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UBC Theses and Dissertations

Disruption of selective autophagy in coxsackievirus B3 infection Shi, Junyan


Coxsackievirus infection induces an abnormal accumulation of ubiquitin aggregates that are generally believed to be harmful to the cells and play a key role in the pathogenesis of coxsackievirus-induced myocarditis and dilated cardiomyopathy. Selective autophagy mediated by autophagy adaptor proteins, including sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 gene 1 protein (NBR1), is an important pathway for disposing of misfolded proteins and damaged organelles. We demonstrated that SQSTM1 was cleaved following CVB3 infection through the proteolytic activity of viral proteinase 2Apro. The resulting cleavage fragments of SQSTM1 were no longer the substrates of autophagy, and their ability to form protein aggregates was greatly decreased due to incapability of interaction with ubiquitinated proteins. We further tested whether NBR1, a functional homolog of SQSTM1, can compensate for SQSTM1 loss-of-function after viral infection. Of interest, we found that NBR1 was also cleaved after coxsackievirus infection, excluding the possible compensation of NBR1 for the loss of SQSTM1. This cleavage took place at two sites mediated by virus-encoded proteinase 2Apro and 3Cpro, respectively. In addition to the loss-of-function, we showed that the C-terminal fragments of SQSTM1 and NBR1 exhibited a dominant-negative effect against native SQSTM1/NBR1, probably by competing for LC3 and ubiquitin chain binding. Apart from the disruption of selective autophagy, CVB3 infection also impaired autophagic flux as confirmed by flux assays with a combination of a tandem fluorescence-tagged LC3 stable cell line and a non-cleavable construct of SQSTM1. Finally, we studied the roles of SQSTM1 and NBR1 in autophagic degradation of depolarized mitochondria (referred to as mitophagy). Following mitochondrial depolarization induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler to trigger mitophagy, we demonstrated that NBR1 did not appear to be required for mitochondrial clustering. Deficiency of NBR1 alone or in concert with SQSTM1 did not block the clearance of damaged mitochondria, suggesting that NBR1 is dispensable for mitophagy regardless of the status of SQSTM1. Taken together, the findings in this study suggest novel mechanisms in coxsackieviral pathogenesis: coxsackievirus infection induces abnormal accumulation of ubiquitin conjugates through the disruption of selective degradation of protein aggregates and blockage of autophagic flux.

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