We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin-protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at aminoacid 327 between glutamine and alanine, resulting in the generation of an N-and C-terminal cleavage fragment of~35 and~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. Coxsackievirus B3 (CVB3) is a small, positive-stranded RNA enterovirus. 1 The single open reading frame of CVB3 is translated into a viral polypeptide that is subsequently cleaved by two virus-encoded proteases 2A and 3C to generate structural and non-structural proteins. 2 In addition to processing viral polyprotein, 2A and 3C target host proteins important for maintenance of protein translation and transcription, antiviral activity, and cellular architecture and signaling, contributing to virus-induced pathogenesis. [3][4][5] Although enteroviral replication takes place exclusively in the cytoplasm, viral infection has been demonstrated to lead to cytoplasmic translocation of nuclear proteins. 6 For example, heterogeneous ribonucleoprotein D (hnRNP D) has been shown to translocate from the nucleus to the cytoplasm during enteroviral infection. 5,7,8 Moreover, hnRNP D is cleaved by 3C and has an antiviral function against enteroviral infection. 5,7,8 Cytoplasmic translocation after enteroviral infection has also been demonstrated for several other hnRNPs (A1, C, and K)...

Coxsackievirus B3 (CVB3), a common human pathogen for viral myocarditis, induces a direct cytopathic effect (CPE) and apoptosis on infected cells. To elucidate the mechanisms that contribute to these processes, we studied the role of glycogen synthase kinase 3b (GSK3b). GSK3b activity was significantly increased after CVB3 infection and addition of tyrosine kinase inhibitors blocked CVB3-triggered GSK3b activation. Inhibition of caspase activity had no inhibitory effect on CVB3-induced CPE; however, blockage of GSK3b activation attenuated both CVB3-induced CPE and apoptosis. We further showed that CVB3 infection resulted in reduced b-catenin protein expression, and GSK3b inhibition led to the accumulation and nuclear translocation of b-catenin. Finally, we found that CVB3-induced CPE and apoptosis were significantly reduced in cells stably overexpressing b-catenin. Taken together, our results demonstrate that CVB3 infection stimulates GSK3b activity via a tyrosine kinasedependent mechanism, which contributes to CVB3-induced CPE and apoptosis through dysregulation of b-catenin.

Coxsackievirus infection induces an abnormal accumulation of ubiquitin aggregates that are generally believed to be noxious to the cells and have a key role in viral pathogenesis. Selective autophagy mediated by autophagy adaptor proteins, including sequestosome 1 (SQSTM1/p62) and neighbor of BRCA1 gene 1 protein (NBR1), are an important pathway for disposing of misfolded/ubiquitin conjugates. We have recently demonstrated that SQSTM1 is cleaved after coxsackievirus infection, resulting in the disruption of SQSTM1 function in selective autophagy. NBR1 is a functional homolog of SQSTM1. In this study, we propose to test whether NBR1 can compensate for the compromise of SQSTM1 after viral infection. Of interest, we found that NBR1 was also cleaved after coxsackievirus infection. This cleavage took place at two sites mediated by virus-encoded protease 2A pro and 3C pro , respectively. In addition to the loss-of-function, we further investigated whether cleavage of SQSTM1/NBR1 leads to the generation of toxic gain-of-function mutants. 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. Finally, we demonstrated a positive, mutual regulatory relationship between SQSTM1 and NBR1 during viral infection. We showed that knockdown of SQSTM1 resulted in reduced expression of NBR1, whereas overexpression of SQSTM1 led to increased level of NBR1, and vice versa, further excluding the possible compensation of NBR1 for the loss of SQSTM1. Taken together, the findings in this study suggest a novel mechanism through which coxsackievirus infection induces increased accumulation of ubiquitin conjugates and subsequent viral damage.