UC San Diego

When the novel coronavirus SARS-CoV-2 infects human cells, it results in the formation of “double-membrane vesicles” (DMVs) upon major rearrangements of the host cell compartment called the endoplasmic reticulum (ER) (Cortese et al., 2020). Previous studies have identified that nonstructural protein 4 (Nsp4), one of SARS-CoV-2 coded proteins, is involved in formation of the DMVs in mammalian cells (Angelini et al., 2013; Hagemeijer et al., 2014; Oudshoorn et al., 2017). Importantly without proper DMV formation, SARS-CoV are unable to replicate and reproduce in the infected cell (Sakai et al., 2017). Little is known about if host cell components are involved and the molecular mechanisms by which the DMV is formed. In order to answer these questions, we used a minimal experimental model and transformed a plasmid that expresses SARS-CoV-2 Nsp4 under the galactose regulatable promoter into wild type Saccharomyces cerevisiae, or budding yeast. These cells carried a prominent ER transmembrane protein fused with GFP (Pho88-GFP) integrated in the genomic pho88 locus. Here, we show that expression of Nsp4 alone in WT cells results in the dramatic rearrangement of ER structure. Using gene knockout cell strains, we found that while upstream elements of ER stress mitigating pathways like the unfolded protein response (UPR) and ER stress surveillance (ERSU) checkpoint were not involved in these rearrangements, proteins Vps4 and Vps24 of the endosomal sorting complexes required for transport (ESCRT) were involved. Finally, we discovered that inducing Nsp4 expression results in a significant decrease of ER inheritance into the daughter cell and is mediated in part by Vps4. This minimal experimental approach allows for a further investigation of host components involved in both morphological and functional changes of the ER and potential drug targets to disrupt DMVs, and thus SARS-CoV-2 reproduction.