Contribution of autophagy machinery factors to HCV and SARS-CoV-2 replication organelle formation

Abstract

Positive-strand RNA viruses replicate in close association with rearranged intracellular membranes. For hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), these rearrangements comprise endoplasmic reticulum (ER)-derived double membrane vesicles (DMVs) serving as RNA replication sites. Cellular factors involved in DMV biogenesis are poorly defined. Here, we show that despite structural similarity of viral DMVs with autophagosomes, conventional macroautophagy is dispensable for HCV and SARS-CoV-2 replication. However, both viruses exploit factors involved in autophagosome formation, most notably class III phosphatidylinositol 3-kinase (PI3K). As revealed with a biosensor, PI3K is activated in cells infected with either virus to produce phosphatidylinositol 3-phosphate (PI3P) while kinase complex inhibition or depletion profoundly reduces replication and viral DMV formation. The PI3P-binding protein DFCP1, recruited to omegasomes in early steps of autophagosome formation, participates in replication and DMV formation of both viruses. These results indicate that phylogenetically unrelated HCV and SARS-CoV-2 exploit similar components of the autophagy machinery to create their replication organelles.

ATPase activity of DFCP1 controls selective autophagy

“…In conclusion, we have identified an evolutionarily conserved Dynamin-related ATPase module that controls the early phase of selective autophagy (Extended Data Fig.9). It is interesting to note that biogenesis of the replication compartments of certain viruses, including SARS-CoV-2, depends on DFCP1 37 , and our data thus imply that constriction of ER-derived membranes is a critical step in SARS-CoV-2 replication. DFCP1 recognizes ubiquitinated cargoes which are included in omegasomes, and ATP binding and hydrolysis of DFCP1 trigger omegasome constriction and thereby the biogenesis of cargo-containing autophagosomes.…”

ATPase activity of DFCP1 controls selective autophagy

“…9). It is interesting to note that biogenesis of the replication compartments of certain viruses, including SARS-CoV-2, depends on DFCP1 37 , and our data thus imply that constriction of ER-derived membranes is a critical step in SARS-CoV-2 replication. DFCP1 recognizes ubiquitinated cargoes which are included in omegasomes, and ATP binding and hydrolysis of DFCP1 trigger omegasome constriction and thereby the biogenesis of cargocontaining autophagosomes.…”

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 ).…”

“…Genome-wide CRISPR/Cas9 screens have identified a subset of autophagy genes essential for SARS-CoV-2 infection. PI(3)P synthesis is required for DMV formation and viral replication of HCV and SARS-CoV-2, while the essential autophagy proteins ATG5 and ATG7 are dispensable for SARS-CoV-2 infection ( Daniloski et al, 2021 ; Kapadia and Chisari, 2005 ; Shang et al, 2021 ; Twu et al, 2021 ; Zhu et al, 2021 ). Class III PI3K inhibitors inhibit SARS-CoV-2 replication ( Yuen et al, 2021 ; Zhu et al, 2021 ).…”