Junyan Shi

Cleavage of sequestosome 1/p62 by an enteroviral protease results in disrupted selective autophagy and impaired NFKB signaling

The adaptor protein, sequestosome 1 (SQSTM1)/p62, plays an essential role in mediating selective autophagy. It serves as an autophagy receptor targeting ubiquitinated proteins to autophagosomes for degradation. In addition, it functions as a scaffold protein to regulate signaling pathways. Here we explored the interplay between coxsackievirus B3 (CVB3) and SQSTM1-mediated selective autophagy. We reported that SQSTM1 was cleaved at glycine 241 following CVB3 infection through the activity of viral protease 2Apro. The resulting cleavage fragments of SQSTM1 were no longer the substrates of autophagy, and their ability to form protein aggregates was greatly decreased. Although the C-terminal truncation sustained the binding activity of SQSTM1 to microtubule-associated protein 1 light chain (LC3), it failed to interact with ubiquitinated proteins. It was also found that colocalization between the C-terminal fragment of SQSTM1 (SQSTM1-C) and LC3 and ubiquitin within the punctate structures was markedly disrupted. Moreover, we observed that SQSTM1-C retained the ability of SQSTM1 to stabilize antioxidant transcription factor NFE2L2 [nuclear factor (erythroid-derived 2)-like 2]; however, both the N-terminal fragment of SQSTM1 (SQSTM1-N) and SQSTM1-C lost the function of SQSTM1 in activating NFKB (the nuclear factor of kappa light polypeptide gene enhancer in B-cells) pathway. Collectively, our results suggest a novel model by which cleavage of SQSTM1 as a result of CVB3 infection impairs the function of SQSTM1 in selective autophagy and host defense signaling.

Production of a dominant-negative fragment due to G3BP1 cleavage contributes to the disruption of mitochondria-associated protective stress granules during CVB3 infection.

Stress granules (SGs) are dynamic cytosolic aggregates containing messenger ribonucleoproteins and target poly-adenylated (A)-mRNA. A key component of SGs is Ras-GAP SH3 domain binding protein-1 (G3BP1), which in part mediates protein-protein and protein-RNA interactions. SGs are modulated during infection by several viruses, however, the function and significance of this process remains poorly understood. In this study, we investigated the interplay between SGs and Coxsackievirus type B3 (CVB3), a member of the Picornaviridae family. Our studies demonstrated that SGs were formed early during CVB3 infection; however, G3BP1-positive SGs were actively disassembled at 5 hrs post-infection, while poly(A)-positive RNA granules persisted. Furthermore, we confirmed G3BP1 cleavage by 3Cpro at Q325. We also demonstrated that overexpression of G3BP1-SGs negatively impacted viral replication at the RNA, protein, and viral progeny levels. Using electron microscopy techniques, we showed that G3BP1-positive SGs localized near mitochondrial surfaces. Finally, we provided evidence that the C-terminal cleavage product of G3BP1 inhibited SG formation and promoted CVB3 replication. Taken together, we conclude that CVB3 infection selectively targets G3BP1-SGs by cleaving G3BP1 to produce a dominant-negative fragment that further inhibits G3BP1-SG formation and facilitates viral replication.

Cytoplasmic redistribution and cleavage of AUF1 during coxsackievirus infection enhance the stability of its viral genome

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, hepatitis, pancreatitis, and meningitis in humans. The adenosine‐uridine (AU)‐rich element RNA binding factor 1 (AUF1) is an integral component in the regulation of gene expression. AUF1 destabilizes mRNAs and targets them for degradation by binding to AU‐rich elements in the 3′ untranslated region (UTR) of mRNAs. The 3′‐UTR of the CVB3 genome contains canonical AU‐rich sequences, raising the possibility that CVB3 RNA may also be subjected to AUF1‐mediated degradation. Here, we reported that CVB3 infection led to cytoplasmic redistribution and cleavage of AUF1. These events are independent of CVB3‐induced caspase activation but require viral protein production. Overexpression of viral protease 2A reproduced CVB3‐induced cytoplasmic redistribution of AUF1, while in vitro cleavage assay revealed that viral protease 3C contributed to AUF1 cleavage. Furthermore, we showed that knockdown of AUF1 facilitated viral RNA, protein, and progeny production, suggesting an antiviral property for AUF1 against CVB3 infection. Finally, an immunoprecipitation study demonstrated the physical interaction between AUF1 and the 3′‐UTR of CVB3, potentially targeting CVB3 genome toward degradation. Together, our results suggest that cleavage of AUF1 may be a strategy employed by CVB3 to enhance the stability of its viral genome.—Wong, J., Si, X., Angeles, A., Zhang, J., Shi, J., Fung, G., Jagdeo, J., Wang, T., Zhong, Z., Jan, E., Luo, H. Cytoplasmic redistribution and cleavage of AUF1 during coxsackievirus infection enhance the stability of its viral genome. FASEB J. 27, 2777‐2787 (2013). www.fasebj.org

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

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 2Apro and 3Cpro, 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.

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

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 amino-acid 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.

Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1

Coxsackievirus B3 (CVB3), an important human causative pathogen for viral myocarditis, pancreatitis, and meningitis, has evolved different strategies to manipulate the host signaling machinery to ensure successful viral infection. We previously revealed a crucial role for the ERK1/2 signaling pathway in regulating viral infectivity. However, the detail mechanism remains largely unknown. Grb2‐associated binder 1 (GAB1) is an important docking protein responsible for intracellular signaling assembly and transduction. In this study, we demonstrated that GAB1 was proteolytically cleaved after CVB3 infection at G175 and G436 by virus‐encoded protease 2Apro, independent of caspase activation. Knockdown of GAB1 resulted in a significant reduction of viral protein expression and virus titers. Moreover, we showed that virus‐induced cleavage of GAB1 is beneficial to viral growth as the N‐terminal proteolytic product of GAB1 (GAB1‐N1‐174) further enhances ERK1/2 activation and promotes viral replication. Our results collectively suggest that CVB3 targets host GAB1 to generate a GAB1‐N1‐174 fragment that enhances viral infectivity, at least in part, via activation of the ERKpathway. The findings in this study suggest a novel mechanism that CVB3 employs to subvert the host signaling and facilitate consequent viral replication.—Deng, H., Fung, G., Shi, J., Xu, S., Wang, C., Yin, M., Hou, J., Zhang, J., Jin, Z.‐G., Luo, H. Enhanced enteroviral infectivity via viral protease‐mediated cleavage of Grb2‐associated binder 1. FASEB J. 29, 4523‐4531 (2015). www.fasebj.org

NBR1 is dispensable for PARK2-mediated mitophagy regardless of the presence or absence of SQSTM1

Degradation of malfunctional mitochondria by mitophagy is a pivotal component of mitochondrial quality control to maintain cellular homeostasis. Mitochondrial clearance through the PINK1/PARK2 pathway is mediated by autophagic adaptor proteins. Previous studies revealed a significant involvement, but not an absolute requirement for SQSTM1 in PARK2-dependent mitophagy, suggesting that the existence of redundant adaptor proteins may compensate for the loss of SQSTM1. Here we investigated whether NBR1, a functional homolog of SQSTM1, has a role in PARK2-mediated mitophagy, either alone or as a compensatory mechanism. We showed that NBR1 does not appear to be required for mitochondrial clustering following mitochondrial depolarization. Moreover, we demonstrated that deletion of NBR1 alone or in combination with SQSTM1 does not prevent the degradation of damaged mitochondria. Our data suggest that NBR1 is dispensable for PARK2-dependent mitophagy and additional autophagic adaptor proteins, other than NBR1, are responsible for mitochondrial degradation in cells depleted of SQSTM1.

Dysferlin deficiency confers increased susceptibility to coxsackievirus‐induced cardiomyopathy

Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb‐girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin‐deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin‐deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus‐induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy.