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

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.

Manipulation of autophagy by (+) RNA viruses

“…As counterstrategy, the host protein HRAS-like suppressor 3 (PLA2G16) is exploited by the virus to enable genome delivery. CVB3, also belonging to the same family, undergoes p62-dependent degradation and uses the viral protease 2A to cleave p62 and inhibit virophagy [94]. Interestingly, although HCV has been demonstrated to induce autophagy to its own advantage by multiple groups, one study demonstrated that an ER transmembrane protein, SCOTIN, interacted with the viral protein NS5A, resulting in its autophagic degradation to suppress viral replication [95].…”

The Intertwined Life Cycles of Enterovirus and Autophagy

“…The first-identified virophagy receptor is SQSTM1/p62 that was discovered to interact with capsid proteins of Sindbis and Chikungunya viruses, which are positive-sense RNA viruses in the Togaviridae family, and target viral particles and/or capsid proteins for autophagic degradation [99,100]. Recent studies on CVB3 have revealed that both SQSTM1/p62 and CALCOCO2/ NDP52 are able to bind capsid protein VP1 to mediate virophagy [101]. Further characterization demonstrated that VP1 undergoes ubiquitination (a common signal for substrate recognition by autophagy receptors [102]) during infection, indicating a possible ubiquitin-dependent virophagy mechanism [101].…”

“…Recent studies on CVB3 have revealed that both SQSTM1/p62 and CALCOCO2/ NDP52 are able to bind capsid protein VP1 to mediate virophagy [101]. Further characterization demonstrated that VP1 undergoes ubiquitination (a common signal for substrate recognition by autophagy receptors [102]) during infection, indicating a possible ubiquitin-dependent virophagy mechanism [101]. Although direct evidence is lacking, the early electron microscopy findings of PV and CVB3 virions inside the double-membraned vesicles suggest the possibility that SQSTM1/p62 and CALCOCO2/ NDP52 can also recruit intact viral particles beyond capsid proteins to autophagosomes for degradation.…”

“…Of note, it was found that cleavage of SQSTM1/p62 and NBR1 not only results in a loss-of-function but also produces a dominantnegative fragment against native proteins [103]. Recently, autophagy receptor CALCOCO2/NDP52 was identified as a novel substrate of CVB3 protease [101].…”

“…In addition to direct targeting of virus, autophagy receptors can also modulate immune signaling [36,37]. Study of CVB3 infection found that knockdown of SQSTM1/p62 results in enhanced viral growth, whereas gene-silencing of CALCOCO2/NDP52 leads to a reduction in viral yields, indicating an opposite role for these two proteins in CVB3 infection [101]. Further investigation has identified the pro-viral mechanism of CALCOCO2/NDP52, which inhibits the anti-viral type I interferon signaling by facilitating the degradation of the mitochondrial anti-viral signaling (MAVS) by autophagy [101].…”