Abstract SARS-CoV-2 exploits the function of the endoplasmic reticulum (ER) to promote its infection life cycle. Despite its strong reliance on the ER, the molecular basis by which SARS-CoV-2 hijacks ER factors to promote defined steps of this life cycle remains unclear. Using infectious SARS-CoV-2, we recently identified two ER membrane proteins – RTN3 and SigmaR1 – as critical host factors that support virus infection. Our findings further reveal that RTN3 plays a role in viral replication, while SigmaR1 exerts a function in viral secretion. However, how SARS-CoV-2 exploits the activities of RTN3 and SigmaR1 to accomplish these two distinct tasks, in mechanistic terms, is completely unknown. Accordingly, the objective of this application is to elucidate the molecular basis by which these two ER membrane factors promote replication and secretion of SARS- CoV-2. We believe these insights will not only illuminate the basic infection mechanism of SARS-CoV-2, but given the continuing global COVID-19 pandemic, may lead to the development of effective anti-virals to blunt the devastating impact of SARS-CoV-2.