Expression of SARS-CoV-2 nsp3 and nsp4 is sufficient to induce double-membrane vesicles, and the host factors FAM149B1, CCAR2, and ZC3HAV1 play a critical role in their formation.
The host factors FAM149B1, CCAR2, and ZC3HAV1 are identified as critical components in the formation of SARS-CoV-2 replication organelles driven by viral proteins nsp3 and nsp4.
ABSTRACT Positive-strand RNA viruses subvert the cellular endomembrane system for the generation of distinct compartments termed replication organelles (ROs) that harbor the site where viral RNAs are generated. In this study, we corroborate that the SARS-CoV-2 non-structural proteins 3 and 4 (nsp3 and nsp4) suffice to remodel the endoplasmic reticulum to form double-membrane vesicles similar to ROs observed in viral infection. Cellular membrane alterations induced by nsp3/4 expression were evaluated through electron tomography and confocal microscopy, and nsp3/4-associated host factors were identified using mass spectrometry. The role of these host factors in virus infection was determined using gene silencing, identifying several host proteins involved in the SARS-CoV-2 replication cycle. Combining the gene silencing approach with ultrastructural analysis of nsp3/4-expressing cells, we found that the host dependency factors FAM149B1, CCAR2, and ZC3HAV1 play a role in the formation of double-membrane vesicles in a replication-independent manner. IMPORTANCE Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle.
Pahmeier et al. (2023) studied SARS-CoV-2. SARS-CoV-2 nsp3/4 expression and host factor gene silencing vs. Empty vector / non-targeting siRNA was evaluated on Formation of double-membrane vesicles (DMVs) and viral replication. Expression of SARS-CoV-2 nsp3 and nsp4 is sufficient to induce double-membrane vesicles, and the host factors FAM149B1, CCAR2, and ZC3HAV1 play a critical role in their formation.