Molecular Mechanisms Of Involvement Of Nonstructural Proteins In Encephalomyocarditis Virus-Induced Autophagy And Apoptosis

Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen with a wide host-range and can infect a wide variety of animal species, including mammals, rodents, non-human primates and even humans, which cause an infectious disease is clinically characterized by encehphalitis, myocarditis or inflammation around cardiac muscles. Pigs are the most extensive and susceptible to EMCV infection, causing sudden death and widely pathological damage of substantive organs in preweaned piglets, and severe reproductive failure such as abortions, stillborns, and weak piglets at birth and mummies in sows. Previous studies have shown that autophagy is induced during EMCV infection in BHK-21cells. However, the roles of viral proteins in autophagy and the mechanisms of viral proteins regulating EMCV replication are still unclear. In the present study, we investigated the viral proteins that are responsible for EMCV-induced autophagy and apoptosis and the associated molecular mechanisms in order to provide valuable scientific evidence for elucidating the pathogenesis of EMCV.By using transmission electron microscopy, western blotting and confocal immunofluorescence analysis, the autophagic activities of the BHK-21cells expressing each viral protein of EMCV were analyzed. Our results showed that2C or3D protein significantly induced a large number of single-and double-membrane vesicles in the cytoplasm of host cells, and meanwhile they clearly enhanced the conversion of LC3-I to LC3-Ⅱ and the degradation of p62, and the2C or3D protein was colocalized with LC3by a subset of positive punctuate foci, suggesting that EMCV2C or3D protein is involved in the EMCV-induced autophagy process. Further results indicated that EMCV2C or3D protein caused endoplasmic reticulum stress and unfolded protein response.Beclinl was identified an interacting partner with EMCV2C protein by coimmunoprecipitation and confocal laser scanning microscopy assays. Moreover, we found that Beclinl was cleaved in EMCV-infected or2C-overexpressing BHK-21cells, and accompanied by the increase of apoptosis level. The effect of knockdown of Beclinl on apoptosis and viral replication in EMCV-infected BHK-21cells was analyzed by RNA interference, western blotting and flow cytometry. Our results showed that the apoptosis level could be enhanced by the reduction of Beclinl expression through triggering translocation of pro-apoptotic protein Bax from the cytosol to mitochondria, and the knockdown of endogenous Beclin1significantly enhanced EMCV growth at the late stage of infection in BHK-21cells, indicating that a potential relation exists between apoptosis and viral replication. Additionally, the apoptosis inhibitor (Z-VAD-FMK) or knockdown of endogenous caspase-3decreased the EMCV yields, suggesting that the apoptosis might play an important role during EMCV infection and have a close relation to EMCV replication.In summary, our findings demonstrated that the nonstructural proteins2C and3D are involved in EMCV-induced autophagy, and moreover, the Beclinl degradation in EMCV-infected or 2C-overexpressed BHK-21cells can enhance the apoptosis and finally promote the viral replication. Our findings provide valuable scientific evidence for further exploring the molecular pathogenesis of EMCV.