Dominant-negative function of the C-terminal fragments of NBR1 and SQSTM1 generated during enteroviral infection

Abstract

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.

Enteroviruses Remodel Autophagic Trafficking through Regulation of Host SNARE Proteins to Promote Virus Replication and Cell Exit

“…We investigated the similarity between this cleavage of SQSTM1 by EV-D68 and the cleavage seen by Shi et al (2014) and obtained SQSTM1-expressing plasmids from the authors. The first plasmid was a SQSTM1 wild-type plasmid, originally sourced from Yu et al (2009).…”

“…Previous work by our group has demonstrated that poliovirus 1 (PV) uses acidic amphisomes to promote virus replication and maturation (Richards and Jackson, 2012). Coxsackievirus B3 induces the autophagic pathway but may inhibit degradation of autophagic cargo (Alirezaei et al, 2015; Shi et al, 2014; Wong et al, 2008). We report here that EV-D68 induces autophagy signaling to benefit its replication and manipulates the autophagosomal SNAREs.…”

Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

“…54,55 HeLa cell lysates (50 μg) were incubated with various concentrations of purified CVB3 2A, 3C, or 3C mutant as indicated in a cleavage reaction buffer (20 mM HEPES (pH 7.4), 150 mM KOAc and 1 mM DTT) at 37˚C for different times as previously described. 20,51 The reaction was stopped by addition of 6 × SDS-PAGE sample buffer and TDP-43 cleavage was assessed by western blot analysis.…”

“…18 However, the role of TDP-43 in virus-induced diseases has not been studied. Recent evidence suggests that CVB3-induced pathogenesis resembles the pathological features of neurodegenative disease, that is, abnormal accumulation of insoluble, misfolded protein aggregates (also known as proteinopathies), 3,19,20 prompting us to hypothesize that dysregulation of TDP-43 during CVB3 infection plays a role in viral pathogenesis, and probably viral infectivity as well. …”

Coxsackievirus B3 Replication and Pathogenesis

“…They function as autophagy receptors targeting ubiquitinated proteins to autophagosomes for degradation [186][187]191]. It was recently reported that both p62 and Nbr1 are cleaved following CVB3 infection, through the action of CVB3-encoded protease 2A and 3C [192,193]. It was further shown that such cleavage leads to disrupted selective autophagy and results in the impaired clearance of ubiquitin aggregates [193].…”