SARS-CoV-2 protein ORF3a induces Atg8ylation of lysosomal membranes

Atg8ylation is an autophagy associated response to membrane damage that recruits mammalian ATG8 proteins (mATG8s) to damaged membranes to promote their repair or removal. Here, we show that the SARS-CoV-2 protein ORF3a induces lysosomal membrane atg8ylation and that this response protects cells from death. mATG8s interact with ORF3a and are required for lysosomal damage induced by ORF3a. ORF3a targets mTOR in an Atg8ylation dependent manner and promotes lysophagy through mATG8 mediated coordination of TRIM16 and Galectin-3. ORF3a triggers apoptosis, necroptosis, and pyroptosis, whereas atg8ylation limits ORF3a-induced cell death. Together, our findings identify mATG8s and the autophagy conjugation machinery as key regulators of lysosomal atg8ylation and lysophagy in response to the SARS-CoV-2 virulence factor ORF3a.