VMP1 and TMEM41B are essential for DMV formation during β-coronavirus infection

Abstract

β-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3–nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.

Endomembrane remodeling in SARS-CoV-2 infection

“…Depletion or mutation of NSP3 and NSP4 causes a defect in DMV formation and dramatically decreases SARS-CoV or MHV replication ( Beachboard et al, 2015 ; Clementz et al, 2008 ; Sakai et al, 2017 ; Stokes et al, 2010 ). Co-expression of MERS-CoV or SARS-CoV-2 NSP3 and NSP4 is required and sufficient to induce DMVs, while NSP6 is dispensable ( Oudshoorn et al, 2017 ; Twu et al, 2021 ; Ji et al, 2022 ). NSP3 and NSP4 form homotypic and heterotypic interactions through their intraluminal domains ( Hagemeijer et al, 2011 ; Neuman et al, 2008 ; Ji et al, 2022 ).…”

“…Co-expression of MERS-CoV or SARS-CoV-2 NSP3 and NSP4 is required and sufficient to induce DMVs, while NSP6 is dispensable ( Oudshoorn et al, 2017 ; Twu et al, 2021 ; Ji et al, 2022 ). NSP3 and NSP4 form homotypic and heterotypic interactions through their intraluminal domains ( Hagemeijer et al, 2011 ; Neuman et al, 2008 ; Ji et al, 2022 ). NSP3 and NSP4 are located in the outer and inner membrane of DMVs, respectively, and this distinct localization is necessary for DMV formation ( Ji et al, 2022 ).…”

“…NSP3 and NSP4 form homotypic and heterotypic interactions through their intraluminal domains ( Hagemeijer et al, 2011 ; Neuman et al, 2008 ; Ji et al, 2022 ). NSP3 and NSP4 are located in the outer and inner membrane of DMVs, respectively, and this distinct localization is necessary for DMV formation ( Ji et al, 2022 ). Other NSPs, such as NSP2/3, NSP8 and NSP12, are also enriched in DMVs or CMs ( Bost et al, 2000 ; Hagemeijer et al 2012 ; van der Meer et al, 1999 ), but their functions during DMV biogenesis are still unknown.…”

“…The ER-localized transmembrane autophagy proteins TMEM41B and VMP1 are essential for a post-entry step in the infection cycle of SARS-CoV-2 ( Morita et al, 2018 ; Zhao et al, 2017 ; Hoffmann et al, 2021a ; Shoemaker et al, 2019 ). Depletion of VMP1 or TMEM41B greatly impairs DMV formation induced by MHV infection or expression of SARS-CoV-2 NSP3/4 ( Ji et al, 2022 ). VMP1 and TMEM41B interact with the NSP3/4 complex and are recruited to DMV formation sites ( Figure 3 ).…”

“…VMP1 and TMEM41B interact with the NSP3/4 complex and are recruited to DMV formation sites ( Figure 3 ). They function at different steps during DMV generation: TMEM41B facilitates NSP3/4 binding, while VMP1 is required for curvature of paired ER membranes into closed DMVs ( Ji et al, 2022 ). TMEM41B and VMP1 have recently been shown to act as ER scramblases for normal cross-membrane distribution of phospholipids, such as cholesterol and phosphatidylserine ( Ghanbarpour et al, 2021 ; Li et al, 2021b ; Huang et al, 2021).…”