CALCOCO2/NDP52 and SQSTM1/p62 differentially regulate coxsackievirus B3 propagation

Yasir Mohamud,Honglin Luo,Yuan Chao Xue,Haoyu Deng,Junyan Qu,Huitao Liu

doi:10.1038/s41418-018-0185-5

Abstract

Cell autonomous immunity is the ability of individual cells to initiate a first line of host defense against invading microbes, such as viruses. Autophagy receptors, a diverse family of multivalent proteins, play a key role in this host response by detecting, sequestering, and eliminating virus in a process termed virophagy. To counteract this, positive-stranded RNA viruses, such as enteroviruses, have evolved strategies to circumvent the host autophagic machinery in an effort to promote viral propagation; however, the underlying mechanisms remain largely unclear. Here we studied the interaction between coxsackievirus B3 (CVB3) and the autophagy receptor SQSTM1 (sequestosome 1)/p62 and CALCOCO2/NDP52 (calcium binding and coiled-coil domain-containing protein 2/nuclear dot 10 protein 52). We demonstrated that SQSTM1 and CALCOCO2 differentially regulate CVB3 infection. We showed that knockdown of SQSTM1 causes increased viral protein production and elevated viral titers, whereas depletion of CALCOCO2 results in a significant inhibition of viral growth. Both receptors appear to have a role in virophagy through direct interaction with the viral capsid protein VP1 that undergoes ubiquitination during infection. Further investigation of the proviral mechanism of CALCOCO2 revealed that CALCOCO2, but not SQSTM1, suppresses the antiviral type I interferon signaling by promoting autophagy-mediated degradation of the mitochondrial antiviral signaling (MAVS) protein. Moreover, we demonstrated that viral proteinase 2A-mediated cleavage of SQSTM1 at glycine 241 impairs its capacity to associate with viral capsid, whereas cleavage of CALCOCO2 by viral proteinase 3C at glutamine 139, generates a stable C-terminal fragment that retains the proviral function of full-length CALCOCO2. Altogether, our study reveals a mechanism by which CVB3 targets selective autophagy receptors to evade host virophagy.

Highlights

Autophagy is paramount to cellular health and its dysfunction is implicated in the pathogenesis of many human diseases
We have previously demonstrated that autophagy receptors sequestosome 1 (SQSTM1) and neighbor of BRCA1 (NBR1) are cleaved following coxsackievirus B3 (CVB3) infection, leading to impaired clearance of host ubiquitin conjugates [15, 16]; the functional significance of this cleavage in CVB3 infection has not been carefully characterized and the role of other autophagy receptors, in particular CALCOCO2/nuclear dot protein 52 (NDP52) that has been extensively studied in bacteria, remains to be investigated in viral propagation
Immunoprecipitation (IP) of viral capsid protein 1 (VP1) was performed using anti- To determine the role of autophagy receptors, SQSTM1 and VP1 antibody (Dako, M706401-1) pre-incubated with CALCOCO2, in coxsackievirus infection, we first from three replicates. c, d HeLa cells were treated with siCALCOCO2 as above, and subjected to infection with different doses of CVB3 for various times as indicated

Summary

Introduction

Autophagy is paramount to cellular health and its dysfunction is implicated in the pathogenesis of many human diseases. Autophagy is widely considered a cellular strategy to remove damaged proteins/organelles and to eliminate invading pathogens, its reciprocal regulation by Edited by H.-U. Selective autophagy is mediated by a family of multivalent autophagy receptors that target cytosolic cargo (e.g., misfolded/aggregate proteins, damaged organelles, and invading microbes) to autophagosomes for degradation through the highly conserved ubiquitin association domains that recognize ubiquitinated substrates and the LC3interaction regions (LIR) that anchor to autophagosomes [11, 12]. All autophagy receptors have been shown to be able to target invading microbes for lysosomal degradation, a process referred to as xenophagy (or virophagy in the context of viral elimination [13]), as part of the cell autonomous innate immune response [14]. We have previously demonstrated that autophagy receptors SQSTM1 and NBR1 are cleaved following CVB3 infection, leading to impaired clearance of host ubiquitin conjugates [15, 16]; the functional significance of this cleavage in CVB3 infection has not been carefully characterized and the role of other autophagy receptors, in particular CALCOCO2/NDP52 that has been extensively studied in bacteria, remains to be investigated in viral propagation

Methods

Results

Conclusion