key: cord-0888463-0swt5xj3
authors: Chen, Di; Zhao, Yan; Zhang, Hong
title: Endomembrane remodeling in SARS-CoV-2 infection
date: 2022-05-17
journal: nan
DOI: 10.1016/j.cellin.2022.100031
sha: c637f60208913a4c04c0a8bb6a77cc33d6074aaf
doc_id: 888463
cord_uid: 0swt5xj3

During severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the viral proteins intimately interact with host factors to remodel the endomembrane system at various steps of the viral lifecycle. The entry of SARS-CoV-2 can be mediated by endocytosis-mediated internalization. Virus-containing endosomes then fuse with lysosomes, in which the viral S protein is cleaved to trigger membrane fusion. Double-membrane vesicles generated from the ER serve as platforms for viral replication and transcription. Virions are assembled at the ER–Golgi intermediate compartment and released through the secretory pathway and/or lysosome-mediated exocytosis. In this review, we will focus on how SARS-CoV-2 viral proteins collaborate with host factors to remodel the endomembrane system for viral entry, replication, assembly and egress. We will also describe how viral proteins hijack the host cell surveillance system — the autophagic degradation pathway — to evade destruction and benefit virus production. Finally, potential antiviral therapies targeting the host cell endomembrane system will be discussed.

lipids and/or other biomacromolecules are internalized at the plasma membrane (PM) into vesicles which then fuse with early endosomes. The internalized cargos are ultimately delivered to lysosomes for degradation or retrieved by endosomal protein sorting/trafficking complexes, such as retromer, for recycling (Naslavsky and Caplan, 2018) . The coronaviral proteins intersect extensively with the endosomal network at various steps of infection ( Figure 1 ). The binding of the surface-anchored viral glycoprotein S with cell-membrane receptors (e.g. angiotensin converting enzyme 2 (ACE2) for SARS-CoV and SARS-CoV-2) facilitates the entry of coronaviruses into host cells via direct membrane fusion or endocytosis-mediated uptake (Bayati et al., 2021; Hoffmann et al., 2020b; Li et al., 2003) . The virus-containing endosomes fuse with lysosomes, resulting in cleavage of S proteins by cathepsin proteases and subsequent viral membrane fusion to release the viral genome into the cytosol ( Figure   1 ). The receptor is recycled back to the PM for further rounds of infection. Doublemembrane vesicles (DMVs), which are generated from the ER, act as platforms for anchoring the RTCs for viral replication (Figure 1 ). Virions are assembled at the ER or the ER-Golgi intermediate compartment (ERGIC) and are thought to use smoothwall secretory vesicles for release ( Figure 1 ) (Knoops et al., 2008; Reggiori et al., 2010; Snijder et al., 2020) . Recent studies of SARS-CoV-2 infection, which induces extensive ER remodeling, Golgi fragmentation and mitochondrial network reshaping (Cortese et al., 2020; Scherer et al., 2022) , have greatly advanced our understanding of the mechanism and function of endomembrane remodeling in coronavirus infection. For example, double-membrane-spanning pores in DMVs transport the viral J o u r n a l P r e -p r o o f RNA products from the interior of DMVs to the cytosol for translation or viral assembly (Wolff et al., 2020) . The secretory pathway and/or the late endosome/lysosome exocytic pathway are involved in the release of coronaviruses such as SARS-CoV-2 and MHV Ghosh et al., 2020) . SARS-CoV-2 also employs a novel mechanism to modulate the autophagic machinery in order to evade destruction (Miao et al., 2021) . Genome-wide CRISPR/Cas9 screens identified numerous host factors that are involved in endomembrane remodeling in the infection cycle of SARS-CoV-2 (Daniloski et al., 2021; Hoffmann et al., 2021a; Morita et al., 2018; Schneider et al., 2021; Wang et al., 2021) . In this review, we will summarize the most recent progress in understanding the involvement of endomembrane remodeling in the entry, replication, assembly and egress of SARS-CoV-2. Finally, we will offer insights into the possibility of targeting the endomembrane system as a therapeutic strategy.

Entry of SARS-CoV-2 is triggered by interaction of the viral S protein with its receptor ACE2 on the host cell surface. ACE2 is expressed in multiple tissues such as the respiratory tract and the cardiovascular and renal systems (Hou et al., 2020) . It was previously identified as a cell surface-localized transmembrane carboxypeptidase which acts in the renin-angiotensin system, an important hormonal axis for blood pressure maintenance and cardiovascular functions (Crackower et al., 2002) . SARS-CoV or SARS-CoV-2 infection requires a suitable cell-surface level of ACE2 but not J o u r n a l P r e -p r o o f its catalytic activities (Gordon et al., 2020; Li et al., 2005; Schneider et al., 2021; Wang et al., 2008; Zhang et al., 2021d) . Newly synthesized ACE2 is N-glycosylated in the ER and then transported to the cis Golgi apparatus, where it is further modified, packaged and translocated to the PM via the vesicular trafficking route (Hirschberg et al., 1998; Lumangtad and Bell, 2020; Vincent et al., 2005) . S glycoprotein forms homotrimers on the virus surface. Each monomer consists of an S1 subunit (extracellular subunit) and an S2 subunit (transmembrane subunit) (Walls et al., 2020; Watanabe et al., 2020; Wrapp et al., 2020) . During its biosynthesis, the S protein is cleaved in the Golgi apparatus by the proprotein convertase Furin at the S1-S2 boundary, where the polybasic cleavage site (PRRAR) is located (Hoffmann et al., 2020a; Shang et al., 2020; Walls et al., 2020) . The S1 subunit remains non-covalently associated with S2. Furin-like cleavage sites are present in the entry glycoproteins of other human viruses, such as HIV, HCoV-OC43 and MERS-CoV (Binley et al., 2002; Le Coupanec et al., 2015; Millet and Whittaker, 2014) . The Furin target site (PRRAR) is absent from the SARS-CoV S protein, even though it displays ~76% amino acid identity and shares a similar entry process with SARS-CoV-2 (Hartenian et al., 2020; Hoffmann et al., 2020a; Zhang et al., 2021a) . The Furin cleavage site is an important determinant of the increased transmissibility of SARS-CoV-2 compared to SARS-CoV (Shang et al., 2020) . At the initial step of SARS-CoV-2 infection, the receptorbinding domain (RBD) of the S1 subunit specifically binds to ACE2, resulting in stable association of the virion with the PM (Hartenian et al., 2020) . The interaction induces conformational changes and subsequent exposure of an internal site in the S2 J o u r n a l P r e -p r o o f subunit (S2' cleavage site), which can be cleaved by cell surface-localized transmembrane protease serine 2 (TMPRSS2) (Hoffmann et al., 2020a; Tang et al., 2021) . In cells expressing TMPRSS2, such as human respiratory epithelium, S2' cleavage usually occurs at the host cell surface. This process subsequently induces S1/ACE2 shedding and dramatic conformational changes of the S2 subunit ( Figure   2A ). The exposed hydrophobic fusion peptide of S2 inserts into the target cell membrane and mediates membrane fusion to release the viral genome into the cytosol for transcription and replication (Hoffmann et al., 2021b; Zhang et al., 2020) .

TMPRSS2 plays important roles in the entry of respiratory viruses, such as SARS-CoV-2, SARS-CoV and Avian Influenza Viruses (Bestle et al., 2021; Glowacka et al., 2011; Song et al., 2020) . Inhibition of TMPRSS2 significantly prevents SARS-CoV and SARS-CoV-2 from entering into target cells (Hoffmann et al., 2021b; Kawase et al., 2012) . Human coronaviruses, such as SARS-CoV-2, SARS-CoV, MERS-CoV and HCoV-229E, also exploit the endocytosis pathway for viral entry ( Figure 2B ) (Bayati et al., 2021; Inoue et al., 2007; Lu et al., 2013; Nomura et al., 2004; Wang et al., 2008) .

Coronaviruses can enter host cells via clathrin-mediated endocytosis and other endocytic processes. For example, SARS-CoV uses both clathrin-mediated and clathrin-and caveola-independent endocytic pathways for viral entry (Inoue et al., 2007; Wang et al., 2008) , while lipid raft/caveola-mediated endocytosis is involved in HCoV-229E entry and probably in mouse hepatitis virus (MHV) entry (Choi et al., 2005; Nomura et al., 2004) . In the absence of TMPRSS2, the ACE2-engaged SARS-J o u r n a l P r e -p r o o f CoV-2 virion is internalized into the cytosol through clathrin-mediated endocytosis (Bayati et al., 2021; Yang et al., 2022) . SARS-CoV-2 pseudovirus infection assays suggest that caveolar/lipid raft-and cytoskeleton-mediated endocytosis are also involved in viral infection (Li et al., 2021a; Zhou et al., 2022) . After fusion of virioncontaining endosomes with lysosomes, S2 subunit is cleaved by cathepsin cysteine proteases (e.g. CTSB and CTSL), resulting in the exposure of the fusion peptide to promote fusion between endosomal and viral membranes for release of the viral genome (Hoffmann et al., 2020b; Mellott et al., 2021) . The S protein of the recently emerged SARS-CoV-2 Omicron variant shows less efficient TMPRSS2-mediated S1/S2 cleavage Zhao et al., 2022) . The entry of Omicron mainly depends on the endocytic pathway, and compared to the Delta variant it thus exhibits lower infectivity in cells expressing high levels of TMPRSS2, and less sensitivity to the TMPRSS2 inhibitor Camostat Zhao et al., 2022) . The different entry routes exploited by Omicron and other variants may contribute to the distinct clinical manifestations.

Genome-wide screens have revealed that host factors involved in endocytic cargo internalization and endosomal trafficking/recycling are essential for SARS-CoV-2 entry (Daniloski et al., 2021; Zhu et al., 2021) . These host factors include endosomal cargo-sorting nexin-27 (SNX27), the retromer complex, the COMMD/CCDC22/CCDC93 (CCC) complex, the actin-related protein 2/3 (ARP2/3) complex, the ARP2/3 activator WASH complex, and the late-endosomal/lysosomal GTPase Rab7a (Daniloski et al., 2021; Zhu et al., 2021) . The ARP2/3 complex acts as J o u r n a l P r e -p r o o f an actin-polymerization/organization factor, initiating actin filament branches (Welch et al., 1997) . During endocytic cargo internalization, the ARP2/3 complex and type I myosin motor provide the force to extend vesicle invagination (Sun et al., 2006) . Inhibiting ARP2/3-mediated actin polymerization blocks endocytic uptake of cell membrane proteins (Park et al., 2013; Rocca et al., 2008) . The endosomal protein sorting and trafficking complex retromer, which consists of vacuolar protein sorting proteins (VPS26, VPS29 and VPS35), mediates the recycling of transmembrane proteins to various compartments. In concert with sorting nexins, retromer recruits the WASH complex to activate ARP2/3-dependent actin polymerization, and facilitates the formation of endosomal actin-enriched subdomains for selective cargo recycling (Gomez and Billadeau, 2009; Puthenveedu et al., 2010) . SNX27 acts as a cargosorting nexin of retromer, mediating the recycling of transmembrane receptors from the endosome to the PM (Gallon et al., 2014; Lauffer et al., 2010) . During SARS-CoV infection, the internalized ACE2 is recycled from endosomes back to the PM (Wang et al., 2008) . ACE2 binds to the PDZ domain of SNX27 through its short linear motifs (SLiMs) (Kliche et al., 2021) , which suggests that the internalized ACE2 might be rapidly recycled back to the PM via the SNX27-retromer complex. Levels of surface ACE2 are dramatically reduced in cells with knockout (KO) of COMMD3, CCDC53

(encoding a component of WASH complex), VPS29 and VPS35 .

Interestingly, the internalized ACE2 is transported to lysosomes for degradation during angiotensin II (Ang-II) treatment (Deshotels et al., 2014) . The mechanism specifying ACE2 degradation or recycling has yet to be determined. SNX27 also J o u r n a l P r e -p r o o f interacts with the SARS-CoV/SARS-CoV-2 S protein, facilitating its intracellular trafficking and cell surface expression and consequently promoting S proteinmediated cell-to-cell fusion and viral entry (Petit et al., 2005; Zhao et al., 2021a) .

Depletion of SNX27, retromer, WASH or components of the ARP2/3 complex (i.e. ACTR2, ACTR3, ARPC3 and ARPC4) effectively prevents SARS-CoV-2 pseudovirus entry and SARS-CoV-2 infection (Daniloski et al., 2021; Zhu et al., 2021) . Rab7a is involved in trafficking and degradation of cell membrane receptors through the endo-lysosome pathway (Shinde and Maddika, 2016) . Rab7a also recruits the retromer complex to late endosomes through interaction with VPS35, thus contributing to endosome maturation and cargo export (Rojas et al., 2008) . Depletion of Rab7a reduces viral entry by sequestering the ACE2 receptor in endosomes (Daniloski et al., 2021) . The SARS-CoV-2 NSP7 interacts with Rab7a, which suggests that NSP7 may manipulate Rab7a to facilitate viral infection (Gordon et al., 2020) . Thus, endocytic trafficking/recycling plays multiple roles in the entry of coronaviruses.

Positive-stranded RNA viruses remodel the endomembrane system to form viral replication organelles (ROs) for RNA synthesis. ROs serve as platforms for docking cellular and viral factors for viral genome replication, and also shield viral RNAs from cellular innate immune defenses or degradation (Harak and Lohmann, 2015; Scutigliani and Kikkert, 2017; Snijder et al., 2020) . The architecture of ROs varies J o u r n a l P r e -p r o o f among different virus families. Alphaviruses, flaviviruses, bromoviruses and nodaviruses generate small invaginations, called spherules/vesicles, in the membrane of specific organelles, such as the ER, mitochondria and endolysosomes. The invaginated spherules/vesicles are often tethered to the endomembrane and possess a narrow channel connecting the spherule interior with the cytosol (Ertel et al., 2017; Fernández de Castro et al., 2017; Gillespie et al., 2010; Kallio et al., 2013; Kujala et al., 2001; Schwartz et al., 2004; Welsch et al., 2009) . In contrast, coronaviruses, noroviruses, hepacivirus, arteriviruses and picornaviruses induce the protrusion of DMVs from the ER (Belov et al., 2012; Doerflinger et al., 2017; Gosert et al., 2003; Knoops et al., 2008; Wolff et al., 2020) . DMVs create a microenvironment for RNA replication and subgenomic mRNA synthesis, and also serve as storage sites for negative-stranded templates and double-stranded viral RNAs. Infection by betacoronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and MHV, generates a reticulovesicular network (RVN) consisting of DMVs (200-300 nm in diameter) that connect to a complex of convoluted membranes (CMs) derived from the ER (Knoops et al., 2008; Ogando et al., 2020; Snijder et al., 2020; Wolff et al., 2020) . DMV biogenesis involves multiple membrane rearrangements, including membrane pairing, curvature induction and membrane fission. The paired ER membrane forms CMs, which are reticular inclusion structures (Angelini et al., 2013; de Wilde et al., 2013; Knoops et al., 2008; Ulasli et al., 2010) . Electron tomography analysis revealed that DMV outer membranes are often connected to the ER or CMs (Knoops et al., 2008; Snijder et al., 2020; Wolff et al., 2020) . The number of DMVs increases at early stages of MHV infection but dramatically decreases at late stages (Mihelc et al., 2021) . Some DMVs are separated from the RVN and trafficked to lysosomes for degradation or exocytosis during MHV infection (Ghosh et al., 2020) .

Unlike invaginated spherules/vesicles, whose "neck-like" channels export nascent positive-sense viral RNAs to the cytosol for translation or virion assembly, DMVs are closed compartments that lack visible connections to the cytosol (Ertel et al., 2017; Knoops et al., 2008) . Cryo-electron microscopy analysis revealed that RNAs of MHV and SARS-CoV-2 are transported from the interior of DMVs to the cytosol via double-membrane-spanning pores (Wolff et al., 2020) . The pore complex in the MHV-induced DMV is a structure with sixfold symmetry. It consists of a crown-like structure extending ~13 nm into the cytosol, a ~24-nm-wide platform embedded in the double membranes, and a ~6-nm-wide channel connecting the DMV interior to the cytosol (Wolff et al., 2020) . NSP3 of MHV is the major component of the cytosolic crown-like structure of the DMV pore complex (Wolff et al., 2020) .

Pore-like complexes are also observed in SARS-CoV-2-induced DMVs, but the role of SARS-CoV-2 NSP3 in DMV pore formation is still obscure. Further investigations will be required to identify the constituents of the inter-membrane platform and also the cytosolic/luminal partners associated with the pore complex that mediate viral RNA synthesis, transcription, import/export, RNA packaging and transport. Hagemeijer et al., 2014) . 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) . NSP3 and NSP4 are located in the outer and inner J o u r n a l P r e -p r o o f 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.

Genome-wide CRISPR/Cas9 screens, proteomics and SARS-CoV-2-human interactomics have identified host factors that interact with viral proteins for DMV formation (Daniloski et al., 2021; Gordon et al., 2020; Stukalov et al., 2021; . The lipid transport protein sigma nonopioid intracellular receptor 1 (SIGMAR1) contributes to viral replication Su, 2004, 2007; Yang et al., 2019) . Depletion or inhibition of SIGMAR1 effectively reduces SARS-CoV-2 infectivity (Daniloski et al., 2021; Gordon et al., 2020) . Additionally, SIGMAR1 mediates the early steps of viral RNA replication during hepatitis C virus (HCV) infection, which also involves the formation of DMVs (Friesland et al., 2013) . As a cholesterol-and sphingolipid-binding protein, SIGMAR1 primarily resides in lipidrich areas of the ER (Hayashi and Su, 2003) . It is possible that viral proteins bind to SIGMAR1 and use constituents (such as cholesterol) of the ER as platforms for the initial steps of DMV formation. Consistent with this, genes involved in cholesterol or lipid synthesis, such as SCAP, MBPTS1 and MBPTS2, have also been identified as critical factors for SARS-CoV-2 infection (Schneider et al., 2021; Wang et al., 2021) . Morita, 2019; Neufeldt et al., 2018) . The role of RTNs and PI(4)P in DMV formation in SARS-CoV-2-infected cells has yet to be determined. In general, generation of DMVs requires interactions between viral transmembrane NSPs and host proteins to trigger ER membrane pairing and curvature.

For coronaviral particle assembly, the genomic RNAs exported from the DMV are recognized by the dimeric nucleocapsid phosphoprotein (N) and tightly packaged into higher-order supercoiled ribonucleoprotein complexes (RNPs) (Dinesh et al., 2020; Gui et al., 2017) . The M protein interacts with RNPs and S glycoproteins at the budding site to promote assembly and to control the viral particle shape (Neuman et al., 2011) . Budding occurs at the ERGIC (Ogando et al., 2020; Stertz et al., 2007; Wolff et al., 2020) . A recent study showed that SARS-CoV-2 induces the formation of single-membrane vesicles (SMVs) as virion assembly and budding sites (Mendonça et al., 2021) . Transport vesicles containing S proteins fuse with the SMVs. The S proteins on the SMV membrane form clusters with viral RNPs to initiate the assembly of viral particles and their subsequent budding and release into the SMV (Mendonça et al., 2021) . Elucidating the nature of the endomembrane structures that facilitate virion assembly requires detailed dynamic analysis of characteristic markers for various compartments.

Autophagy is a cellular metabolic process in which cytoplasmic contents are J o u r n a l P r e -p r o o f sequestered within a double-membrane autophagosome, which eventually fuses with lysosomes for degradation (Zhao et al., 2021b) . The core step in autophagy is the generation of the double-membrane autophagosome from a precursor called an isolation membrane (IM). Upon autophagy induction, the FIP200/ULK1/ATG13/ATG101 complex and vesicles carrying the multispanning membrane protein Atg9 are targeted to the ER. This is followed by recruitment of the class III VPS34 phosphatidylinositol 3-kinase (PI3K) complex to generate PI(3)Penriched ER subdomains, which serve as platforms for subsequent recruitment of other ATG proteins for autophagosome formation (Ktistakis and Tooze, 2016) . The integral ER membrane proteins VAPA/B (VAPs) and Atlastin 2/3 (ATLs) facilitate and stabilize the association of the FIP200/ULK1 complex with the ER (Zhao et al., 2018; Liu et al., 2021) . The origin of the initial membrane seed for the IM remains unknown. The phospholipid transfer activity of ATG2, which transports lipids from the ER, and the lipid scramblase activity of ATG9 are involved in IM expansion (Matoba et al., 2020; Maeda et al., 2020; Zhang, 2020) . Two ubiquitin-like conjugation systems (conjugation of Atg8 to phosphatidylethanolamine (PE) by the E1-like enzyme ATG7 and the E2-like enzyme ATG3; and conjugation of ATG12 to ATG5 by ATG7 and the E2-like enzyme ATG10, which further interacts with ATG16) act at several steps of autophagosome formation, including IM expansion and closure (Mizushima, 2020; Nakatogawa, 2020) . During expansion, the IM forms dynamic contacts with the ER. The ER transmembrane autophagy protein VMP1 controls the dynamics of the IM-ER contact (Tian et al., 2010; Zhao et al., 2017) .

DMVs and autophagosomes are both double-membrane structures, but the membrane remodeling processes are distinct. The DMV is directly generated via remodeling of the ER, while autophagosomes requires the ER as a platform and also as a lipid source for IM expansion (Snijder et al., 2020; Zhao and Zhang, 2019) . The ER does not directly constitute the membrane of autophagosomes (Zhao and Zhang, 2019) . DMVs in cells infected with coronaviruses, such as MHV, SARS-CoV and SARS-CoV-2, are not generated via the canonical autophagic machinery (Prentice et al., 2004; Reggiori et al., 2010; Shang et al., 2021) . In the canonical autophagy pathway, the mammalian Atg8 family protein LC3 is conjugated to PE in autophagosome membranes. Interestingly, non-lipidated LC3 is colocalized with

lipidation is not required for the replication of MHV or SARS-CoV Schneider et al., 2012; Zhao et al., 2007) . Instead, components which modulate the ER-associated degradation (ERAD) pathway are involved in MHV-induced DMV formation and viral replication . To tightly control ERAD activity, effectors, including mannosidase alpha-like 1 (EDEM1), osteosarcoma amplified 9 (OS9), and ERAD component SEL1, are delivered from the ER to late endosomes/lysosomes through ER-derived vesicles named EDEMosomes (Noack et al., 2014) . The non-lipidated form of LC3 (LC3-І) interacts with SEL1 and is (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 crossmembrane distribution of phospholipids, such as cholesterol and phosphatidylserine (Ghanbarpour et al., 2021; Li et al., 2021b; Huang et al., 2021) . Depletion of PS synthetase PTDSS1 partially suppresses the DMV defects in VMP1 KO cells (Ji et al., 2022) , which indicates that VMP1 may participate in DMV formation by regulating PS levels. During autophagosome formation, VMP1 promotes the activity of the ER-J o u r n a l P r e -p r o o f localized Ca 2+ pump SERCA to control ER-IM contacts (Zhao et al., 2017) . However, inhibition of SERCA activity by thapsigargin has no effect on DMV formation (Ji et al., 2022) , which indicates that VMP1 functions in autophagosome and DMV formation via distinct mechanisms. Thus, SARS-CoV-2 exploits a subset of autophagy machinery components to facilitate viral replication.

Similar to other enveloped RNA viruses, such as HCV and DENV, coronaviruses use smooth-wall secretory vesicles for release. In this process, trans-Golgi network (TGN)-localized viral particles are trafficked to the PM for egress (Machamer, 2013; Robinson et al., 2018) . SARS-CoV-2 modulates the Golgi apparatus to facilitate viral trafficking. SARS-CoV-2 infection or expression of certain viral proteins induces Golgi fragmentation by modulating the expression level of GRASP55 and TGN46 . Interference with the dysregulated GRASP55/TGN46 expression reduces SARS-CoV-2 infection in Huh7-ACE2 cells (Scherer et al., 2022; Zhang et al., 2022) . Transmission electron microscopy analysis revealed the presence of a large number of small secretory vesicles containing a single virion in SARS-CoV-2-infected Vero cells (Eymieux et al., 2021) . Consistent with this, multiple subunits of the exocyst, an octameric protein complex that interacts with the ARP2/3 complex and facilitates the tethering of secretory vesicles to the PM, have been isolated as host factors essential for the SARS-CoV-2 infection cycle (Schneider et al., 2021; Wang et al., 2021) . Coronaviruses such as SARS-CoV-2 and MHV also J o u r n a l P r e -p r o o f appear to use the late endosome/lysosome exocytic pathway for release (Ghosh et al., 2020) . SARS-CoV-2 and MHV virions are enriched in endosomes labeled by the late endosomal/lysosomal marker LAMP1. The level of LAMP1 is dramatically increased after SARS-CoV-2 infection Ghosh et al., 2020) . The Rab7a inhibitor CID1067700, which inhibits the maturation of endo-lysosomes, but not the biosynthetic secretory inhibitor Brefeldin A (BFA), significantly reduces betacoronavirus egress (Ghosh et al., 2020) . Late endosome/lysosome-localized SARS-CoV-2 ORF3a promotes the recruitment of BORC-ARL8b complex and exocytosis-related SNARE proteins to facilitate retrograde transport of lysosomes and subsequent fusion with the PM . Overexpression of SARS-CoV-2

ORF3a promotes egress of MHV, which itself does not contain an ORF3a counterpart . During SARS-CoV-2 infection, lysosomes are deacidified (Ghosh et al., 2020) . Lysosomal neutralization promotes their exocytosis (Carnell et al., 2011; Miao et al., 2015) . SARS-CoV-2 ORF3a also causes a certain amount of lysosomal damage and deacidification. The number of Galectin 3-labeled damaged lysosomal structures is slightly increased and the fluorescence intensities of puncta labeled by LysoTracker, a pH-sensitive acid dye, are weaker in ORF3a-expressing cells Miao et al., 2021) . ORF7a expression also causes deacidification of lysosomes (Hayn et al., 2021) . Indirect causes of lysosomal deacidification may include excessive loading with viral particles or impaired function of lysosomal proton pumps or ion channels (Westerbeck and Machamer, 2019) . The lysosomal membrane protein TMEM106B, components of the vacuolar ATPase proton pump, J o u r n a l P r e -p r o o f and cathepsins are essential host factors for SARS-CoV-2 infection (Baggen et al., 2021; Daniloski et al., 2021; Wang et al., 2021) . Lysosomal exocytosis requires lysosomal Ca 2+ release, which is mediated by lysosomal/endosomal membranelocalized Ca 2+ channel mucolipins (TRPML1, TRPML2 and TRPML3) (Rosato et al., 2021) . Depletion of TRPML3 effectively inhibits ORF3a-mediated lysosomal exocytosis . Knocking down TRPMLs prevents SARS-CoV-2 invasion . Berbamine inhibits SARS-CoV-2 infection by inhibiting TRPML-mediated lysosomal Ca 2+ release . Berbamine treatment inhibits endolysosomal retrograde trafficking and fusion with the PM, and also causes an increase in alternative secretion of ACE2 via exosomes, resulting in the reduction of cell surface-localized ACE2 .

Autophagy acts as a surveillance system by selectively removing damaged/obsolete organelles, protein aggregates, and invading pathogens (Deretic et al., 2013; Sharma et al., 2018) . Autophagy has different and even opposite functions during the infection of different viruses. Autophagy mediates viral clearance by degrading virions or nascent viral proteins/genomes and also enhances the immune response through antigen presentation (Deretic et al., 2013) . Many viruses, such as MERS-CoV, Sindbis virus and herpes simplex virus 1 (HSV-1), have evolved various mechanisms to inhibit autophagy and even to exploit autophagy components to benefit viral replication (English et al., 2009; Orvedahl et al., 2010) . MERS-CoV J o u r n a l P r e -p r o o f infection leads to a reduced level of the autophagy protein BECN1 and blocks the fusion of autophagosomes with lysosomes (Gassen et al., 2019) . Inhibition of SKP2, which increases the BECN1 level and induces autophagy, effectively inhibits MERS-CoV replication (Gassen et al., 2019) . The autophagy pathway has also been shown to positively regulate infection of many positive-stranded RNA viruses, including poliovirus (Taylor and Kirkegaard, 2008) , coxsackievirus (Yoon et al., 2008) , enterovirus (Huang et al., 2009) , HCV (Dreux et al., 2009) , and coronaviruses (Prentice et al., 2004; Shang et al., 2021) . Autophagy can facilitate viral egress. For example, the release of poliovirus and other enteroviruses, including human rhinovirus (HRV) and coxsackievirus B3 (CVB3), depends on non-lytic secretory autophagy, in which autophagosomes envelop the virions and fuse with the PM instead of lysosomes (Bird et al., 2014; Chen et al., 2015; Robinson et al., 2014) .

Depletion of autophagy genes, such as those encoding LC3 and Beclin1, leads to significantly decreased viral release (Bird et al., 2014; Chen et al., 2015; Robinson et al., 2014) . Autophagy also inhibits immune surveillance in SARS-CoV-2-infected cells. The SARS-CoV-2 ORF8 interacts with major histocompatibility complex class Ι (MHC-I) proteins and leads to their autophagic degradation, protecting SARS-CoV-2infected cells from being recognized and lysed by cytotoxic T lymphocytes . Inhibiting autophagy restores MHC-I expression in ORF8-expressing cells . NSP13 promotes autophagic degradation of TANKbinding kinase 1 (TBK1) by targeting it to the autophagy substrate p62 (Sui et al., 2022) . Removal of TBK1 inhibits type I interferon (IFN-I) production (Sui et al., J o u r n a l P r e -p r o o f 2022).

SARS-CoV-2 hijacks the autophagic machinery at multiple steps of the autophagy pathway to benefit viral infection (Figure 4) . SARS-CoV-2 infection promotes autophagosome generation but inhibits autophagic degradation by preventing formation of degradative autolysosomes (Miao et al., 2021) . Nascent autophagosomes undergo a maturation process by fusing with early endosomes, late endosomes and lysosomes to generate single-membrane amphisomes, which eventually form degradative autolysosomes (Zhao and Zhang, 2019; . SARS-CoV-2 blocks fusion of autophagosomes/amphisomes with lysosomes by sequestering the homotypic fusion and protein sorting complex (HOPS) component VPS39 on late endosomes (Mendonça et al., 2021; Miao et al., 2021) .

SARS-CoV-2 ORF3a interacts with and sequesters VPS39 on late endosomes/lysosomes and subsequently prevents the assembly of the STX17-SNAP29-VAMP8 SNARE complex that mediates autolysosome formation (Figure 4 ) (Miao et al., 2021) . SARS-CoV-2 ORF3a also sequesters the autophagy regulator UVRAG to facilitate autophagosome formation but inhibit autophagosome maturation (Qu et al., 2021) . Meanwhile, ORF3a impairs lysosomal degradation (Miao et al., 2021; Zhang et al., 2021c) . Other SARS-CoV-2 viral proteins also modulate autophagy. NSP15 of SARS-CoV-2 blocks autophagy induction, while ORF7a reduces the acidity of lysosomes, resulting in inhibition of autolysosome degradation ( Figure 4) (Hayn et al., 2021; Miao et al., 2021) . The viral M and E proteins also inhibit formation of acidified autolysosomes (Hayn et al., 2021; Miao et al., 2021) .

The viral particles are detected in amphisomes in SARS-CoV-2-infected cells (Miao et al., 2021) . Amphisomes also participate in unconventional secretion (Ganesan and Cai, 2021) . Therefore, multiple SARS-CoV-2 viral proteins causing accumulation of amphisomes may simultaneously facilitate exocytosis for viral release.

Autophagosomes formed in SARS-CoV-2-infected cells are smaller than autophagosomes induced by starvation in uninfected cells (Miao et al., 2021) . The NSP6 proteins of coronaviruses, including IBV, MHV, and SARS-CoV, have been shown to induce generation of ER-derived vesicles positive for ATG5 and lipidated LC3 (LC3-II). These vesicles possess properties of nascent autophagosomes but with a smaller diameter than starvation-induced autophagosomes (Cottam et al., 2011; . Autophagosome biogenesis involves the formation of a hybrid preautophagosomal structure (HyPAS), which is derived from the fusion of cis Golgi and endosomal membranes . The SARS-CoV-2 NSP6 has been shown to effectively inhibit HyPAS formation . The small autophagosomes may degrade viral elements less efficiently (Cottam et al., 2014; Gassen et al., 2019) . NSP6 also impairs lysosomal acidification through interaction with the lysosomal ATPase component ATP6AP1, which inhibits its activation . The autophagy pathway appears to be differentially modulated during Our improved understanding of endomembrane system remodeling during SARS-CoV-2 infection also provides potential drug targets and diagnostic approaches.

The current strategies for preventing SARS-CoV-2 infection mainly focus on blocking virus entry by therapeutic antibodies, inhibiting the viral RNA polymerase and protease, and enhancing the immune system. The emergence of SARS-CoV-2 variants, such as Omicron, with enhanced transmissibility and evasion of neutralizing antibodies (Hoffmann et al., 2022; Yin et al., 2022) , highlights the importance of developing approaches to explore cellular mechanisms for treatment. Several inhibitors targeting the endomembrane system have been shown to effectively prevent SARS-CoV-2 infection. For example, FDA-approved drugs such as Haloperidol target the interaction between SIGMAR1 and NSP6 (Gordon et al., 2020) . Chloroquine, which inhibits autolysosomal/endolysosomal protease activities by increasing the lysosomal pH, exerts antivirus function in SARS-CoV-2-infected Vero E6 cells . Chloroquine fails to block SARS-CoV-2 infection in the TMPRSS2expressing human lung cell line Calu-3 (Hoffmann et al., 2020c) . Inhibitors of PI3K, which effectively prevent DMV formation and viral replication, are also promising antiviral candidates (Twu et al., 2021) . More importantly, the interaction between viral spike proteins and receptors appears to be virus-specific. Endomembrane remodeling, such as generation of DMVs for viral replication and modulation of the secretory and lysosome pathways for egress, is generally required for coronavirus infection. The endomembrane system can be targeted as a therapeutic strategy to combat SARS-CoV-2, its yet-to-emerge variants, and even new coronaviruses. 

Sense RNA Viruses

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response

Genome-wide CRISPR screening identifies TMEM106B as a proviral host factor for SARS-CoV-2

SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis

Mutations across murine hepatitis virus nsp4 alter virus fitness and membrane modifications

Complex dynamic development of poliovirus membranous replication complexes

Hemagglutinins of Avian Influenza Viruses Are Proteolytically Activated by TMPRSS2 in Human and Murine Airway Cells

Enhancing the proteolytic maturation of human immunodeficiency virus type 1 envelope glycoproteins

Nonlytic viral spread enhanced by autophagy components

Four proteins processed from the replicase gene polyprotein of mouse hepatitis virus colocalize in the cell periphery and adjacent to sites of virion assembly

Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress

Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses

Emerging coronaviruses: Genome structure, replication, and pathogenesis

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Mutation in murine coronavirus replication protein nsp4 alters assembly of double membrane vesicles

Integrative Imaging Reveals SARS-CoV-2-Induced Reshaping of Subcellular Morphologies

Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate

Coronavirus NSP6 restricts autophagosome expansion

Angiotensin-converting enzyme 2 is an essential regulator of heart function

The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2

Origin and evolution of pathogenic coronaviruses

Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment

Autophagy in infection, inflammation and immunity

Angiotensin II mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin II type I receptor-dependent mechanism

Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein

Membrane alterations induced by nonstructural proteins of human norovirus

The autophagy machinery is required to initiate hepatitis C virus replication

Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection

Cryo-electron tomography reveals novel features of a viral RNA replication compartment

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress

Three-dimensional imaging of the intracellular assembly of a functional viral RNA replicase complex

Sigma-1 receptor regulates early steps of viral RNA replication at the onset of hepatitis C virus infection

A unique PDZ domain and arrestinlike fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer

Understanding amphisomes

SKP2 attenuates autophagy through Beclin1-ubiquitination and its inhibition reduces MERS-Coronavirus infection

A model for a partnership of lipid transfer proteins and scramblases in membrane expansion and organelle biogenesis

β-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway

The endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex

Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response

A FAM21-containing WASH complex regulates retromer-dependent sorting

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons

Electron microscopy studies of the coronavirus ribonucleoprotein complex

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4. Virology 458-459

Mobility and interactions of coronavirus nonstructural protein 4

Visualizing coronavirus RNA synthesis in time by using click chemistry

Ultrastructure of the replication sites of positivestrand RNA viruses

The molecular virology of coronaviruses

Sigma-1 receptors (sigma(1) binding sites) form raftlike microdomains and target lipid droplets on the endoplasmic reticulum: roles in endoplasmic reticulum lipid compartmentalization and export

Sigma-1 receptors at galactosylceramide-enriched lipid microdomains regulate oligodendrocyte differentiation

Sigma-1 receptor chaperones at the ERmitochondrion interface regulate Ca(2+) signaling and cell survival

Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities

Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells

TMEM41B Is a Pan-flavivirus Host Factor

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

The Omicron variant is highly resistant against antibody-mediated neutralizationimplications for control of the COVID-19 pandemic

Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract

TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis

Berbamine inhibits SARS-CoV-2 infection by compromising TRPMLs-mediated endolysosomal trafficking of ACE2

Enterovirus 71-induced autophagy detected in vitro and in vivo promotes viral replication

Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted

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

Template RNA length determines the size of replication complex spherules for Semliki Forest virus

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids

Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry

Cytoplasmic short linear motifs in ACE2 and integrin β(3) link SARS-CoV-2 host cell receptors to mediators of endocytosis and autophagy

Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis

SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum

Digesting the Expanding Mechanisms of Autophagy

Biogenesis of the Semliki Forest virus RNA replication complex

Mammalian hybrid preautophagosomal structure HyPAS generates autophagosomes

SNX27 mediates PDZ-directed sorting from endosomes to the plasma membrane

Cleavage of a Neuroinvasive Human Respiratory Virus Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System

Angiotensinconverting enzyme 2 is a functional receptor for the SARS coronavirus

Receptor and viral determinants of SARScoronavirus adaptation to human ACE2

Dependence of SARS-CoV-2 infection on cholesterol-rich lipid raft and endosomal acidification

TMEM41B and VMP1 are scramblases and regulate the distribution of cholesterol and phosphatidylserine

Atlastin 2/3 regulate ER targeting of the ULK1 complex to initiate autophagy

Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26

The signal peptide as a new target for drug design

Accommodation of large cargo within Golgi cisternae

Structure, lipid scrambling activity and role in autophagosome formation of ATG9A

Atg9 is a lipid scramblase that mediates autophagosomal membrane expansion

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells

Correlative multi-scale cryo-imaging unveils SARS-CoV-2 assembly and egress

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity

ORF3a of the COVID-19 virus SARS-CoV-2 blocks HOPS complexmediated assembly of the SNARE complex required for autolysosome formation

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles

Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein

The ATG conjugation systems in autophagy

Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

Mechanisms governing autophagosome biogenesis

The enigmatic endosome -sorting the ins and outs of endocytic trafficking

Rewiring cellular networks by members of the Flaviviridae family

Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3

A structural analysis of M protein in coronavirus assembly and morphology

How viruses hijack the ERAD tuning machinery

Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae

SARS-coronavirus-2 replication in Vero E6 cells: replication kinetics, rapid adaptation and cytopathology

Autophagy protects against Sindbis virus infection of the central nervous system

Expression and Cleavage of Middle East Respiratory Syndrome Coronavirus nsp3-4 Polyprotein Induce the Formation of Double-Membrane Vesicles That Mimic Those Associated with Coronaviral RNA Replication

Methamphetamine-induced occludin endocytosis is mediated by the Arp2/3 complex-regulated actin rearrangement

Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

Coronavirus replication complex formation utilizes components of cellular autophagy

Sequencedependent sorting of recycling proteins by actin-stabilized endosomal microdomains

ORF3a-Mediated Incomplete Autophagy Facilitates Severe Acute Respiratory Syndrome Coronavirus-2 Replication

Coronaviruses Hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication

Viral journeys on the intracellular highways

Coxsackievirus B exits the host cell in shed microvesicles displaying autophagosomal markers

Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

Two-pore and TRPML cation channels: Regulators of phagocytosis, autophagy and lysosomal exocytosis

Two-amino acids change in the nsp4 of SARS coronavirus abolishes viral replication

SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Severe acute respiratory syndrome coronavirus replication is severely impaired by MG132 due to proteasome-independent inhibition of Mcalpain

Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks

Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication

Interaction of the innate immune system with positive-strand RNA virus replication organelles

Inhibition of Autophagy Suppresses SARS-CoV-2 Replication and Ameliorates Pneumonia in hACE2 Transgenic Mice and Xenografted Human Lung Tissues

Cell entry mechanisms of SARS-CoV-2

Selective Autophagy and Xenophagy in Infection and Disease

PTEN modulates EGFR late endocytic trafficking and degradation by dephosphorylating Rab7

CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor

A unifying structural and functional model of the coronavirus replication organelle: Tracking down RNA synthesis

Powering membrane traffic in endocytosis and recycling

Expression of ACE2, the SARS-CoV-2 Receptor, and TMPRSS2 in Prostate Epithelial Cells

The intracellular sites of early replication and budding of SARS-coronavirus

A new cistron in the murine hepatitis virus replicase gene

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV

SARS-CoV-2 NSP13 Inhibits Type I IFN Production by Degradation of TBK1 via p62-Dependent Selective Autophagy

SARS-CoV-2 non-structural protein 6 triggers NLRP3-dependent pyroptosis by targeting ATP6AP1

Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity

Proteolytic Activation of SARS-CoV-2 Spike at the S1/S2 Boundary: Potential Role of Proteases beyond Furin

Potential subversion of autophagosomal pathway by picornaviruses

C. elegans screen identifies autophagy genes specific to multicellular organisms

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

Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus

Biogenesis and architecture of arterivirus replication organelles

Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication

Chloroquine is a potent inhibitor of SARS coronavirus infection and spread

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

SARS coronavirus entry into host cells through a novel clathrin-and caveolaeindependent endocytic pathway

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

Sitespecific glycan analysis of the SARS-CoV-2 spike

Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes

Composition and three-dimensional architecture of the dengue virus replication and assembly sites

The Infectious Bronchitis Coronavirus Envelope Protein Alters Golgi pH To Protect the Spike Protein and Promote the Release of Infectious Virus

A molecular pore spans the double membrane of the coronavirus replication organelle

Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

SIGMAR1/Sigma-1 receptor ablation impairs autophagosome clearance

Cytoplasmic domain and enzymatic activity of ACE2 is not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody

Coxsackievirus B4 uses autophagy for replication after calpain activation in rat primary neurons

Suppression of SARS-CoV-2 infection in ex-vivo human lung tissues by targeting class III phosphoinositide 3-kinase

Lipid transfer at ER-isolation membrane contacts

SARS-CoV-2 triggers Golgi fragmentation via down-regulation of GRASP55 to facilitate viral trafficking

SARS-CoV-2 spikeprotein D614G mutation increases virion spike density and infectivity

Furin cleavage of the SARS-CoV-2 spike is modulated by O-glycosylation

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι

The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

Potent prophylactic and therapeutic efficacy of recombinant human ACE2-Fc against SARS-CoV-2 infection in vivo

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells

SARS-CoV-2 spike protein harnesses SNX27-mediated endocytic recycling pathway

The ER-Localized Transmembrane Protein EPG-3/VMP1 Regulates SERCA Activity to Control ER-Isolation Membrane Contacts for Autophagosome Formation

Machinery, regulation and pathophysiological implications of autophagosome maturation

The ER Contact Proteins VAPA/B Interact with Multiple Autophagy Proteins to Modulate Autophagosome Biogenesis

Autophagosome maturation: An epic journey from the ER to lysosomes

Coronavirus replication does not require the autophagy gene ATG5

SARS-CoV-2 pseudovirus enters the host cells through spike protein-CD147 in Arf6-dependent manner

A genome-wide CRISPR screen identifies host factors that regulate SARS-CoV-2 entry

We are grateful to Dr. Isabel Hanson for editing work.