Host ER Membrane Proteins Promote Non-Structural Protein Biosynthesis and Genome Replication of SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the etiologic agent for the global COVID-19 pandemic responsible for millions of deaths worldwide. Despite the global impact of COVID-19 on human health, the cellular basis of SARS-CoV-2 infection is not fully understood. After binding to the host cell surface, SARS-CoV-2 is endocytosed and reaches the endosome where the viral and endosome membranes fuse, releasing the viral RNA genome into the cytosol. Translation of the viral genome leads to the formation of two polyproteins, PP1A and PP1AB, which are embedded in the endoplasmic reticulum (ER) membrane and subsequently proteolytically cleaved into distinct viral non-structural proteins (NSPs). A critical function of these NSPs, most prominently the transmembrane proteins NSP3 and NSP4, is to facilitate viral genome replication. Specifically, NSP3 and NSP4 promote formation of ER-derived structures known as double-membrane vesicles (DMVs) that serve as replication organelles for the viral genomic RNA (gRNA) and subgenomic RNA (sgRNA). After the replicated gRNA and sgRNA are released from the DMV into the cytosol, the sgRNA undergoes co-translational translocation at the ER membrane, generating viral structural transmembrane proteins (S, E, and M) which are packaged with the gRNA at the ER-Golgi intermediate compartment (ERGIC). From the ERGIC, the mature virions are thought to transport along the secretory pathway, leading to egress from the cell to cause infection.

Two enigmatic steps during SARS-CoV-2 entry are: 1) how the DMVs are generated, and 2) how NSP3 and NSP4 viral proteins responsible for DMV formation are synthesized at the ER membrane. This dissertation addresses these two questions. Specifically, Chapter 2 of this dissertation demonstrates that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 support DMV formation, enabling viral replication which leads to productive infection. We show that different SARS-CoV-2 variants rely on the RTN-dependent pathway to promote infection. Mechanistically, our findings further reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to promote viral replication and physically interact with NSP3 and NSP4.

Chapter 3 of this thesis demonstrates that the multi-subunit ER membrane complex (EMC) is critical for biosynthesis of NSP3, which promote formation of the DMV replication organelles that enable proper viral replication and infection. Importantly, computational analysis suggests that NSP3 has marginally hydrophobic transmembrane segments, consistent with the established function of the EMC in assisting biosynthesis of cellular transmembrane proteins with marginally hydrophobic transmembrane domains. In sum, my thesis identifies two different ER transmembrane proteins (EMC and RTNs) in supporting two distinct steps (translation and replication) of SARS-CoV-2 infection.