Enterovirus 71 3C Promotes Apoptosis through Cleavage of PinX1, a Telomere Binding Protein

ABSTRACT

Enterovirus 71 (EV71) is an emerging pathogen causing hand, foot, and mouth disease (HFMD) and fatal neurological diseases in infants and young children due to their underdeveloped immunocompetence. EV71 infection can induce cellular apoptosis through a variety of pathways, which promotes EV71 release. The viral protease 3C plays an important role in EV71-induced apoptosis. However, the molecular mechanism responsible for 3C-triggered apoptosis remains elusive. Here, we found that EV71 3C directly interacted with PinX1, a telomere binding protein. Furthermore, 3C cleaved PinX1 at the site of Q50-G51 pair through its protease activity. Overexpression of PinX1 reduced the level of EV71-induced apoptosis and EV71 release, whereas depletion of PinX1 by small interfering RNA promoted apoptosis induced by etoposide and increased EV71 release. Taken together, our study uncovered a mechanism that EV71 utilizes to promote host cell apoptosis through cleavage of cellular protein PinX1 by 3C.

IMPORTANCE EV71 3C plays an important role in processing viral proteins and interacting with host cells. In this study, we showed that 3C promoted apoptosis through cleaving PinX1, a telomere binding protein, and that this cleavage facilitated EV71 release. Our study demonstrated that PinX1 plays an important role in EV71 release and revealed a novel mechanism that EV71 utilizes to induce apoptosis. This finding is important in understanding EV71-host cell interactions and has potential impact on understanding other enterovirus-host cell interactions.

INTRODUCTION

Enterovirus 71 (EV71) is an emerging pathogen that was originally isolated from an infant with central nervous system disease in California (1). Since then, EV71 has caused several large-scale epidemic outbreaks throughout the world, especially in the Asia-Pacific region (2–4). These outbreaks caused significant morbidity and mortality and severely endangered the public health (5). EV71 infection causes hand, foot, and mouth disease (HFMD) and fatal neurological diseases, such as aseptic meningitis, brainstem encephalitis, and neurogenic pulmonary edema (NPE), in infants and young children due to their underdeveloped immunocompetence (6). To date, there are no effective therapeutic medicines or vaccines for EV71.

EV71 belongs to the Picornaviridae family with a single positive-stranded RNA genome. Translation of the RNA genome produces a single polyprotein precursor that is subsequently processed into structural (VP1, VP2, VP3, and VP4) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (7). In addition to its role in viral precursor processing (8), 3C is also involved in a number of biological processes. It has been reported that 3C cleaves cellular CstF-64 protein, which inhibits host RNA processing and polyadenylation (9). Interferon-regulatory factor 7 (IRF7) (10), TIR domain-containing adaptor inducing beta interferon (TRIF) (11) and the TAK1/TAB1/TAB2/TAB3 complex (12) are also substrates of 3C, and the cleavage of these factors plays important roles in antiviral immune evasion.

EV71-induced cytopathic effect (CPE) usually includes cell swelling, plasma membrane breaks, chromatin condensation in the nucleus, and nuclear degeneration (13, 14), indicating apoptosis and tissue inflammation. Apoptosis, also called programmed cell death, is an important cell regulation mechanism in many biological processes, including viral infections (15–17). EV71 infection can induce apoptosis in various cell types through different mechanisms (13, 15, 18–20). For example, EV71 infection regulates the expression of miR-146a or miR-370, coordinating apoptosis through targeting SOS1 and GADD45β (16). EV71 also activates calpain via Ca²⁺ flux, playing an essential role in the caspase-independent apoptotic pathway (21). Furthermore, it was found that the cleavage of eukaryotic initiation factor 4G (eIF4G) by EV71 2A, which shuts off host translation, also induces apoptosis (22, 23). EV71 3C triggers apoptosis through caspase activation (8); however, the molecular events in 3C triggering of apoptosis remain elusive.

PinX1 is a conserved nuclear protein and was originally identified as a Pin2/TRF1-interacting protein through yeast two-hybrid screening (24). It is an intrinsic telomerase inhibitor and a putative tumor suppressor (25–27). It also plays an important role in telomere integrity maintenance (28) and regulation of cell growth and mitosis (29, 30). PinX1 also is involved in cellular apoptosis, and this is likely due to its role in telomere maintenance. It has been demonstrated that reduced PinX1 protein expression enhances apoptosis, while increased PinX1 expression inhibits apoptosis (31, 32). In this study, we first identified and demonstrated PinX1 as a novel 3C-interacting protein. Further experiments demonstrated that EV71 3C cleaved PinX1 at the Q51-G52 pair through its protease activity. The depletion of PinX1 or EV71 3C cleavage promoted cell apoptosis, subsequently facilitating EV71 release.

RESULTS

EV71 3C interacts with PinX1.

To identify potential proteins that interact with EV71 3C, a yeast two-hybrid screening was performed using a human universal cDNA library and 3C as the bait. Sixty positive clones were obtained. One positive clone contained an in-frame 471-bp partial cDNA (GenBank accession no. AY523569.1) encoding amino acids 68 to 224 of the human PinX1 protein. Yeast cells transformed with pGADT7-PinX1 and pGBKT7-3C were able to grow on selective medium and exhibited robust β-galactosidase activity (Fig. 1A), indicating interaction between PinX1 and 3C in yeast. T7 interacts with p53 but not lam, so pGADT7-p53 and pGBKT7-lam were used as a positive control and a negative control, respectively. Next, we confirmed the interaction of 3C and PinX1 in mammalian cells by transfection of HEK293T cells with green fluorescent protein (GFP)-3C and Flag-PinX1, followed by coimmunoprecipitation with an anti-Flag or anti-GFP antibody. The 3C was coimmunoprecipitated with Flag-PinX1 (Fig. 1B and C). To further examine whether the 3C-PinX1 interaction is direct, a glutathione S-transferase (GST) pulldown assay was performed using bacterially expressed GST-PinX1 and His-3C. The results clearly showed that His-3C directly interacted with GST-PinX1 but not with GST, the negative control (Fig. 1D).

FIG 1.

EV71 3C interacts with PinX1. (A) Yeast AH109 cells were transformed with empty vector or pGBK-T7-3C and pGAD-T7-PinX1, spread onto yeast selective medium without Trp and Leu, and then streaked onto selective medium without Ade, His, Trp, and Leu supplemented with 5-bromo-4-chloro-3-indolyl-α-d-galactopyranoside (X-α-Gal). β-Galactosidase activity of lysates of AH109 transformed with both pGBKT7-3C and pGADT7-PinX1 was examined by liquid culture assay using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate. em, empty vector; lam, pGBKT7-lam; T, pGADT7-T; p53, pGADT7-p53. T7 interacts with p53 but not lam. pGADT7-T and pGADT7-p53 were used as positive controls, and pGADT7-T and pGBKT7-lam were used as negative controls. (B and C) HEK293T cells (4 × 10⁶) were cotransfected with empty vector or Flag-PinX1 and GFP-3C (B) or GFP or GFP-3C and Flag-PinX1 (C). Coimmunoprecipitation was performed with the indicated antibodies. Samples of both cell lysates and immunoprecipitates were subjected to Western blotting and probed with mouse anti-GFP and mouse anti-Flag antibodies. (D) Recombinant His-3C as well as GST-PinX1 was expressed and purified from E. coli. GST pulldown assay was performed to confirm the direct interaction of PinX1 and 3C in vitro.

EV71 3C cleaves PinX1, and 3C protease activity is required for the cleavage.

Interestingly, the amount of the full-length PinX1 was reduced when 3C and PinX1 was coexpressed (Fig. 1C). Because EV71 3C is a protease and has been shown to cleave multiple host proteins (9–12), we carried out cleavage assays to examine whether 3C could cleave PinX1. HEK293T cells were transfected with increasing amounts of Myc-3C, along with Flag-PinX1. At 48 h after transfection, PinX1 was detected by Western blotting. As shown in Fig. 2A, 3C reduced PinX1 accumulation in a dose-dependent manner compared to C147S, a 3C mutant with impaired protease activity. The reduction of full-length PinX1 paralleled the appearance of a smaller protein of approximately 40 kDa (Fig. 2A). Next, a cleavage assay was carried out by incubating recombinant wild-type EV71 3C or the C147S mutant of 3C produced from Escherichia coli with PinX1 expressed in HEK293T cells. The results clearly showed that 3C cleaved PinX1 in a concentration- and time-dependent manner (Fig. 2B and C). To further exclude any effect of undefined factors in cell lysates on PinX1 cleavage, we carried out an in vitro cleavage assay using bacterially expressed recombinant His-PinX1 and His-3C proteins. Recombinant His-PinX1 was cleaved by His-3C but not GST or His-C147S (Fig. 2D). Furthermore, we assessed the impact of EV71 3C on endogenous PinX1. We found that PinX1 is constitutively expressed in HEK293T, HeLa, RD, and, Jurkat cells (data not shown). HeLa cells transfected with increasing amounts of 3C or C147S expression plasmid were subjected to Western blotting. Again, the results showed that endogenous PinX1 was cleaved by 3C in a dose-dependent manner (Fig. 2E). Taken together, these results demonstrated that EV71 3C cleaved PinX1 and that the protease activity of 3C was required.

FIG 2.

3C cleaves PinX1 through protease activity but not RNA binding activity. (A) Lysates of HEK293T cells expressing Flag-PinX1 and increasing amounts of pQCXIP-3C (0, 0.2 μg, 0.4 μg, and 0.8 μg for lanes 1 to 4, respectively) or pQC-XIP-C147S were subjected to Western blotting with antibodies against PinX1, Myc, or tubulin. Tubulin was used as a protein loading control. (B) Flag-PinX1 expressed in HEK293T cells and His-3C expressed in E. coli were tested in an in vitro cleavage assay. (C) Flag-PinX1 and His-3C were tested in an in vitro cleavage assay for different times. (D) In vitro cleavage assay was performed with recombinant His-PinX1 and GST, His-3C, or His-C147S. Samples were subjected to SDS-PAGE and Coomassie blue staining. (E) Endogenous PinX1 was cleaved by 3C. HeLa cells were transfected with Myc-3C or Myc-C147S. After 48 h, cells were subjected to Western blotting. (F) Cotransfection analysis of Flag-PinX1 and different 3C variants (3C, C147S, and R84Q). Lysates were tested by Western blotting. Important blots were quantified with ImageJ software.

3C also possesses RNA binding activity in addition to its protease activity (33). The 3C mutant R84Q, which abolishes the RNA binding activity, still cleaved PinX1 (Fig. 2F, lane 4), while the C147S with impaired protease activity failed to cleave PinX1 (Fig. 2F, lane 3). Thus, the protease activity of 3C, but not the RNA binding activity, is essential for PinX1 cleavage.

The cleavage site(s) of 3C is localized at Q50-G51 in PinX1.

In order to identify the 3C cleavage site(s) in PinX1, we performed mutational analysis of PinX1. EV71 3C preferentially cleaves polypeptides with Gln-Gly (Q-G) or Gln-Ser (Q-S) junctions (34). There are three such junctions in PinX1, Q50-G51, Q151-S152 and Q267-S268 (Fig. 3A). As PinX1 cleavage by 3C produced an approximately 40-kDa specific fragment, we inferred that Q50-G51 or Q267-S268A was the potential cleavage site(s) of 3C. To test this, we analyzed the PinX1 G51A and S268A mutants. These mutants were expressed with 3C or C147S in HEK293T cells. Western blotting showed that the wild-type PinX1 and S268A were cleaved by 3C but not C147S (Fig. 3B and C), while G51A was resistant to cleavage, suggesting that Q50-G51 is the cleavage site of 3C in PinX1. This also explained why the cleaved PinX1 was not detected using anti-Flag antibody, because the Flag tag was at the N terminus of PinX1 and PinX1-1-50 was too small to be detected.

FIG 3.

Q50-G51 is the site of PinX1 cleavage by 3C. (A) Schematic illustration of the potential sites of cleavage by 3C in PinX1. (B) Cotransfection analysis of PinX1 variants and 3C. Lysates were tested by Western blotting. (C) In vitro cleavage assay of PinX1 variants and 3C. PinX1 variants were in vitro cleaved by His-3C or His-C147S and then detected by Western blotting.

Cleavage of PinX1 by 3C promotes apoptosis.

PinX1 contributes to telomere maintenance, and depletion of PinX1 results in the DNA damage response and apoptosis (28). To investigate the impact of PinX1 cleavage by 3C, we generated a PinX1 mutant, PinX1 G51A, that cannot be cleaved by EV71 3C. HeLa cells were cotransfected with PinX1 or the G51A PinX1 mutant along with 3C or treated with 25 μM etoposide. Etoposide is a DNA-damaging reagent and induces apoptosis in a variety of cell lines (35–38). The apoptosis was measured by annexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) staining and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. Fluorochrome-labeled annexin V binds to phosphatidylserine translocated to the external leaflet during early apoptosis and can be used to specifically target and identify apoptotic cells. TUNEL staining is a method for detecting DNA fragmentation that results from apoptosis or severe DNA damage by labeling the terminal ends of nucleic acids. The results indicated that both etoposide and 3C could induce apoptosis in HeLa cells and that G51A but not PinX1 could inhibit 3C-induced apoptosis (Fig. 4A to D). We also examined the effect of 3C on etoposide-induced apoptosis using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (a tetrazolium inner salt for determining the number of viable cells in proliferation or chemosensitivity assays) assay and nuclear staining assay (apoptotic cells present bright condensed fluorescence). HeLa cells were transfected with the empty vector, 3C, or C147S and then treated with etoposide for 24 h. The results showed that 3C, but not its C147S mutant, enhanced the etoposide-induced apoptosis (Fig. 4E to H).

FIG 4.

Cleavage of PinX1 by 3C promotes apoptosis. (A to D) HeLa cells were cotransfected with empty vector or Myc-3C and empty vector, PinX1, or G51A. Forty-eight hours later, cells were subjected to annexin V-FITC-PI (A) or TUNEL (C) staining. Fluorescence results were quantified with the Image J software and were analyzed using GraphPad Prism software (B and D). In annexin V-FITC staining, green (FITC) indicates the degree of apoptosis, and red (PI) indicates the degree of necrocytosis. In TUNEL staining, green (dUTP-FITC) indicates the degree of apoptosis, and red (PI) indicates the degree of necrocytosis. (E and F) HeLa cells transfected with empty vector, Myc-3C, or Myc-C147S were untreated or treated with the indicated dosage of etoposide and then were stained with DAPI (4′,6′-diamidino-2-phenylindole). (G and H) HeLa cells were transfected with empty vector, Myc-3C, Myc-C147S, and then treated with increasing doses of etoposide. Cell viability was measured by MTS assay, and data were analyzed using GraphPad Prism software.

Cleavage of PinX1 enhances apoptosis induced by etoposide and EV71 infection.

Next, we generated three PinX1 short hairpin RNA (shRNA) knockdown cell lines to further test whether PinX1 deficiency induces apoptosis. The control cell line expressed a random sequence that is not complementary with any sequence in the human genome. We successfully silenced PinX1 in both HeLa and Jurkat cells (Fig. 5A). The shRNA control and shRNA PinX1#2 cells were treated with increasing doses of etoposide. After 24 h, the treated cells were subjected to MTS assay and Western blotting to assess poly(ADP-ribose) polymerase (PARP) cleavage, a molecular indicator of cellular apoptosis. As shown in Fig. 5B to D, although etoposide could induce apoptosis in a dose-dependent manner in both shRNA PinX1 and shRNA control cells, PinX1 silenced cells were more sensitive to etoposide treatment at all dosages used. Nuclear staining assay also confirmed this observation (Fig. 5E to H). Therefore, silencing of PinX1 enhanced the cellular apoptosis induced by etoposide.

FIG 5.

Knockdown of PinX1 promotes apoptosis triggered by etoposide. (A) Three shRNAs targeting different sites of PinX1 and a random sequence were inserted into lentivirus vectors pSIRE-RetroQ. HeLa and Jurkat cells were infected with lentiviruses packaging these vectors for 48 h and then screened in the presence of 2 μg/ml puromycin. The efficiency of PinX1 knockdown was examined by Western blotting with anti-PinX1. (B to D) Both HeLa-shRNA control and HeLa-shRNA PinX1#2 cells were treated with increasing doses of etoposide for 24 h, cell viability was tested by MTS assay (B and C), and lysates were subjected to Western blotting with the indicated antibodies (D). (E and F) HeLa-shRNA control, HeLa-shRNA-PinX1#2, and HeLa-shRNA-PinX1#3 cells were mock treated or treated with 25 μM etoposide. Twenty-four hours later, cells were stained with DAPI. (G and H) Jurkat-shRNA control, Jurkat-shRNA-PinX1#2, and Jurkat-shRNA-PinX1#3 cells were untreated or treated with 5 μM etoposide. Twenty-four hours later, cells were stained with Hoechst 33258.

We next examined whether PinX1 cleavage also increases EV71 infection-triggered apoptosis. As shown in Fig. 6A to D, similar to etoposide treatment, EV71 infection also induced apoptosis. PinX1 depletion enhanced EV71-induced apoptosis. We further examined whether PinX1 or its G51A mutant could antagonize EV71-induced apoptosis using annexin V-FITC-PI staining and TUNEL staining. As shown in Fig. 6E to H, etoposide treatment and EV71 infection both induced apoptosis, and G51A but not PinX1 expression reduced EV71-triggered apoptosis. These findings demonstrated that PinX1 cleavage induces and promotes apoptosis during EV71 infection.

FIG 6.

Cleavage of PinX1 promotes apoptosis triggered by EV71 infection. (A and B) HeLa-shRNA control, HeLa-shRNA-PinX1#2, and HeLa-shRNA-PinX1#3 cells were mock infected or infected with EV71. Twenty-four hours later, cells were stained with DAPI. (C and D) Jurkat-shRNA control, Jurkat-shRNA-PinX1#2, and Jurkat-shRNA-PinX1#3 cells were mock infected or infected with EV71. Twenty-four hours later, cells were stained with Hoechst 33258. (E to H) HeLa cells transfected with PinX1 or G51A were infected with EV71 and then subjected to annexin V-FITC-PI (E and F) and TUNEL (G and H) staining.

Cleavage of PinX1 facilitates EV71 release.

To determine the effect of PinX1 cleavage on EV71 replication, HeLa and Jurkat cells with shRNA control and shRNA PinX1 were infected or mock infected with EV71 at the indicated multiplicity of infection (MOI). At 24 h postinfection, supernatants and cells lysates were subjected to Western blotting. As shown in Fig. 7A and C, although there was little difference in cell lysates, the PinX1 knockdown cells produced more progeny viruses in supernatant than did shRNA control cells. Knockdown of PinX1 resulted in an approximately 2.5-fold increase in virus titers compared to those for control cells (Fig. 7B and D). To further confirm the mechanism that PinX1 cleavage enhances viral release, HeLa cells were transfected with PinX1 or PinX1 G51A, followed by infection with EV71. Western blotting showed that EV71 infection cleaved the endogenous PinX1, while G51A but not PinX1 overexpression inhibited EV71 release (Fig. 7E). Our results suggested that PinX1 cleavage promoted EV71 release.

FIG 7.

PinX1 cleavage by EV71 3C facilitates EV71 release. (A and C) HeLa (A) and Jurkat (C) shRNA control and shRNA PinX1 cells were mock infected or infected with EV71 at an MOI of 5 for HeLa and 0.5 for Jurkat. Twenty-four hours postinfection, cells were processed for Western blotting with the indicated antibodies. (B and D) TCID₅₀ assay was performed to measure viral titers of supernatants from the Jurkat cells used for panels A and B, and TCID₅₀ was converted to PFU. (E) HeLa cells overexpressing PinX1 or G51A were infected with EV71. Twenty-four hours later, cell lysates and supernatant were subjected to Western blotting with the indicated antibodies.

DISCUSSION

As one of the seven nonstructural proteins, 3C plays an important role in viral protein maturation and virus-host cell interaction (33). Several host factors have been found to be cleaved by 3C (9–12). In this study, we identified PinX1 as a novel target of EV71 3C. Furthermore, PinX1 cleavage by 3C promoted apoptosis and facilitated EV71 release. Although 3C has numerous cell fate-relevant targets and its inactivation surely affects the apoptotic machinery not only due to its effect on PinX1, we concentrated on its effect on PinX1, and we confirmed that PinX1 cleavage plays a role in 3C induction of apoptosis.

Similar to the case for other 3C substrates, 3C cleaves PinX1 through its protease activity but not its RNA binding activity. Mutational analysis reveals that PinX1 cleavage occurs at the Q50-G51 pair, which results in two cleaved products. PinX1 Q51A mutation abolished cleavage, suggesting that this is a likely EV71 3C cleavage site. This cleavage site of PinX1 is localized in the G-patch domain, which has a highly conserved Gly-rich signature: hhx(3)Gax(2)GxGhGx(4)G. Thus, perhaps 3C selects host proteins that bear specific sequences as substrates.

PinX1 is a telomere binding protein and plays an essential role in telomerase activity inhibition, telomere maintenance, and regulation and control of mitosis and the cell cycle (28, 31). PinX1 is expressed in various tissues and cells, and silencing of PinX1 induces DNA damage responses and enhances sensitivity of cells to apoptosis inducers (31, 39). In this study, we found that silencing of PinX1 enhances apoptosis induced by etoposide and EV71 infection in HeLa and Jurkat cells. Because EV71 infection or overexpression of 3C could cleave PinX1, the PinX1 Q51A but not wild-type PinX1 could inhibit apoptosis induced by EV71 infection or overexpression of 3C. Furthermore, silencing of PinX1 enhanced cell apoptosis induced by EV71 infection. Therefore, we unveiled a novel function of PinX1 in this study.

Apoptosis can serve as an active defense mechanism of cells to restrict virus replication and spread, especially at the early stage. However, viruses are also able to induce apoptosis to benefit viral assembly, release, and spread at the late stage (40, 41). For example, HIV-1 Vpr protein induces apoptosis via targeting the mitochondrial permeability transition pore, and this might benefit the depletion of CD4⁺ lymphocytes (42). HBV X protein also induces apoptosis through sustained activation of cyclin B1-CDK1 kinase (43), and this is suggested to contribute to cell growth inhibition. Wang et al. showed that the virulence of coxsackie virus B3 was markedly inhibited when apoptosis was inhibited (44). Xi et al. also demonstrated that the inhibition of apoptosis prevents EV71 viral particle release (45), and our data also confirmed this finding. In this study, we found that EV71 3C induced apoptosis through cleaving PinX1 and that knockdown of PinX1 promotes apoptosis, which facilitates EV71 release. Once apoptosis was inhibited by PinX1 Q51A, EV71 release was inhibited as well. Our study suggests that EV71 3C-triggered apoptosis is an important strategy employed by EV71 to boost viral release and spread.

In conclusion, we find that PinX1, a telomere binding protein, is a new target of EV71 3C cleavage. The cleavage promotes EV71-induced apoptosis and viral release from infected cells.

MATERIALS AND METHODS

Cell lines and viruses.

Adherent (HEK293T, HeLa, and RD) and suspension (Jurkat) cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (high glucose; Gibco) or RPMI 1640 (HyClone) supplemented with 10% heated-inactivated fetal bovine serum (FBS) (HyClone) and 100 U/ml penicillin-streptomycin (GIBCO/BRL) at 37°C in a 5% CO₂ humidified atmosphere. EV71 was recovered from pSVA-EV71. Briefly, the EV71 infectious clone was linearized with SalI, and the resultant linearized DNA was in vitro transcribed to RNA, which subsequently was transfected into RD cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Viruses were propagated in RD cells, and the titers of viruses were measured using a 50% tissue culture infective dose (TCID₅₀) assay. EV71 at the indicated multiplicity of infection (MOI) was incubated with cells in DMEM without FBS for 1 h, and the medium was then replaced with DMEM containing 2% FBS.

Plasmids and transfection.

PinX1 cDNA obtained by reverse transcription-PCR from HeLa total RNA was inserted into pCMV-Tag-2B (Stratagene). The EV71 infectious clone pSVA-EV71 was a gift from Zhiyong Lou (Tsinghua University). The plasmids expressing C147S (a 3C variant with impaired protease activity), R84Q (a 3C variant with impaired RNA binding activity), and PinX1 variants G51A and S268A were generated by site-directed mutagenesis (QuikChange; Stratagene). PinX1, 3C, and 3C variants were all subcloned into the indicated vectors, including pQCXIP-2.0 (reconstructed from pQCXIP [Clontech]) and pEGFP-C3 (Clontech). For E. coli expression, PinX1, 3C, and C147S cDNAs were inserted into prokaryotic expression vector pET-H (reconstructed from pET-32a [Novagen]) and pGEX-6P-1 (GE Healthcare). HeLa, HEK293T, and RD cells were transfected with polyethyleneimine (PEI) (Polysciences) or Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's instructions.

Yeast two-hybrid screening.

A fragment encoding EV71 3C was cloned into pGBKT7 (Clontech) and used as the bait. The screening was performed using a human universal cDNA library. The Matchmaker GAL4 two-hybrid system 3 (PT3247-1) was used according to the manufacturer's instructions (Clontech).

Preparation and purification of recombinant proteins.

To express recombinant PinX1 and 3C, pET-H-3C, pET-H-PinX1, or pGEX-6P-1-PinX1 was transformed into competent E. coli BL21(DE3), and protein expression was induced by treatment with 200 μM IPTG (isopropyl-β-d-thiogalactopyranoside) at 16°C overnight. His-tagged or GST-tagged recombinant proteins were purified by affinity chromatography.

Generation of PinX1 knockdown cell lines.

To construct PinX1 shRNA cell lines, three pairs of oligonucleotides targeting PinX1 were designed by shRNA Sequence Designer (Clontech). Double-stranded oligonucleotides corresponding to the target sequences were synthesized, annealed, and cloned into pSIREN-RetroQ (Clontech). The pairs of targeting oligonucleotides were as follows: 5′-GATCCAGAAGATGGGGTGGTCTAATTCAAGAGATTAGACCACCCCATCTTCTTTTTTTACGCGTG-3′ and 5′-AATTCACGCGTAAAAAAAGAAGATGGGGTGGTCTAATCTCTTGAATTAGACCACCCCATCTTCTG-3′, 5′-GATCCGCAAGGAGCCACAGATCATATTCAAGAGATATGATCTGTGGCTCCTTGTTTTTTACGCGTG-3′ and 5′-AATTCACGCGTAAAAAACAAGGAGCCACAGATCATATCTCTTGAATATGATCTGTGGCTCCTTGCG-3′, and 5′-GATCCGCTCGGAGCTACCATCAATATTCAAGAGATATTGATGGTAGCTCCGAGTTTTTTACGCGTG-3′ and 5′-AATTCACGCGTAAAAAACTCGGAGCTACCATCAATATCTCTTGAATATTGATGGTAGCTCCGAGCG-3′.

Retrovirus particles were prepared by transfecting HEK293T cells with 1 μg pMLV-Gag-Pol, 0.5 μg pVSV-G, and 1 μg pSIREN-RetroQ DNA constructs. After 48 h, supernatants were collected and centrifuged at 3,000 rpm to remove cell debris. HeLa or Jurkat cells were infected with the harvested virus particles in the presence of 5 μg/ml Polybrene by spinoculation at 450 × g for 30 min at room temperature. Forty-eight hours after infection, cells were subcultured in selection medium containing 2 μg/ml puromycin (Sigma). Knockdown efficiency was confirmed by Western blotting using specific antibodies.

Western blotting.

Cell lysates or immunoprecipitated materials were subjected to SDS-PAGE (10% or 12% polyacrylamide) and then transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare). The membranes were blocked in 5% nonfat milk (in 1× phosphate-buffered saline [PBS]) for at least 45 min at room temperature. The proteins were probed with primary antibodies overnight at 4°C. After incubation with appropriate secondary antibodies, the membranes were treated with enhanced chemiluminescence reagents (Millipore), and the protein signals were detected by exposure to X-ray films.

Coimmunoprecipitation.

Transfected cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl buffer [pH 7.4] containing 150 mM NaCl, 1% NP-40, and 0.25% sodium deoxycholate) containing protease inhibitor cocktail (Roche, Indianapolis, IN). Lysates of cells were incubated with indicated antibody (Sigma, St. Louis, MO) in 500 μl RIPA buffer at 4°C overnight on a rotator in the presence of protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). The immunocomplex captured on the protein A-agarose was fractionated by 10 to 12% SDS-PAGE and subjected to Western blotting.

GST pulldown assay.

Twenty micrograms of GST or GST-PinX1 recombinant protein produced by E. coli expression was incubated in 600 μl PBS at 4°C for 2 h with glutathione-Sepharose 4B beads (GE Healthcare) pretreated with prechilled PBS three times. Unbound protein was washed away using PBS and washing buffer. Ten micrograms of His-3C recombinant protein was added to the tubes and incubated in 600 μl PBS at 4°C for 1 h. After washing 6 times using wash buffer, the complex captured was subjected to Western blotting.

In vitro cleavage assays.

Recombinant EV71 3C^pro protease was produced with an E. coli expression system. To examine PinX1 cleavage in vitro, aliquots of recombinant His-3C and His-PinX1 or lysates of cells overexpressing PinX1 were incubated at 30°C in 50 mM Tris-HCl buffer (pH 7.0) containing 200 mM NaCl. After 2 h, samples were subjected to Western blotting.

MTS assay.

shRNA control or shRNA PINX1 cells in 96-well plates were treated with increasing dose of etoposide. Twenty-four hours later, 20 μl MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] reagent (Promega) was added to the treated cells at left at 37°C in a 5% CO₂ humidified atmosphere for 1 to 4 h. The optical density at 490 nm (OD₄₉₀) of the cell culture was detected as cell viability, and the data were analyzed using the GraphPad Prism software.

Annexin V-FITC-PI apoptosis detection assay.

Treated cells were washed twice in ice-cold PBS and suspended in ice-cold 1× binding buffer. To the cell suspensions were added 3 μl of the annexin conjugate ApopNexin-FITC and 2 μl of 100× PI, and the mixtures were incubated for 15 min at room temperature in the dark. Double-staining cells were analyzed with a fluorescence microscope.

Statistical analysis.

Data were expressed as the mean ± standard deviation (SD) from three independent experiments, in which each assay was performed in triplicate. Data were compared using the unpaired two-tailed t test. A P value of <0.05 was considered significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant).