Biochem Biophys Res Commun. 2013 Nov 29;441(4):856-61.

Coxsackievirus A16 infection triggers apoptosis of RD cells by inducing ER stress

Guoguo Zhu 1†, Yingcheng Zheng 1†, Yingying Shi1, Lianglu Zhang1, Wenhua Li2, Zhongchun Liu3, Biwen Peng1, Jun Yin1, Wanhong Liu1*, and Xiaohua He1

1Hubei Province Key Laboratory of Allergy and Immunology, School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China

2College of Life Sciences, Wuhan University, Wuhan 430072, P.R. China

3Department of Psychiatry, Renmin Hospital, Wuhan University, Wuhan 430060, China



Coxsackievirus A16 (CA16) infection, which is responsible for hand, foot, and mouth disease (HMFD), has become a common health problem in Asia due to the prevalence of the virus. Thus, it is important to understand the pathogenesis of CA16 infection. Viruses that induce endoplasmic reticulum (ER) stress are confronted with the consequent unfolded protein response (UPR), which may lead to apoptotic cell death and may influence viral replication. In this study, we found that CA16 infection could induce apoptosis and ER stress in RD cells. Interestingly, apoptosis via the activation of caspase-3, -8, and -9 in the extrinsic or intrinsic apoptotic pathways in RD cells was inhibited by 4-phenyl butyric acid (4PBA), a chemical chaperone that reduces ER stress. These results suggest that CA16 infection could lead to ER stress and then to prolonged ER stress-induced apoptosis. This study provides a new basis for understanding CA16 infection and host responses.



Coxsackievirus A16 (CA16), one of the major causative agents of human hand, foot and mouth disease (HFMD), belongs to the enterovirus genus of the Picornaviridae family, is a positive single-stranded RNA virus with approximately 7410 bases in its genome, and has one predominant serotype (1). Despite the established association of CA16 infection with HFMD, the pathogenic mechanisms of this viral infection are still unclear. Infection with single stranded RNA (ssRNA) viruses induces apoptosis by different signaling pathways including ER stress. And the killing of infected cells is a strategy that host cells use to suppress viral replication. Our understanding of CA16-induced apoptosis and the relationship between CA16 and ER stress is incomplete.

In this study, we show that CA16 infection not only triggers ER stress by inducing all three signaling pathways but also that CA16 infection efficiently induces apoptosis in RD cells. This host cell response to virus may allow these cells to prevent the spread of the virus. We discovered that CA16 infection activates caspase-3, -8, and -9 in RD cells, and the CA16-induced apoptosis could be inhibited by the pan-caspase inhibitor z-VAD-fmk. The activation of caspase-8 by CA16 indicated that CA16 infection could activate the extrinsic apoptosis pathway in RD cells, and the inhibition of z-VAD-fmk confirmed that CA16 infection activates caspases and triggers apoptosis in RD cells. Using the dye TMRM, an acute decrease in MMP was observed in CA16-infected RD cells, which means that CA16 induces the opening of the MPTP following the dysfunction of mitochondria. In addition to facilitating the opening of MPTP as previously described, the increase in the amount of Bax and the activation of caspase-9 and caspase-3 suggests that CA16 infection triggers apoptosis in RD cells through the intrinsic pathway. Moreover, we found that following CA16 infection GRP78 and CHOP expression increased and IRE1/JNK pathway activated along with ATF6 and PERK pathway, which suggested the occurrence of ER stress in RD cells. Using the ER stress inhibitor 4PBA, we have confirmed that ER stress is an event upstream of CA16 infection-triggered apoptosis.

Collectively, CA16 infection triggered both extrinsic and intrinsic apoptosis in RD cells, and this apoptosis was partially inhibited by the pan-caspase inhibitor z-VAD-fmk. In addition, the expression of GRP78 and CHOP was increased following infection with CA16, while the three ER stress transducers were all activated, as was the splicing of XBP1 and the phosphorylation of JNK. The activation of ER stress is a partial mechanism for RD cell apoptosis.


  1. Zhang, Y., Wang, D., Yan, D., Zhu, S., Liu, J., Wang, H., Zhao, S., Yu, D., Nan, L., An, J., Chen, L., An, H., Xu, A., and Xu, W. (2010). Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China. J Clin Microbiol 48(2), 619-22.


Acknowledgments: This work was supported by the National Natural Sciences Foundation of China (No. 81171577, 81371790 and 81171127) and the Fundamental Research Funds for the Central Universities of China (No. 2012301020207, 201130102020001, and 201130102020002).



Wanhong Liu-fig1Wanhong Liu, Ph.D., Professor

Department of Immunology

School of Basic Medical Sciences, Wuhan University

Donghu Road No. 185, Wuchang, Wuhan 430071, P.R. China



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