ABSTRACTThe ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understandbetacoronavirusesas a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected more than 29 million individuals worldwide with nearly ~1 million fatalities. Understanding how SARS-CoV-2 initiates viral pathogenesis is of the utmost importance for development of antiviral drugs. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks underline the urgent need for better understanding the mechanism of viral subversion of autophagy. Using murine hepatitis virus-A59 (MHV-A59) as a modelbetacoronavirus, time-course infections revealed a significant loss in the protein level of ULK1, a canonical autophagy regulating serine-threonine kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target this protein, we conductedin vitroand cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PLpro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PLprorecognition sequence (LGGG) after G499, separating its N-terminal kinase domain from the C-terminal substrate recognition region. Consistent with this, over-expression of SARS-CoV-2 PLprois sufficient to impair starvation-induced canonical autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PLproofbetacoronavirusesinduces viral pathogenesis by targeting cellular autophagic pathway (Word count=250)IMPORTANCEThe recent COVID-19 global pandemic alongside the 2003 SARS and 2012 MERS outbreaks underscore an urgent need to better understandbetacoronavirusesas pathogens that pose global challenge to human health. Studying the underlying biology of howbetacoronavirusessubvert innate cellular defense pathways such as autophagy will help to guide future efforts to develop anti-viral therapy. (Word count= 55)