hnRNP M-HA, the FLAG and HA signals were predominantly nuclear localized in mock-infected cells and was localized to the cytoplasm in poliovirus-infected cells to the same extent and time as the wild-type protein (data not shown). In summary, these results indicate that although 3C^pro cleaves between amino acid pair ³⁸⁹Q↓G in vitro, hnRNP M is likely cleaved at another site nearby during poliovirus-infected cells. Currently, it is unclear whether the secondary site(s) is cleaved by 3C^pro or by another protease.

FIG 6.

Expression of mutant FLAG-hnRNP M-HA in poliovirus-infected cells. HeLa cells were transfected with either wild-type or mutant Q389E/G390P (A) or E337K/E350K (B) FLAG-hnRNP M-HA expression plasmids for 48 h, followed by mock or poliovirus infection (MOI of 1), for the indicated times. Lysates were immunoblotted with anti-FLAG.

hnRNP M facilitates poliovirus infection.

We next explored the significance of hnRNP M during poliovirus infection using a siRNA knockdown approach. Transfection of hnRNP M-specific siRNAs but not scrambled siRNAs in HeLa cells resulted in the loss of hnRNP M protein expression (Fig. 7A) and did not significantly affect cell viability (Fig. 7B). Cells transfected with scrambled or hnRNP M siRNAs for 72 h were then mock or poliovirus infected, and virus production was monitored by immunoblot and Northern blot analysis. In scrambled-siRNA-treated cells, cleavage of hnRNP M was detected in poliovirus-infected cells at 5 and 7 h p.i. (Fig. 2 and 7C). As expected, hnRNP M was not detected in infected cells treated with hnRNP M siRNAs (Fig. 7C). Interestingly, the viral structural protein VP1 was significantly reduced in poliovirus-infected cells treated with hnRNP M siRNAs compared to the scrambled control at 5 and 7 h p.i. (Fig. 7C). By 9 and 11 h p.i., VP1 expression in hnRNP siRNA-treated cells accumulated to similar levels as in scrambled treated cells (Fig. 7C). These results suggest that loss of hnRNP M inhibits and delays poliovirus infection. Furthermore, knockdown of hnRNP M decreased poliovirus genomic RNA at 5 and 7 h p.i. (Fig. 7D) and resulted in a 5- to 6-fold decrease in virus titer of both intracellular (5 h p.i.) and extracellular viral yield (7 h p.i.) compared to the scrambled control (Fig. 7E). These results collectively demonstrate that hnRNP M facilitates poliovirus infection in HeLa cells.

FIG 7.

Poliovirus infection is inhibited in HeLa cells lacking hnRNP M. (A) hnRNP M knockdown by transfection of siRNA in HeLa cells. hnRNP M and α-tubulin were assessed by immunoblotting. (B) Cell viability of cells treated with siRNAs for 72 h was calculated by determining the percentage of cells that were not stained with trypan blue. Averages ± the SD are shown. N.D., no statistically significant difference (P = 0.415). (C) HeLa cells were transfected with either scrambled (siSCX) or hnRNP M (sihnRNP M) siRNA for 72 h, followed by poliovirus infection (MOI of 1), for the indicated times. Immunoblots of hnRNP M, poliovirus structural protein VP1 and α-tubulin are shown. (D) Northern blot analysis of poliovirus genomic RNA in poliovirus-infected HeLa cells (MOI of 1) treated with siSCX or sihnRNP M. (E) Virus titers of intracellular (5 h p.i.) and extracellular (7 h p.i.) virus from poliovirus-infected cells (MOI of 0.1) pretreated with siSCX or sihnRNP M. Titers were calculated as PFU (PFU/ml ± the SD; *, P < 0.05) from three independent experiments.

Role of hnRNP M in poliovirus IRES translation.

We have shown that hnRNP M promotes poliovirus infection. Given that hnRNP M does not colocalize with replication complexes under infection, we next investigated a role for hnRNP M in viral translation. To examine this further, we investigated whether hnRNP M affects host translation and viral protein synthesis during infection. Mock- or poliovirus-infected cells that were pretreated with hnRNP M siRNAs for 72 h were pulse-labeled with [³⁵S]methionine-cysteine for 30 min prior to harvesting at each time point. Knockdown of hnRNP M did not significantly affect (94% ± 11%) overall protein synthesis compared to cells treated with scrambled siRNAs, a finding which is in agreement with our observation that knockdown of hnRNP M does not affect cell viability (Fig. 8A, mock-infected lanes). Host translational shutoff was observed beginning at 3 to 5 h p.i. in poliovirus-infected scrambled siRNA-treated cells at an MOI of 5, which is concomitant with viral protein synthesis (Fig. 8A). Synthesis of viral proteins such as P1, VP0, VP3, VP1, and 2BC was clearly observed in infected cells at MOIs of 1 and 5 at 5 h p.i. (Fig. 8A). In contrast, in cells depleted of hnRNP M, host translational shutoff was delayed at 5 and 7 h p.i. in infected cells at an MOI of 5 (Fig. 8A). Moreover, viral protein synthesis was reduced in these cells, which is consistent with the observation that VP1 expression is decreased in hnRNP M siRNA-treated, virus-infected cells (Fig. 8A and Fig. 7C). This result is most evident in infected cells at an MOI of 1 where viral protein synthesis is barely detected at 5 and 7 h p.i. (Fig. 8A). Thus, depletion of hnRNP M results in a decrease in either viral protein synthesis or replication in infected HeLa cells.

FIG 8.

Role of hnRNP M in poliovirus IRES translation. (A) Pulse-labeling using [³⁵S]methionine-cysteine at the indicated times after poliovirus infection (MOIs of 1 and 5) in cells treated with siSCX or sihnRNP M for 72 h prior to infection. The cells were pulse-labeled for 30 min prior to harvesting at each time point. A representative gel is shown from at least two independent experiments. (B) Flow chart of the transfection protocol to monitor poliovirus IRES translation. A schematic of the bicistronic reporter construct containing the poliovirus IRES within the intergenic region is shown below. (C and D) Cap-dependent Renilla and IRES-mediated firefly luciferase activities of the PV IRES bicistronic reporter construct (C) and mutant EMCV IRES bicistronic reporter construct (D). The relative luminescence was calculated as the mean ± the SD of three independent experiments. N.D., no statistically significant difference (P > 0.09). *, P < 0.05.

To examine more closely whether hnRNP M has a role in viral translation, we monitored poliovirus IRES translation directly by using an IRES-containing reporter construct (Fig. 8B). The bicistronic reporter construct contains the poliovirus IRES within the intergenic region between the Renilla and firefly luciferase genes, which monitor cap-dependent and poliovirus IRES-mediated translation, respectively (Fig. 8B). Because hnRNP M redistributes to the cytoplasm during poliovirus infection, we monitored poliovirus IRES translation by transfecting the IRES-containing reporter construct in poliovirus-infected cells. Briefly, cells treated with scrambled or hnRNP M siRNAs for 72 h were transfected with the bicistronic construct for 1 h, followed by mock or poliovirus infection (Fig. 8B, flowchart). Cells were then harvested 5 h later, and the luciferase activities were measured.

In mock-infected cells, hnRNP M siRNA treatment decreased Renilla luciferase activity by ca. 12% compared to scrambled siRNA treatment, indicating that depletion of hnRNP M had a slight effect on cap-dependent translation using this transfection reporter approach (Fig. 8C). In contrast, firefly luciferase activity was detected at similar levels in both the scrambled and hnRNP M siRNA treatments, suggesting that hnRNP M does not have a role in IRES-dependent translation under basal conditions (Fig. 8C). In poliovirus-infected cells, Renilla luciferase activity was inhibited to the same extent in both the scrambled and hnRNP M siRNA treatments, which is a reflection of shutoff of host translation during infection (Fig. 8C). In contrast, firefly luciferase activity was still detected at similar levels in the hnRNP M siRNA-treated cells compared to the scrambled controls (Fig. 8C), suggesting that hnRNP M is not required for poliovirus IRES translation. A bicistronic construct containing an inactive IRES did not result in firefly luciferase expression, indicating that IRES activity is being measured (Fig. 8D). In summary, the results suggest that hnRNP M is not required for poliovirus IRES translation during infection and likely participates in another step of the viral life cycle.

hnRNP M is not required for poliovirus genomic RNA stability.

The decrease in viral RNA in poliovirus-infected cells that are depleted of hnRNP M may be due to an effect on viral replication or viral RNA stability. To address whether hnRNP M is involved in viral RNA stability, we monitored the fate of viral RNA in scrambled or hnRNP M siRNA-treated poliovirus-infected cells after treating the cells at 4 h p.i. with 2 mM guanidine hydrochloride (GuHCl), a known inhibitor of poliovirus RNA synthesis (54). No significant difference was observed between the stabilities of viral RNA between the scrambled and hnRNP M siRNA-treated cells following the addition of guanidine hydrochloride (Fig. 9). Thus, hnRNP M is not required for maintaining poliovirus RNA stability during infection.

FIG 9.

Stability of viral genomic RNA in poliovirus-infected cells. HeLa cells pretreated with siSCX or sihnRNP M for 72 h were infected with poliovirus (MOI of 5) and, at 4 h p.i., treated with 2 mM guanidine hydrochloride (GuHCl). Cells were harvested at the indicated times, and the viral RNA and GAPDH mRNA was assayed by Northern blotting. The ratio of viral RNA to GAPDH at 4 h p.i. was set as 100%. Shown are the averages of three independent experiments ± the SD.

Role of hnRNP M in CVB3 infection.

Our results have established a novel role of hnRNP M in poliovirus infection. We next determined whether the requirement of hnRNP M is specific to poliovirus infection. To address this, we sought to determine whether hnRNP M facilitates infection of another picornavirus, CVB3. Similar to that observed with poliovirus 3C^pro, immunoblot analysis detected the accumulation a cleavage product of ∼34 kDa in HeLa lysates incubated with purified CVB3 3C^pro but not a catalytically inactive mutant CVB3 3C^pro (Fig. 10A). The cleavage product of hnRNP M by CVB3 3C^pro migrated slightly ahead than the cleavage product produced by poliovirus 3C^pro, suggesting the CVB3 3C^pro may target another site within hnRNP M (Fig. 10A). When monitored during CVB3 infection, a single cleavage 55-kDa protein was observed at 5 h p.i., followed by multiple cleavage products observed at 7 and 9 h p.i., demonstrating the hnRNP M is cleaved at multiple sites under CVB3 infection (Fig. 10B). Cleavage of hnRNP M was still observed during CVB3 infection in the presence of zVAD-FMK, demonstrating that targeting of hnRNP M is not due to caspase activity (data not shown). The significance of hnRNP M in CVB3 infection was also explored by measuring virus titers after siRNA-mediated knockdown of hnRNP M. We observed an ∼7-fold decrease in CVB3 titer in hnRNP M siRNA-treated cells (Fig. 10C).

FIG 10.

hnRNP M in CVB3-infected cells. (A) Cleavage of hnRNP M in CVB3-infected cells. (A) Immunoblots of HeLa lysates incubated with purified wild-type or mutant (C147A) CVB3 3C proteinase. (B and D) Immunoblots of lysates from mock- or CVB3-infected HeLa cells (MOI of 10) (B) or hearts of CVB3-infected mice (D) (results from two independent experiments are shown). (C) Virus titers of CVB3-infected (MOI of 1, 16 h p.i.) HeLa cells that were pretreated with siSCX or sihnRNP M (n = 3; mean ± the SD; *, P < 0.05).

CVB3 is a prevalent contributor to dilated cardiomyopathy among young children by targeting and ultimately destroying cardiomyocytes (55). To assess whether cleavage of hnRNP M occurs under more physiologically relevant conditions, we monitored hnRNP M in cardiomyocytes from mice infected with CVB3. Cleavage products of hnRNP M were detected in the CVB3-treated mice but not in the mock-treated mice (Fig. 10D). Altogether, these data indicate that cleavage of hnRNP M and the requirement of this protein for infection may be a conserved strategy among picornaviruses to facilitate virus infection.

DISCUSSION

We have identified hnRNP M as a direct substrate of poliovirus 3C^pro and 3CD^pro in vitro and that hnRNP M is cleaved in both poliovirus- and CVB3-infected cells. We also demonstrate that hnRNP M promotes both poliovirus and CVB3 infection. Several members of the hnRNP family, including hnRNP A1, PCBP1/2, AUF1, and PTB, are important host factors for picornavirus infection (2–9). A subset of these hnRNPs are modified through cleavage by picornavirus proteinases in infected cells and, as a result, picornaviruses either can inhibit or alter the function of these proteins or exploit the function of their cleavage products. Our work suggests that cleavage of hnRNP M is common strategy of picornavirus infections and that picornaviruses hijack hnRNP M to facilitate infection.

We demonstrated here conclusively that hnRNP M is cleaved by poliovirus 3C^pro and 3CD^pro between ³⁸⁹Q↓G³⁹⁰ (M4 isoform numbering) to produce a 36-kDa cleavage protein that is detected by the 1D8 hnRNP M antibody (Fig. 1). The 1D8 antibody recognizes an epitope within the C-terminal fragment of hnRNP M, which is based on the observation that both 1D8 and HA antibodies detect the same C-terminal cleavage product of the tagged FLAG-hnRNP M-HA in poliovirus-infected cells (Fig. 4B). We posit that all variants of hnRNP M are targeted, since all isoforms harbor the region containing the identified cleavage site, and thus cleavage by 3C^pro would produce the same C-terminal end of hnRNP M. Furthermore, hnRNP M is completely cleaved by 7 h p.i. in poliovirus-infected HeLa cells, which suggests that all four isoforms are being targeted during infection (Fig. 2A).

We detected a single 36-kDa cleavage product of hnRNP M in vitro (Fig. 1B); however, two cleavage products of approximately 36 and 39 kDa were consistently detected in poliovirus-infected cells (Fig. 2A). Accumulation of a second cleavage product under infection may occur through more than one mechanism. First, hnRNP M may be cleaved twice at a second site close in proximity to the ³⁸⁹Q↓G³⁹⁰ cleavage site identified in vitro, through either 3C^pro or another protease. We have determined that the viral 2A^pro is probably not involved and ruled out the possibility of caspase-induced cleavage (Fig. 2B). Furthermore, while mutating Q389E/G390P prevented direct cleavage by 3C^pro in vitro, we still observed cleavage of this mutant under poliovirus infection (Fig. 1H and 6A). These results suggest that hnRNP M is cleaved at one or more sites in close proximity to the 3C^{pro 389}Q↓G³⁹⁰ in vitro cleavage site. Several candidate proteinase cleavage sites were tested, but all failed to prevent cleavage (Fig. 6B; data not shown). Further mapping of the cleavage sites will provide insights into the proteases that target hnRNP M during poliovirus infection. Interestingly, expression of FLAG-hnRNP M-HA generates two cleavage products detected by the FLAG epitope under poliovirus infection and only one cleavage product detected by the HA epitope. This may indicate that a second cleavage event is occurring on a cleavage product after destabilization of the hnRNP M structure following the initial cleavage. Alternatively, the two cleavage products may be due to two distinct isoforms that are cleaved during infection, or that the cleaved fragment is subject to posttranslational modification. Further investigation is required to determine the cleavage activity of hnRNP M under infection.

During poliovirus and CVB3 infections, hnRNP M is relocalized to the cytoplasm and is cleaved by the viral 3C^pro (Fig. 3 and data not shown). The subcellular relocalization of hnRNP M is similar to that observed of other hnRNP proteins during poliovirus infection, thus suggesting a general strategy of picornaviruses to redistribute RNA-binding proteins (53). Poliovirus and CVB3 infections lead to remodeling of the nuclear pore complex by viral proteinases, which contributes to the inhibition of nuclear import of specific proteins (53, 56–58). Interestingly, it has previously been demonstrated that 3C in its precursor form as 3CD is capable of entering the nucleus of virus-infected cells through a 3D nuclear localization signal (59). Although we demonstrate that hnRNP M can also serve as a substrate of 3CD^pro (Fig. 1I), we observed that FLAG-hnRNP M-HA begins to redistribute from the nucleus to the cytoplasm at 3 h p.i. prior to being cleaved (Fig. 4B and 5). It is likely that hnRNP M relocalizes to the cytoplasm due to blockage of nuclear import mediated by the 2A proteinase and is then targeted by the 3C proteinase.

Like other hnRNPs, hnRNP M is a RNA-binding protein that associates with G-U-rich regions of pre-mRNA (48). hnRNP M is part of pre-spliceosome assembly complexes and functions in splice-site recognition and alternative splicing (43–45, 52, 60, 61). In addition, hnRNP M has also been implicated in transcriptional controls, heat shock stress responses, and cell signaling (62–64). Our work shows for the first time that the function of hnRNP M is subverted during poliovirus infection and diverted toward a step in the viral life cycle. An indirect effect of relocalization of hnRNP M to the cytoplasm is that splicing will cease or be altered in the nucleus. Previous reports have shown that inhibition of splicing may be a strategy utilized by picornaviruses to subvert host antiviral responses (28, 65, 66). Thus, redistribution of hnRNP M contributes to this effect.

Although hnRNP M is cleaved during infection, our study shows that hnRNP M is required for optimal picornavirus infection (Fig. 7E and 10C), suggesting that hnRNP M and/or its cleavage products contribute to a specific step of the viral life cycle. Several hnRNPs are exploited by picornaviruses to aid in viral translation, replication, or stability of the viral genome. For example, inhibition of nucleocytoplasmic transport of protein during picornavirus infection leads to relocalization of nuclear proteins, such as PTB and PCBP2, to the cytoplasm that then aid in viral translation (33, 53, 57). Although several hnRNP proteins have specifically been identified as mediators of poliovirus translation through direct interaction with the IRES (2, 4–7), our translation assays in hnRNP M-depleted cells do not show an effect on poliovirus IRES-mediated translation (Fig. 8B).

Given that viral RNA levels are dampened in hnRNP M siRNA-treated cells, the simplest hypotheses are that the defect is at the step of RNA metabolism replication or viral RNA stability (Fig. 7D). We showed that hnRNP M does not have role in maintaining viral RNA stability in infected HeLa cells treated with GuHCl (Fig. 9). Previous studies have implicated a role for hnRNP M in replication of influenza A virus and Semliki Forest virus (SFV) (67, 68). Moreover, depletion of hnRNP M enhances SFV gene expression and replication, suggesting that hnRNP M may be antiviral. Both the N- and C-terminal cleavage proteins of hnRNP M contain at least one RRM and thus presumably have the ability to bind RNA. Moreover, because both the N- and C-terminal fragment persists at least until 5 h p.i. (Fig. 2 and 4), we hypothesized that the cleavage products of hnRNP M act in viral replication. However, quantitation of the confocal images showed no colocalization of the tagged hnRNP M and dsRNA antibody signals, which mark sites of replication (Fig. 5; data not shown) (69). Moreover, viral RNA was not detected in hnRNP M immunoprecipitation experiments (data not shown). Thus, hnRNP M does not have a direct role in poliovirus replication. It is still possible that hnRNP M has an indirect role in replication, possibly by interacting with and affecting the function of a specific protein or an mRNA that encodes a protein involved in viral replication. Alternatively, hnRNP M may interact with a protein or mRNA that encodes a protein that have a role in the innate immune response or in stress granules or P body formation, host processes that could affect poliovirus RNA accumulation during infection (36, 70, 71). Further experiments to identify the proteins and/or mRNAs that interact with the cleavage products of hnRNP M in infected cells will undoubtedly shed light into the functions of hnRNP M in infected cells.

Our findings are in line with the general theme that the RNA-binding family of hnRNPs is targeted by picornavirus infections. However, not all hnRNPs function similarly in infected cells. Depletion of a subset of hnRNPs does not have an effect on virus infection, whereas others do, thus highlighting that each hnRNP has specific roles in picornavirus-host interactions (2–9). Our work demonstrates that hnRNP M plays in important role in poliovirus and CVB3 infections. It will be important to determine how the cleavage products contribute to a specific step of the viral life cycle. Lastly, we identified hnRNP M through an unbiased proteomics approach using TAILS N-terminome enrichment to identify substrates of viral proteinases. This proof-in-principle study provides added confidence that TAILS has great potential to reveal other proteinase substrates that are important for picornavirus infection.