3C protease of enterovirus 71 cleaves promyelocytic leukemia protein and impairs PML-NBs production

HEK293T and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (high glucose; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA). HeLa-Scrambled^KO and HeLa-PML^KO cells were also cultured in the same medium supplemented with puromycin (5 μg/mL). All cells were cultured at 37 °C in a 5% CO₂ container. EV71 was recovered from pSVA-EV71 infectious clone (strain SK-EV006/Malaysia/97, Accession code: AB469182.1), gifted by Professor Zhiyong Lou (Tsinghua University, Beijing, China). 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, then the cell supernatants were collected by centrifugation at 3000×g for 5 min, and the target viruses were stored at − 80 °C. The titers were measured by median tissue culture infective dose (TCID₅₀) assay. To the point, seeded 1 × 10⁴ RD cells per well in 96-well plates; after overnight culture, the viruses were serially diluted 10-fold with DMEM containing 10% FBS (10^–1- to 10^–8-fold dilutions) and added to RD cell, the plates were then incubated at 37 °C in 5% CO₂; cytopathic effects (CPE) was observed under the microscope after 3 to 4 days post infection; calculated the TCID₅₀ of EV71 virus. 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.

pRK5-Flag series plasmids (Flag-PMLI–Flag-PMLVI) were kindly provided by Professor Jun Tang (State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, China). pRK5-Flag vector plasmid was generated from pRK5-Flag-PMLI in our laboratory. The plasmids expressing Myc-3C (E71A), with impaired proteolytic activity, Myc-3C (R84Q) and Myc-3C (V154Q), with impaired RNA-binding activity, were generated using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) based on pQCXIP-Myc-3C, which expressed wildtype EV71 3C protein with a Myc tag on its C-terminal. Briefly, prepared the needed segments and added into the tubes in a certain dose according to the instruction, then run the thermal cycle reaction for 16 cycles, and finally placed the reaction on ice for 2 min to cool the reaction. The vector pQCXIP-Myc was reconstructed from pQCXIP (Clontech) in our laboratory. pRK5-Flag-PMLVI variants (Q430A, Q444A) were also generated by the same method based on pRK5-Flag-PMLVI. pRK5-Flag-PMLVI (Q430A/Q444A) was generated based on pRK5-Flag-PMLVI (Q444A). The sequence of QuikChange Primer used were as follows: 5′-GAACAAGGGGTTAATTTGGCGCTAACCCTAATC, and 5′-GATTAGGGTTAGCGCCAAATTAACCCCTTGTTC for Myc-3C (E71A), 5′-CTAATGAGAAATTCCAAGACATTACTAAGTTC, and 5′-GAACTTAGTAATGTCTTGGAATTTCTCATTAG for Myc-3C (R84Q), 5′-GTGGTGACATCATCTGGAAAGGTCATTG, and 5′-CAATGACCTTTCCAGATGATGTCACCAC for Myc-3C (V154Q), 5′-GTGAAGGCCCAGGTTGCGGCCCTGGGGCTGGCTGAAG, and 5′-CTTCAGCCAGCCCCAGGGCCGCAACCTGGGCCTTCAC for PMLVI (Q430A), 5′-GCCCAGCCTATGGCTGTGGTAGCGTCAGTGCCCGGGGCACACCCCGTG, and 5′-CACGGGGTGTGCCCCGGGCACTGACGCTACCACAGCCATAGGCTGGGC for PMLVI (Q444A). Plasmid transfection was performed using the polyethyleneimine (PEI) reagent (Polyscience, Niles, IL, USA) following the manufacturer's instructions.

Transfected cells were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) on ice for 10 min. Lysates 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, USA). The immunocomplex captured on the protein A-agarose was fractionated by 10% or 12% SDS-PAGE and subjected to Western blotting.

HeLa cells were seeded onto 22 mm diameter coverslips and harvested at 36 h post-transfection. The cells were washed two times with PBS, fixed with 4% paraformaldehyde diluted by PBS at room temperature for 10 min, and permeabilized using 0.1% Triton X-100 for 10 min at room temperature. Then cells were incubated in a blocking solution (5% bovine serum albumin, BSA in PBS) for 1 h at room temperature and washed with PBS. The cells were then incubated at room temperature with anti-Flag (1:200) or anti-Myc (1:200) diluted in PBS containing 0.1% BSA and 0.02% sodium azide for 2 h. After they had been washed three times with PBS containing 0.1% Tween 20® (PBS-T), the cells were incubated with fluorescein (FITC)-conjugated AffiniPure Goat Anti-Mouse IgG (diluted 1:500) or Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-Rabbit IgG (diluted 1:500) for 1 h at room temperature. Finally, the cells were stained with DAPI (diluted 1:2000 in PBS) and mounted with Antifade Mounting Medium (Beyotime, Shanghai, China) for confocal microscopy analysis. The images were captured by Leica TCS SP5 Confocal system, with 488 nm and 550 nm laser lines.

Promyelocytic leukemia (PML) protein has seven isoforms, which consist mainly of six nuclear isoforms, including NLS designated PMLI to PMLVI, and one cytoplasmic isoform, the shortest PMLVIIb (Fig. 1A). It has been reported that enterovirus 71 3C protease could mediate PMLIII and IV degradation instead of 2A protease [28]. Considered that EV71 3C protein was mainly functioned as a protease to cleave virus precursor protein and multiple host proteins, we hypothesized that EV71 3C might cleave nuclear PML isoforms which are major organizers of PML-NBs. HEK293T cells were transfected with Flag-PMLI–VI separately along with Myc-3C or Myc-3C (E71A), a mutant that had been proved to abolish the protease activity only. At 36 h after the transfection, the cells were harvested to imply Western blotting assays. As shown in Fig. 1B, compared with 3C (E71A), EV71 3C reduced all the six nuclear isoforms of PML and yield low molecular weight products, approximately 60 kDa, specifically. These results support that the decrease of PML caused by 3C protease cleaved PML.

Now that EV71 3C might cleave all the six nuclear PML isoforms, we want to determine whether this cleavage function is specific between 3C protease and PML exactly. The previous study has demonstrated that cellular PMLIII and PMLIV could suppress EV71 infection effectively [28]. Therefore, we chose PMLIII and PMLIV and transfected them together with increasing amounts of Myc-3C in HEK293T cells, while 3C E71A mutant was the negative control. It was clear to see that both full-length proteins gradually decreased with the gradual increase of 3C concentration (Fig. 2A, B), which means the cleavage was conducted in a protease dose-dependent manner. 3C protease of enterovirus 71 also possesses RNA-binding activity, and a previous study indicated that mutations in the RNA-binding regions (amino acid positions 82–86 and 154–156) could influence 3C proteolytic activity [15]. So next, we checked whether RNA-binding activity would influence cleavage. The Western blotting assays showed that EV71 3C R84Q and V154Q mutants could still cleave PMLIII and PMLIV, which is distinct from E71A mutant obviously (Fig. 2C, D). This result implied that R84Q and V154Q did not affect protease function, and proteolytic activity but not RNA-binding activity of EV71 3C was vital to PML cleavage.

Since 3C could mediate the reduction of PMLIV dramatically, we wondered whether the proteasome pathway and lysosomal pathway participated in this degradation. Still, HEK293T cells were transfected with PMLIV and Myc-3C, Myc-3C (E71A), or vector. Different from the former experiments, MG-132 (proteasome/calpain inhibitor) and Chloroquine (CQ, autophagy-lysosome inhibitor) were applied as the experimental group. DMSO was the organic dissolvent of the two drugs and was used as another negative control. From Fig. 2E, F, we observed that neither MG-132 nor CQ could impair the cleavage because the reduction of PMLIV and ~ 60 kDa products still existed. Last, we confirmed the interaction of EV71 3C protease and PMLIV in host cells by transfection of HEK293T cells with Myc-3C or Myc-3C (E71A) and Flag-PMLIV, followed by coimmunoprecipitation with an anti-Flag or anti-Myc antibody. The 3C protease was coimmunoprecipitated with PMLIV and vice versa (Fig. 2G, H). Altogether, EV71 3C protease cleaved PMLIV directly and, specifically, irrelevant of its RNA-binding activity and proteasome or lysosomal pathway in host cells.

To clarify the cleavage specificity, we identified the definite cleavage site(s) of EV71 3C in PMLIV. Firstly, we analyzed the potential site(s) according to its amino acid sequence (NP_002666.1) (Fig. 3A). As shown in the cleavage assay earlier (Fig. 1B), all six PML nuclear isoforms share the same cleaved products with an approximate molecular weight of 60 kDa, indicating that the cleavage site(s) might be located in the conservative regions of PML. We also noticed PMLIV was composed of 634 amino acids and the size of the cleavage was about two-thirds of the total molecular weight. Therefore, we predict the site(s) was located between 400aa–440aa. On the other hand, enterovirus 3C protease had Q–X (X was mostly referenced to G or S or A) sequence preference according to previous research [29], and we found Q430–A431 and Q444–S445 in the predicted area. The pRK5-Flag-PMLIV (Q430A) mutant and pRK5-Flag-PMLIV (Q444A) mutant were constructed to characterize these two amino acids. HEK293T cells were transfected with PMLIV wild type or mutants along with Myc-3C or Myc-3C (E71A) for 36 h. The Western blotting result was shown in Fig. 3B, and we surprisingly found that both were still be cleaved by EV71 3C protease. However, either of the PMLIV mutants could not be cleaved completely. Then we postulated logically that both sites were responsible for 3C protease cleavage. The pRK5-Flag-PMLIV (Q430A/Q444A) dual-mutation variant plasmid was constructed and transfected with Myc-3C or Myc-3C (E71A) in HEK293T cells. As demonstrated in Fig. 3C, the cleavage blotting sizing about 60 kDa disappeared, and the PMLIV (Q430A/Q444A) dual-mutation variant was resistant to cleavage totally, illustrating that both Q430–A431 and Q444–S445 were the cleavage sites of 3C in PMLIV (Fig. 3D). This evidence also elucidated why we detected only one cleaved blotting of PMLIV using an anti-Flag antibody. The two cleavage sites are too closed to show only one blotting in the Western blotting assay. We also applied coimmunoprecipitation using an anti-Flag antibody and confirmed that amino acid mutation could not affect the interaction between 3C protease and PMLIV (Fig. 3E). Collectively, these results suggested 3C protease cleaved PMLIV at Q430–A431 and Q444–S445 sites.

To further investigate the impact of PMLIV cleavage by EV71 3C protease, we set virus infection experiment. The wild-type PMLIV or Q430A/Q444A dual-mutation variant was transfected into PML knockout cell lines (HeLa-PML^KO) and non-knockout cell lines (HeLa-Scrambled^KO). Vector was transfected as control. 24 h after transfection, all cells were infected with EV71 at the multiplicity of infection (MOI) of 1 for 8 h. Then Western blotting was applied using an anti-VP1 antibody to assess virus replication. As shown in Fig. 4, at the same conditions, knockout of PML renders the cells more susceptible to EV71 infection (VP1 blot band in lane 1 was bolder than lane 4, which was the same condition in lane 2 and5, 3 and 6). Meanwhile, EV71 replication had a lower level in PMLIV dual-mutation-variant-expressed cells than wildtype-expressed cells (comparing VP1 blot bands in lane 3 and 2, 6 and 5), suggesting that 3C protease cleaved PMLIV to antagonize its antiviral activity in favor of viral replication; however, 3C could not cleave PMLIV dual-mutation variant, which had stronger antiviral potential.

Given that PML functions as a major organizer of PML nuclear bodies (NBs) in most mammalian cells and plays an important role in antiviral activities [30], we applied immunofluorescence analysis to inspect the interfere of 3C protease to PML-NBs directly (Fig. 5). Both PMLIV and PMLIV (Q430A/Q444A) dual-mutant could form normal PML-NBs. While, EV71 3C protein and its mutant were localized in both nucleus and cytoplasm, but more in the nucleus with forming of dots. When 3C protease and PMLIV were co-transfected in HeLa cells, the large clustered spots turned into smaller dots which means PML-NBs were disintegrated by 3C protease. However, this phenomenon was not observed when we co-transfected either PMLIV and 3C (E71A) or PMLIV (Q430A/Q444A) dual-mutation variant and 3C. Overall, our findings suggest that 3C might impair PML-NBs production via PMLIV cleavage and thereby counter its antiviral activities.