ABSTRACT
Objective To analyze how enterovirus D68 (EV-D68) protease 2A affects the anti-vi-ral interferon typeⅠ(IFN-Ⅰ) pathway in 293T cells following infection. Methods Western blot was used to detect the expression of recombinant protease 2A, IFN-α and signal transducers and activators of tran-scription 1 (STAT1) at protein level. Expression of EV-D68 viral protein (VP1) and protease 2A was ana-lyzed by immunofluorescence at different time points. Cytopathic effects were recorded to calculate 50％ cell culture infective dose ( CCID50 ) . Expression of the genes involved in the anti-viral IFN-Ⅰ pathway was measured by real-time PCR (RT-PCR). Results The recombinant plasmid pCLIPf-2A was successfully constructed and the expression of recombinant protease 2A could be detected by Western blot 24 h after transfection. The recombinant protease 2A promoted the proliferation of EV-D68 at the late stage of infection and induced the production of IFN-α. Expression of the genes involved in the anti-viral IFN-Ⅰ pathway at mRNA level was up- or down-regulated to different degrees with various trends in different groups following infection. Expression of STAT1 was enhanced in all groups. Conclusions EV-D68 protease 2A promoted the activation of anti-viral IFN-Ⅰpathway in response to viral infection and enhanced the proliferation of virus at the late stage of infection.
ABSTRACT
It has been previously demonstrated that dystrophin is cleaved in the cardiac myocyte by the viral protease 2A following infection with Coxsackievirus B3 (CVB3). The viral protease 2A mediated cardiomyopathy can be prevented by inhibiting cleavage of dystrophin. However, it is less clear whether uncleaved dysdrophin have other heart protective effect in coxsackievirus infection. To address this, we generated a Balb/C background mouse that had a point mutation in dystrophin that prevents cleavage by protease 2A (KI). We show here that when mice expressing cleavage-resistant dystrophin were infected with CVB3, there was increased cardiac myocyte apoptosis. Bax and Bcl-X(L) mRNA ratio was significantly increased in KI mice heart compare to wild type mice heart. We found cleavage-resistant dystrophin induced the apoptosis related enzyme capspase-3 and caspase-8 activity. In addition, TUNEL stain was observed many TUNEL positive cardiac myocyte in KI mice heart compare to wild type mice heart (3.7% vs 0.3%). However, zVAD treatment for apoptosis blocking was significantly decreased myocardium damage and fibrosis in KI mice heart. These findings indicated that uncleaved dystrophin may have a critical role in cardiac myocyte viral propagation. Uncleaved dystrophin mutant induced cardiac myocyte apoptosis. It delayed coxsackievirus propagation in cardiac myocyte and could prevent cardiac myocyte death.