HSP27 Protein Dampens Encephalomyocarditis Virus Replication by Stabilizing Melanoma Differentiation-Associated Gene 5

Heat shock proteins (HSPs) are a protein family that respond to physiological stress, such as heat, starvation, and infection. As cellular protein chaperones, they play an important role in protein folding, assembly, and degradation. Though it is well known that HSP27 is involved in a range of viral infections, its role during an encephalomyocarditis virus (EMCV) infection is not known. Here, we report that EMCV degrades HSP27 and that EMCV proteins 2Cpro and 3Apro are primarily responsible for its degradation. Consequently, loss of cellular HSP27 augmented EMCV proliferation, an effect that could be reversed upon HSP27 overexpression. Importantly, we found that HSP27 positively regulated EMCV-triggered type I interferon (IFN) production. Moreover, overexpression of 2Cpro and 3Apro significantly blocked type I IFN production. We also found for the first time that HSP27, as a molecular chaperone, can specifically interact with MDA5 and stabilize the expression of MDA5. Collectively, this study shows that HSP27 dampens EMCV infectivity by positively regulating EMCV-triggered retinoic acid-inducible gene (RIG)-I-like receptor (RLR)/melanoma differentiation-associated gene 5 (MDA5) signal pathway, while EMCV proteins 2Cpro and 3Apro interact with HSP27 and degrade HSP27 protein expression to allow EMCV proliferation. Our findings provide further mechanistic evidence for EMCV partaking in immune escape mechanisms, and that 2Cpro and 3Apro could serve as potential antiviral targets.

Heat shock proteins (HSPs) are molecular chaperones that play an important role in protein folding, assembly, and degradation (Tutar and Tutar, 2010;Ikwegbue et al., 2017;Zininga et al., 2018;Bolhassani and Agi, 2019). Many viral proteins have been shown to interact with HSP27 to regulate virus-induced autophagy, type I IFN and NF-κB signaling pathways (Ling et al., 2018;Sun et al., 2018). However, it is not known whether HSP27 plays a role in EMCV infection. We reported that HSP27 was degraded during an EMCV infection via EMCV proteins 2C pro and 3A pro . We found that HSP27, as a host factor against EMCV infection, positively regulated the production of EMCV-triggered type I IFNs. Moreover, this study shows for the first time that HSP27, as a molecular chaperone, positively regulates EMCV-triggered RLR/MDA5 signal pathway by stabilizing the expression of MDA5. This study reveals a novel role of EMCV 2C pro and 3A pro in antagonizing host antiviral responses, and elucidates the role of EMCV in modulating HSP27 function. Our findings provide further mechanistic evidence for EMCV partaking in immune escape mechanisms, and that 2C pro and 3A pro could serve as potential antiviral targets.

Virus Infection and Titration of Viral Progeny
For in vitro virus infection, the untreated A549 cells or A549 cells transfected with different plasmids were infected with EMCV at a multiplicity of infection (MOI) of 0.01 or 0.1 and cultured at 37 • C for 2 h. Then cells were washed with phosphate-buffered saline (PBS) three times, replaced with DMEM containing 3% NBS, and incubated at 37 • C for the indicated time. The cells and supernatants were repeatedly frozen and thawed to detect TCID 50 in BHK-21 cells (using the Reed-Muench method), while the cells collected separately were used for subsequent quantitative real-time polymerase chain reaction (RT-qPCR) or immunoblotting analysis.

RNA Extraction, Reverse Transcription, and Quantitative Real-Time Polymerase Chain Reaction
According to manufacturers' protocol, total RNA was extracted from cells using RNAiso Plus (Takara, 9109). Then, 1 µg of total RNA was reverse transcribed using Evo M-MLV RT kit with gDNA clean for qPCR II (Accurate biology, AG11711) to synthesis cDNA. Relative RT-qPCR was performed using the TransStart R Top Green qPCR SuperMix (+Dye II) (Transgen, AQ132-24), and target gene expression was calculated by normalization to GAPDH using the 2 − Ct method. The copy number of the polymerase 3D gene was detected by absolute RT-qPCR using the Premix Ex Taq Probe qPCR (Takara, RR390A). The probe and primer sequences used in this study are described in Table 2.

Co-immunoprecipitation and Immunoblotting
The transfected cells were lysed in RIPA or NP-40 lysis buffer containing a protease and phosphatase inhibitor cocktail (Beyotime, P1045) for 30 min on ice. And then, the cell lysates were centrifuged at 12,000 × g for 30 min at 4 • C to remove the debris. For Immunoprecipitation, the cell supernatant was incubated with corresponding antibodies at 4 • C overnight, then added Protein G Agarose (Beyotime, P2009) and incubated at 4 • C for another 3 h. The bead complexes were washed five times with ice-cold phosphate-buffered salinetween (PBST) and subjected to immunoblotting, target proteins were fractionated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck, ISEQ00010) separately, and these membranes were blocked and incubated with the corresponding primary antibodies and peroxidase affinipure goat anti-rabbit or mouse IgG (H + L). Finally, the bound protein bands were detected using Western lightning Plus-ECL kit (PerkinElmer, NEL105001EA) in membranes and visualized with Amersham Imager 600 imaging system (GE).

Statistical Analysis
Data are displayed as the mean ± standard deviation (SD) of three independent experiments. Statistical significance was determined with one-way analysis of variance (ANOVA) or Student's t-test ( * P < 0.05; * * P < 0.01; * * * P < 0.001).

Encephalomyocarditis Virus 2C pro and 3A pro Directly Bind and Degrade HSP27
Lung epithelial A549 cells were infected with EMCV (MOI = 0.1) to explore the expression profile of HSPs in EMCV-infected  The expression of HA tag and HSP27 protein were detected by immunoblotting. GAPDH was used as a loading control. Data were represented as mean ± SD of three independent experiments and were measured in technical duplicate, **P < 0.01 and ***P < 0.001.
Frontiers in Microbiology | www.frontiersin.org  The protein expression of Myc tag and VP1 were analyzed by immunoblotting. GAPDH was used as a loading control. (F) Cells and supernatant were harvested, and EMCV copy number was measured by RT-qPCR. Data were represented as mean ± SD of three independent experiments and were measured in technical duplicate, **P < 0.01 and ***P < 0.001. cells, and the dynamics of HSPs expression were evaluated over a 24 h period. Out of all HSPs tested, HSP27 expression decreased only at the protein level from 9 h post-infection (hpi) onward, suggesting that HSP27 protein expression is lost, presumably via degradation during an EMCV infection (Figures 1A-C). We hypothesize that EMCV proteins are responsible for HSP27 degradation. A549 cells were transfected with increasing doses of HA or Myc-labeled EMCV viral proteins, and endogenous HSP27 protein expression was assessed. Only viral proteins 2C pro and 3A pro reduced HSP27 protein levels (Figures 2A,B), whereas VP1 pro , VP2 pro , VP3 pro , 2A pro , and 3C pro had no effect (Supplementary Figures 1A-E). We further verified this in stable A549 cell lines engineered to express short hairpin RNA (shRNA) targeted toward viral 2C pro and 3A pro . Here, we showed that both shRNA-2C-002 and shRNA-3A-001 significantly reversed the loss of HSP27 expression during EMCV infection (Figures 2C,D). Moreover, when we attenuated the expression of 2C pro and 3A pro , respectively, the EMCV VP1 protein and viral titers were significantly reduced (Figures 2C-F), indicating that the expression of 2C pro and 3A pro are significantly positively correlated with EMCV proliferation. To see whether the proteasomal, lysosomal, or caspase-dependent apoptosis pathways play a role in 2C pro and 3A pro -mediated HSP27 degradation, we used the proteasome inhibitor MG132, the autolysosome inhibitor chloroquine (CQ), or the pan-caspase inhibitor, Z-VAD-FMK, to evaluate the inhibitory effects of 2C pro and 3A pro on HSP27 protein expression, respectively. Nearly all tested inhibitors failed to reverse the 2C pro -mediated reduction of HSP27 (Figure 2G), suggesting that the inhibitory effect of 2C pro on HSP27 was independent of these pathways. However, we found that the reduction of HSP27 protein expression by 3A pro could be reversed upon treatment with Z-VAD-FMK (Figure 2H), indicating that 3A pro -mediated HSP27 degradation is dependent on caspase activity. We then wondered whether HSP27 directly interacts with 2C pro and 3A pro . Indeed, co-immunoprecipitation (Co-IP) assays confirmed an interaction between HSP27 and 2C pro or 3A pro (Figures 3A,B). Collectively, these results show that 2C pro and 3A pro directly bind and degrade HSP27.

HSP27 Plays an Antiviral Role Against Encephalomyocarditis Virus
In light of the observation that EMCV infection and its viral proteins 2C pro and 3A pro could lead to the degradation of HSP27 suggests that HSP27 is involved in dampening EMCV infection.
To evaluate this, we designed two siRNAs targeting HSP27 ( Figure 4A). Combining both siRNAs enhanced viral growth (Figures 4B,C), corresponding to elevated VP1 expression ( Figure 4D). To further confirm the antiviral role of HSP27, A549 cells were transfected with increasing doses of an HSP27 expressing plasmid and then infected with EMCV over 24 h. We also overexpressed 2 ,5 -oligoadenylate synthase 1 (OAS1) as a positive control as OAS1 is known to inhibit EMCV proliferation (Kristiansen et al., 2010). Overexpression of either OAS1 or HSP27 reduced VP1 levels in EMCV infected cells (Figure 4E), consistent with lower EMCV copy number and titers ( Figure 4F and Supplementary Figure 2), suggesting that HSP27 can dampen EMCV proliferation. Collectively, our data show that HSP27 plays a negative regulatory role in the proliferation of EMCV.

HSP27 Positively Regulates the Production of Encephalomyocarditis Virus-Triggered Interferon-I
Given that HSP27 can dampen EMCV infectivity, we wanted to see whether this is due to HSP27 affecting EMCV-triggered IFN response. Indeed, siRNA knockdown of HSP27 reduced EMCV-triggered IFN-α and IFN-β ( Figure 5A). Consistent with this, HSP27 overexpression reversed this effect ( Figure 5B). Since HSP27 can positively regulate IFN-I production triggered by EMCV, we predicted that 2C pro and 3A pro would also impact IFN-I production. Indeed, overexpression of 2C pro and 3A pro significantly blocked type I IFN production activated by poly (I:C) stimulation, synthetic dsRNA mimic (Figures 5C,D). Altogether, these findings suggest that HSP27 interacts with the type I IFN signaling cascade.

HSP27 Enhances Encephalomyocarditis Virus-Triggered Retinoic Acid-Inducible Gene-I-Like Receptor/Melanoma Differentiation-Associated Gene 5 Signaling Pathway by Specifically Stabilizing Melanoma Differentiation-Associated Gene 5 Expression
Given that EMCV mainly activates the IFN-I via the MDA5 signaling pathway (Gitlin et al., 2006;Kato et al., 2006), we assessed whether the positive regulatory effect of HSP27 on EMCV-triggered IFN response is due to its assistance to MDA5 signaling pathway components. Indeed, HSP27 knockdown led to a parallel loss of EMCV-triggered MDA5, MAVS, TBK1 protein expression levels ( Figure 6A). Though the effect of HSP27 loss on EMCV-triggered IRF3 expression and its phosphorylation were not evident (Figure 6A), HSP27 overexpression did increase the expression of IRF3 and its phosphorylation, as well as the expression of other adaptor molecules in the MDA5 signaling pathway (Figure 6B). Consistent with the  inhibitory role of HSP27 on EMCV infectivity (Figure 4), loss of HSP27 increased EMCV viral load by enhanced VP1 expression ( Figure 6A). In summary, HSP27 positively regulates EMCVtriggered RLR/MDA5 signaling cascade. Given that MDA5, MAVS, TBK1, and IRF3 are activated by the same promoter, we then overexpressed HSP27 to verify the changes in the protein expression of endogenous MDA5, MAVS, TBK1, and IRF3 to reveal the specific targets of HSP27. Interestingly, we found that over-expression of HSP27 only significantly increased the protein expression of endogenous MDA5 and had no notable effect on the protein expression of endogenous MAVS, TBK1, and IRF3 ( Figure 7A), indicating that HSP27 specifically stabilizes MDA5 protein expression. Co-IP assays also revealed that MDA5 directly binds to HSP27 (Figure 7B). In conclusion, we show for the first time that HSP27, as a molecular chaperone, positively regulates EMCV-triggered RLR/MDA5 signal pathway by stabilizing the expression of MDA5.

DISCUSSION
As a member of the Cardiovirus genus of the Picornaviridae family, EMCV is widely used as a model virus to study the mechanism of RNA virus-mediated host immune response, viral myocarditis, and insulin-dependent diabetes (Carocci and Bakkali-Kassimi, 2012). There is a growing need to elucidate the infection mechanism of EMCV for the prevention and control of EMCV. Recent studies have shown that EMCV can use its non-structural proteins L pro , 2B pro , 2C pro , 3C pro , and structural protein VP2 pro to inhibit type I IFN responses and degrade the key adaptor molecules in the RLRs or NF-κB pathways to escape host immune response (Li et al., 1998(Li et al., , 2019Ito et al., 2012;Huang et al., 2015Huang et al., , 2017. Our results suggest that EMCV degrades the expression of HSP27 in host cells to evade the host immune response via its proteins, 2C and 3A. Studies have shown that many members of the HSP family can affect the proliferation of a range of viruses by participating in cell growth, apoptosis, transcriptional regulation, carcinogenesis, autophagy, and innate immunity of the host (Arrigo et al., 2007;Ikwegbue et al., 2017;Zininga et al., 2018;Bolhassani and Agi, 2019;Yun et al., 2019). As one of the important members of this family of proteins, HSP27 has attracted much attention in recent years for its role in innate immune defense, viral proliferation, tumor immunity, and other immunoregulatory processes (Kumano et al., 2012;Rajaiya et al., 2012;Tong et al., 2013;Arrigo, 2017;Sun et al., 2017Sun et al., , 2018Ling et al., 2018). The non-structural protein NS5A of classical swine fever virus (CSFV) can directly interact with HSP27 and thus affect the proliferation of the virus by promoting the activation of the NF-κB signaling pathway (Ling et al., 2018). It has also been suggested that HSP27 is an intracellular anti-porcine epidemic diarrhea virus factor dependent on the IFN-β signaling pathway (Sun et al., 2017). We found that overexpression of HSP27 can promote the expression of MDA5, MAVS, TBK1, and IRF3 during EMCV infection ( Figure 6B), but the increased expression of MDA5, MAVS, TBK1, and IRF3 may be due to the expression of a certain plasmid in the same promoter. To verify whether HSP27 has a specific role on the expression of MDA5, MAVS, TBK1, and IRF3, we overexpressed HSP27 to detect the expression of endogenous proteins and found that HSP27 can only significantly promote the protein expression of endogenous MDA5, but did not affect the expression of endogenous MAVS, TBK1, and IRF3 ( Figure 7A). Our results showed that HSP27 plays a clear antiviral role by specifically binding to MDA5 and stabilizing its expression. HSP27 exists in two forms; phosphorylated and nonphosphorylated. Non-phosphorylated HSP27 recognizes and binds to proteins damaged by oxidative stress or that have misfolded and ultimately degrades these proteins through the proteasomal pathway. On the other hand, phosphorylated HSP27 is closely related to the immune response of host cells, viral replication, proliferation, cell cycle, apoptosis, and autophagy (Arrigo, 2018). Future work aims to determine which HSP27 form is involved in EMCV pathobiology.
In this study, we found that EMCV 2C pro and 3A pro directly bind to HSP27 and reduce HSP27 protein expression, revealing a novel function of 2C pro and 3A pro in antagonizing the host antiviral response. We have shown for the first time that HSP27 is an important antiviral factor of EMCV infection and can be used as a potential therapeutic target against EMCV infection. In addition, it is obvious that HSP27 directly binds to and stabilizes the expression of the sensor molecule MDA5 in the RLRs signaling pathway, thereby promoting the MDA5-mediated type I IFN signaling cascade during EMCV infection (Figure 8).
These findings provide new reference into the regulatory role of host factors in EMCV infection and the development of antiviral drugs and provide strong evidence for further elucidation of the molecular mechanism of EMCV pathogenesis and its immune escape strategy.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.