1. Introduction
Encephalomyocarditis virus (EMCV) is a small non-enveloped single-stranded RNA virus, which can cause myocarditis, encephalitis, neurological diseases, reproductive disorders, and diabetes in several mammalian species [
1]. The genome of EMCV is approximately 7.8 kb and encodes a polyprotein. The EMCV 2A protein is a small protein of approximately 17 kDa and is considered an important virulence protein [
1,
2]. Previous studies have shown that the 2A protein plays an important role in inhibiting protein synthesis and apoptosis in host cells [
1]. It was reported that BHK21 cells infected by EMCV with the deletion of 2A causes apoptosis through caspase 3 activation [
3], and it was suggested that the 2A protein is required for the inhibition of apoptosis. However, the specific molecular mechanisms by which the 2A protein inhibits apoptosis remain unclear. In this study, we demonstrated that the EMCV 2A protein inhibits apoptosis, and annexin A2 of the host cells plays an important role in the mechanism of 2A protein inhibiting apoptosis.
Annexin A2 is a 36 kDa protein and contains three distinct functional regions, including the N terminus, the core domain, and the C terminus [
4]. Annexin A2 exists in cells in two forms. One is a monomer that mainly exists in the cytoplasm [
5] and participates in the assembly, dissolution, and repair of intracellular organelle membranes [
6]. The other is a heterotetramer which mainly exists on the surface of cell membranes, and is formed by two molecules of annexin A2 and two molecules of P11 [
5]. Annexin A2 has been implicated in multiple diseases, immune function, and viral infection. Previous studies have shown that it can regulate cell cycles by inhibiting apoptosis. In non-small-cell lung cancer (NSCLC) cells, annexin A2 activated JNK/c-Jun signaling, which, in turn, led to a decrease in p53 transcription [
7]. Our research demonstrates that the 2A protein inhibits apoptosis by interacting with annexin A2 via the activation of the JNK/c-Jun pathway during the early stage of EMCV replication in BHK21 cells.
2. Methods
2.1. Cells, Plasmids and Viruses
PK15 and BHK21 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA), supplemented with 5% and 8% heat-inactivated fetal bovine serum (FBS) (Gibco, California USA) respectively, at 37 °C in a 5 v/v CO2 humidified atmosphere. The EMCV-HB10 strain (GenBank accession number: JQ864080.1) and pcDNA3.1-2A plasmid were kept in laboratory.
2.2. Prokaryotic Expression of 2A Protein
Using the pcDN3.1-2A plasmid as a template, the 2A gene was amplified using the PCR primer pair His-2A (
Table 1). Recombinant plasmid His-2A was constructed by cloning the 2A gene into the pET-28a-sumo vector. The His-2A plasmid was transformed into
E. coli BL21 (DE3) cells to obtain the recombinant fusion protein His-2A. The His-2A protein was purified using His-tag protein purification beads. The purified recombinant fusion protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
2.3. His Pull Down Assay
PK15 cells cultured in flasks were washed twice with PBS. A lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and protease inhibitor were added. After incubating the cells at 4 °C for 30 min, the lysed cells were centrifuged at 3000× g for 15 min at 4 °C. The supernatant was harvested for His pull-down assay. The purified His-2A protein was incubated with the supernatant at 4 °C overnight. After incubation, the mixture was conjugated to His-tag beads for 1.5 h at room temperature. The beads were washed twice with washing buffer. Then, the His-2A protein and interacting proteins were eluted with elution buffer containing 20 mM phosphate buffer, 500 mM NaCl, and 500 mM imidazole, followed by the detection of the proteins by SDS-PAGE.
2.4. Mass Spectrometry
Binding specificity to the 2A protein by SDS-PAGE was analyzed by mass spectrometry. Protein slices in fresh CCB-stained gel were excised, destained twice with 50 v/v acetonitrile and 50 mM NH4HCO3, and dried with acetonitrile three times. Then, the dried gel slices were incubated in ice-cold digestion solution (20 mM NH4HCO3 and trypsin 12.5 ng μL−1) at 37 °C overnight. Finally, peptides in the supernatant were collected after extraction twice with extract solution (5 v/v formic acid in 50 v/v acetonitrile). The peptides collected in the previous step were analyzed by Nano-HPLC-MS/MS. Tandem mass spectra were extracted by Proteome Discoverer software (version 2.4; Thermo Fisher Scientific, Waltham, MA, USA). Peptide confidence was set to high, and peptide ion score was set to >20.
2.5. Localization of 2A
The eukaryotic expression plasmid, pEGFP-2A, was transfected into PK15 cells with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and cultured at 37 °C for 48 h. The cell membranes were labeled with DiI stain (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate) (C1036; Beyotime, Shanghai, China), and the cell nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole) (F6057; Sigma-Aldrich, St. Louis, MO, USA).
2.6. Flow Cytometry
The eukaryotic expression plasmid, pcDNA3.1-2A, was transfected into PK15 cells treated with kevetrin hydrochloride (KH, product number T3184; TargetMol, Shanghai, China). KH-treated and untreated cells were used as positive and negative controls, respectively. Apoptosis of cells was detected using an ANNEXINV-FITC/PI apoptosis detection kit (CA1020; Solarbio, Beijing, China). First, cells were washed with cold PBS and resuspended in 1× binding buffer. Next, annexin V-FITC was added into the suspension at room temperature for 10 min in the dark. Then, PI was added by incubation for 5 min. Finally, cell apoptosis was evaluated using the FACSVerse flow cytometer. The annexin V+ plus annexin V+ PI+ population was gated for apoptosis analysis, and at least 1 × 104 cells event−1 were evaluated for each analysis.
2.7. Caspase 3 Activity Detection
The eukaryotic expression plasmid, pcDNA3.1-2A, was transfected into PK15 cells. Apoptosis of cells was detected using a Caspase 3 Activity Assay kit (C1115; Beyotime, Shanghai, China). Cells were lysed with lysis buffer at 0 °C for 15 min. And then the lysates were centrifuged at 15,000× g for 15 min at 4 °C. The protein concentrations in the supernatant were determined by BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts (30 μg) were incubated in a 96-well microtitre plate with 20 ng Ac-DEVD-pNA for 2 h at 37 °C. Caspase 3 activity was measured by cleavage of the Ac-DEVD-pNA subsatrate to pNA, the absorbance of which was measured at 405 nm. Relative caspase activity was calculated as the ratio of emission of treated cells to untreated cells.
2.8. Confocal Imaging and Co-IP
The pDsRed-ANXA2 and HA-ANXA2 plasmids encoding annexin A2 (GenBank accession number XM_013992912.2) and the pEGFP-2A and Flag-2A plasmids were constructed (
Table 1). To further identify interactions involving 2A and annexin A2 proteins, the appropriate plasmids were transfected into PK15 cells using Lipofectamine 2000. After 48 h, cells analyzed for confocal microscopy and cell nuclei were labeled with DAPI.
PK15 cells were transfected with the HA-ANXA2 and Flag-2A plasmids. After washing with cold PBS, cells were lysed with IP lysis buffer (26149; Thermo Pierce, Shanghai, China), PMSF, and protease inhibitor cocktail (26149; Thermo Pierce, Shanghai, China) at 4 °C for 1 h. The obtained cellular proteins were pre-cleared with protein A/G beads (26149; Thermo Pierce, Shanghai, China) and incubated with protein A/G beads plus and rabbit anti-HA-specific (C29F4; Cell Signaling Technology, Boston, USA) antibody at 4 °C overnight. Then, the beads were washed with elution buffer and the eluted proteins were analyzed by SDS-PAGE. Finally, immunoblotting analysis of the interacting proteins was performed with mouse anti-Flag (F1804; Sigma-aldrich, St. Louis, MO, USA) pcAb and HRP-conjugated goat anti-mouse secondary antibodies.
2.9. siRNA Interference
siRNAs specific for annexin A2 were designed and synthesized by Sangon Biotech (
Table 2) (employing the Rosetta algorithm in their pipeline to design siRNAs; National Center for Biotechnology Information (NCBI) BLAST was used for off-target analysis). The final concentration of the siRNAs used were 100 pM. A non-targeting siRNA was used as the negative control. The siRNAs were transfected into cells by Lipofectamine 2000. The cells (5 × 10
6 cells mL
−1) were treated with siRNA1 for 24 h, and pcDNA3.1-2A at different concentrations were transfected in the cells; the results were observed after 24 h. Kevetrin hydrochloride-treated (10 mM) cells were used as a positive control.
2.10. Replication Kinetics of EMCV
The titers of EMCV HB10 were determined by endpoint dilution assays, and the 50% cell culture infection dose (TCID50) was calculated to determine the replication kinetics of the virus in BHK21 cells. Briefly, cells were seeded onto 96-well microtiter plates and infected with 10-fold serial dilutions of EMCV (1:10−1 to 1:10−8) separately after siRNA treatment for 24 h. The cytopathic effect (CPE) of the cultures was observed at different time intervals up to 48 h post infection (hp.i.). Assays were performed in triplicate. Each data point represents the average of three independent experiments.
2.11. RNA Extraction and Quantitative Real-Time PCR
The mRNAs of cells were obtained by the Cell RNA Rapid Extraction Kit (Aidlab, Beijing, China). Fastking one-step genomic cDNA first strand synthesis Kit (TIANGEN, Beijing, China) was used for reverse transcription. Quantitative real-time PCR assay (RT-PCR) was performed using Universal SYBR
® qPCR (Vazyme, Nanjing, China) on the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA). The relative expression levels of mRNAs were analyzed using the 2
−ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal standard for quantitative analysis of mRNAs. The primers used in the RT-PCR assay are listed in
Table 3, and all reactions were performed in triplicate.
2.12. Western Blot
The cells were lysed with lysis buffer (Thermo Fisher Scientific), PMSF, and a protease inhibitor cocktail (Thermo Fisher Scientific) at 4 °C for 30 min. Then, the lysates were centrifuged at 3000× g for 15 min at 4 °C. The protein concentrations in the supernatant were determined by BCA protein assay (Thermo Fisher Scientific). Protein extracts (40 μg) were detected by SDS-PAGE and western blot. Polyvinylidene fluoride membranes (PVDF) (Millipore A) were blocked with 5% nonfat milk in TBST at room temperature for 1 h. In turn, the membranes were immunoblotted with primary antibodies at room temperature for 2 h and incubated with secondary horseradish peroxidase-conjugated antibody at room temperature for 1 h. Finally, protein bands were detected by the enhanced chemiluminescence western blot detection kit (Solarbio, Beijing, China). The monoclonal rabbit anti-mouse caspase-3, caspase-8, caspase-9, p-c-Jun (Affinity), and p53, JNK (phospho-Thr183/Tyr185) Abs (Sangon Biotech, Shanghai, China) and the monoclonal goat anti-mouse β-actin Abs (Beyotime, Shanghai, China) were used.
2.13. Statistical Analyses
Data are reported as means ± SD of 3 independent experiments and analyzed using GraphPadTM Prism software (version 6; GraphPad Software, San Diego, CA, USA).
4. Discussion
EMCV is a small non-enveloped single-stranded RNA virus that belongs to the
Picornaviridae (pico = small, RNA = ribonucleic acid) family. It can cause myocarditis, encephalitis, and neurological diseases in many mammalian species, and even lead to reproductive disorders and diabetes [
1]. The genome of the virus is approximately 7.8 kb, including three unique domains: the VPg protein binding sequence, a poly (c) region, and an internal ribosome entry site (IRES). The open reading frame (ORF) of the EMCV genome encodes a large polyprotein (L-1ABCD-2ABC-3ABCD). The 2A protein is a nonstructural viral protein (approximately 17 kDa and 143 amino acids), and it is an important virulence factor of EMCV [
1]. The present study was conducted to enlighten the underlined mechanism associated with EMCV pathogenesis. We specifically targeted the 2A protein, because this protein plays an important role in the pathogenesis of EMCV by different mechanisms. One of most important mechanisms is to break host innate immune response by inhibiting the EMCV infected host cells apoptosis. This protein can also competitively inhibit the synthesis of host protein. Involvement of the 2A protein in host cell apoptosis has already been studied and proven by many researchers [
3,
11]. However, the underlined, specific mechanism of the 2A protein to inhibit apoptosis is unclear, and it is not related to the ability to shut off codependent translation [
3].
Our results indicated that the 2A protein of EMCV strain HB10 inhibits the apoptosis of PK15 cells as indicated by flow cytometry and caspase 3 activity assay kit. Our results are in agreement with Carocci’s [
3]. The inhibition of host cell apoptosis by the 2A protein is complemented by annexin A2. Annexin A2, a calcium dependent phospholipid binding protein, is involved in many membrane-related events, such as proliferation, cell-cell adhesion, exocytosis, and endocytosis [
12,
13,
14,
15]. Studies have shown that annexin A2 is closely related to the regulation of apoptosis induced by viral infection [
16]. Ma et al. confirmed that the NS1 protein of the highly pathogenic H5N1 avian influenza virus interacts with the annexin A2 protein to inhibit apoptosis [
8]. Ying et al. showed that the GroEL protein of Mycoplasma gallisepticum induces apoptosis in host cells by interacting with annexin A2 [
17]. In our research, results of flow cytometry exhibited that the inhibition of apoptosis by 2A protein was reduced, when annexin A2 expression was inhibited by siRNA. This data indicates that the interaction between annexin A2 and the 2A protein is necessary for apoptosis inhibition by the 2A protein and subsequent EMCV pathogenesis.
By comparing the changes of apoptosis pathway factors under different expression levels of annexin A2 and the 2A protein, the results showed that the 2A protein can promote the increase of c-Jun and inhibit the synthesis of p53. In other words, the 2A protein inhibited apoptosis by interacting with annexin A2 via JNK/c-Jun pathway. During EMCV infecting BHK21 cells, the increase of c-Jun and the degradation of p53 showed that EMCV inhibited apoptosis through JNK/c-Jun pathway in the early stage of infection. It is proved that in the early replication stage of EMCV, the interaction between the 2A protein and annexin A2 inhibits apoptosis to promote virus reproduction. C-Jun N-terminal kinase (JNK) can regulate apoptosis, which is a subfamily of mitogen-activated protein kinases (MAPK). Studies indicate that JNK plays an antiapoptotic role in regulating the survival of cancer cell. The JNK/c-Jun pathway facilitates the invasion of triple negative breast cancer (TNBC) cells [
18]. In addition, annexin A2 conducts roles in p53 induced apoptosis in non-small cell lung cancer (NSCLC), it can negatively regulate p53 mRNA expression by activating JNK [
7,
19]. Our results demonstrate for the first time that annexin A2 is involved in viral regulation of apoptosis through JNK/c-Jun molecular pathway in the early stage of virus replication. The inhibition of apoptosis was eliminated at 36 h of infection. After 24 h, the rapid elimination of c-Jun and the increase of p53 showed that the inhibitory effect of the 2A protein on apoptosis was disappearing, and other factors were involved in accelerating apoptosis.
Furthermore, annexin A2 is closely related to multiple virus replications by affecting attachment, invasiveness, assembly, and release [
20,
21,
22]. The release mechanism of EMCV is not clear. Whether annexin A2 is involved in the release process of newly formed virions needs to be further studied. In addition, whether the decrease in viral titers observed in this study related to the effect of annexin A2 on virus release needs more research to prove. At present, most antiviral drugs are designed to target virus structure and enzymes, which is easy to produce drug resistance. Targeting virus receptors on host cells is an alternative strategy to solve this problem, which can play an antiviral role in the early stage of virus infection. Compounds or short peptides targeting annexin A2 monomers may become the direction of antiviral therapy in the future.
In conclusion, the key finding of our study was the identification of cellular annexin A2 as a novel interaction partner of the 2A protein during inhibition of apoptosis. These results confirmed that the 2A protein could protect EMCV from cellular defense mechanisms by inhibiting cell apoptosis and promoting the spread of virus particles, which is closely related to the pathogenicity of the virus. The mode and molecular mechanisms involving EMCV 2A protein interactions with annexin A2 are still unknown and require further study; exploring these aspects will provide valuable insights for the development of new antiviral target drugs.