key: cord-0791638-baqjwq6l authors: Yang, Hang; Zhao, Xiaohui; Xun, Meng; Xu, Lingjie; Liu, Bing; Wang, Hongliang title: Cytoplasmic domain and enzymatic activity of ACE2 is not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells date: 2021-03-02 journal: bioRxiv DOI: 10.1101/2021.03.01.433503 sha: 97518c9c9c6fdb7ce570a9a3fb19f81b07919329 doc_id: 791638 cord_uid: baqjwq6l The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor- human angiotensin converting enzyme 2 (ACE2). Here, we used lentivirus based pseudotypes bearing spike protein to demonstrate that entry of SARS-CoV-2 into host cells is dependent on clathrin-mediated endocytosis, and phosphoinositides play essential role during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 is not required for efficient virus entry. These results provide new insights into SARS-CoV-2 cellular entry and present potential targets for drug development. Since the beginning of this century, there have been three beta-coronaviruses crossed the species barrier to cause severe respiratory diseases. In 2002-2003, sever acute respiratory syndrome coronavirus (SARS-CoV) infected 8096 people and caused 774 deaths (1) . In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) was discovered as causative agent of a severe respiratory syndrome in the middle east area and as of January 2020, more than 2500 people was diagnosed with MERS, with 866 associated deaths (WHO). In late 2019, a novel coronavirus, named SARS-CoV-2 emerged and soon transmitted to cause a globally pandemic. As of January 2021, there were over 100 million confirmed cases of COVID-19 worldwide, resulting in more than 2 million deaths (coronavirus.jhu.edu). All three coronaviruses are believed to originate from bats but zoonotic transmission involved different intermediate hosts. With numerous coronaviruses in bats, it is likely that coronavirus crises will continue to occur in the foreseeable future (2) . Cellular entry of coronavirus is mediated by its spike glycoprotein (S) by interacting with cellular receptors. The sequences of SARS-CoV and SARS-CoV-2 S protein are conserved and numerous studies have shown that they exploited the same receptor, angiotensin-converting enzyme 2 (ACE2) (3) (4) (5) (6) . Different from SARS-CoV, the S protein of SARS-CoV-2 was efficiently processed into two subunits, S1 and S2, which mediates attachment and membrane fusion, respectively (3, 6) . Structural analysis showed that S protein formed trimer with the receptor-binding domain exposed up for easy receptor accessibility (5, 6) . ACE2 is a type I transmembrane protein with an extracellular domain with homology to ACE and a short cytoplasmic tail (7, 8) . ACE2 is a Zinc metalloprotease that catalyzes the cleavage of Ang I to Ang1-9, but its catalytic activity is not required for spike induced syncytia formation (9) . The entry of enveloped viruses into cells is known to occur via two primary pathways, i.e. direct membrane fusion at the cell surface, or endocytosis, with the latter being a pHsensitive process (10) . The endocytic pathways exploited by animal viruses to gain entry into host cells include clathrin-mediated endocytosis (CME), caveolae-dependent endocytosis, as well as poorly characterized routes such as clathrin-and caveolaeindependent endocytosis (11) . SARS-CoV and MERS-CoV S can be triggered to fuse either at the plasma membrane or the endosomal membrane depending on protease availability (12) . In addition, SARS-CoV has been shown to enter host cells via direct membrane fusion, clathrin-mediated endocytosis or non-clathrin, non-caveolae-mediated endocytosis (13) (14) (15) , suggesting the virus might employ various strategies to expand its cellular tropism. The specific endocytic pathways SARS-CoV-2 exploited have not been fully characterized. Phosphoinositides(PI) are known to be involved in the whole process of endocytosis (16) , of note, PI(4,5)P2 has long been thought to be the most important PI during CME (17) (18) (19) (20) (21) . A recent study demonstrated that a successive PI molecular conversions accompanies the clathrin-coated pit assembly, budding and uncoating with PI(4,5)P2, PI4P, PI(3,4)P2 function at different stages of CME (22) . Interestingly, SARS-CoV has been reported to require PI4KIII for cell entry and depletion of PI4P by Sac1 inhibited viral entry(23), suggesting a role of PI4P during viral entry. In contrast, inhibitors targeting PI(3,5)P2 blocked SARS-CoV-2 viral entry (4) . Whether other PI molecules are required for SARS-CoV-2 entry is still unknown. Here we demonstrated that SARS-CoV-2 entered cells via clathrin-mediated endocytosis, but independent of caveolae-mediated endocytosis. Distinct from SARS-CoV, SARS-CoV-2 did not seem to require lipid rafts for infection. In addition, phosphatidylinositol molecules PI4P and PI(4,5)P2 also played essential roles during virus entry. Finally, we showed that the cytoplasmic domain and the enzymatic activity of ACE2 were not required for SARS-CoV-2 entry. Our data provided new insights into SARS-CoV-2 cellular entry and presented potential targets for drug development. Pseudovirus has been widely used to mimic the entry of real virus and is a powerful tool for studying early events in the life cycle of a virus. We here employed a lentivirus-based pseudovirus to study the entry of SARS-CoV-2. To facilitate the incorporation of SARS-CoV-2 S protein into pseudovirus, we employed a method similar as previously reported in (4) . A codon-optimized cDNA encoding the S protein was synthesized and the C-terminal last 19 amino acids, which contains an endoplasmic reticulum retention signal was replaced with FLAG tag (Fig S1A) . Immunoblotting showed that codon-optimized S cDNA expressed at a much higher level than that of native spike in 293T cells (Fig S1B) . In addition, consistent with previous reports (3, 4, 6) , the S protein was processed and two major bands were observed, reflecting the full-length and cleaved S proteins respectively( Fig S1B) . To confirm S protein was efficiently incorporated into the pseudovirus, spike and VSV-G protein-bearing pseudoviruses were pelleted by ultracentrifugation and tested against anti-spike antibody and anti-HIV-p24 antibody ( Fig 1A) . The results showed that the majority of S proteins on pseudovirus were cleaved, suggesting the S protein was efficiently processed in host cells. cells can be transduced by S pseudovirus, Huh 7 gave more than two-fold increase in luciferase activity than Vero E6 cells ( Figure 1B ). Thus, Huh 7 was chosen for the following viral entry studies. Similarly, GFP-expressing pseudoviruses also transduced the above cells lines successfully (Fig 1C) , further suggesting that the S pseudovirus we prepared can be used for viral entry studies. We next tested whether SARS-CoV-2 enters cells via endocytosis or direct membrane fusion at plasma membrane. In contrast to direct membrane fusion, endocytosis is thought to be pH-dependent (24) . When Huh 7 cells were pretreated with lysosomotropic agents, like chloroquine, ammonium chloride or bafilomycin A1, significant decreases of transduction were observed for both VSV-G and SARS-CoV-2 S pseudovirus (Fig 2A) , suggesting that SARS-CoV-2 entry of Huh 7 cells is pH-dependent. Consistent with this, dual immunostaining of S protein and early endosome marker EEA1 showed colocalization ( Fig 2B) , supporting that S pseudovirus infected Huh 7 cells via endocytosis. Importantly, we observed that S pseudovirus transduction correlated with higher EEA1 expression in cells ( Fig. S2 ) and immunoblotting showed that S pseudovirus transduction up-regulated EEA1 expression in Huh 7 cells ( Fig 2B) . All these suggested that SARS-CoV-2 enters cells via pH-dependent endocytosis. Clathrin-mediated endocytosis is the best documented mode of endocytosis, to test whether SARS-CoV-2 gain entry into cells via CME, we first pretreated cells with clathrin inhibitor, pitstop (25) . Pitstop inhibited the entry of SARS-CoV-2 in Huh 7 cells in a dosedependent manner (Fig 2C) . Similarly, chlorpromazine(CPZ), which prevents the assembly of coated pits at the cell surface, also inhibited the entry of SARS-CoV-2 in a dosedependent manner ( Fig 2D) . To overcome any nonspecific effect the chemical compounds may have, we also carried out an orthogonal CRISPR/Cas9 knockout experiment. Compared to control cells, uptake of cholera toxin subunit B (CTB), which has been reported to be internalized via caveolae-mediated endocytosis (28) (29) (30) , was blocked at cell surface when cells were treated with filipin or MCD (Fig S2) , suggesting these drugs can block caveolae-mediated endocytosis. However, neither drug inhibited the huh 7 transduction of S pseudovirus (Fig 3A, 3B ), indicating that SARS-CoV-2 entry of Huh 7 cells was independent of cholesterol-enriched membrane microdomains. To further study whether the caveolae-dependent pathway was involved in the endocytosis of this virus, we knocked out the expression of caveolin-1(CAV-1, Figure 3C ), which is the main scaffolding protein of the caveolae membranes in most cell types (31) . Figure 3D showed that S pseudovirus could infect Huh 7 cells in the absence of CAV-1, suggesting the viral entry is independent of caveolae. This finding was further corroborated by the immunostaining experiment. The infected cells immune-labelled for SARS-CoV-2 spike protein and for caveolin-1 showed little colocalization ( Figure 3E ). Taken together, these results indicated that SARS-CoV-2 is able to enter cells in a caveolae-independent manner. Phosphoinositides (PI) are known to be involved throughout the process of endocytosis. Of note, PI(4,5)P2, PI4P and PI (3, 4) P2 are essential molecules in endocytosis. We found that overexpression of either inositol polyphosphate-5-phosphatase E (INPP5E), which converts PI (4,5) P2 to PI4P, or the sac1 phosphatase, which dephosphorylates PI4P Receptor-mediated endocytosis involves the interaction of the cytoplasmic tails of the receptor proteins with intracellular proteins to initiate internalization (32) (33) (34) , and studies have shown that cytoplasmic domain of cell surface receptors could control endocytosis (35, 36) . ACE2 is a type I transmembrane protein with a short cytoplasmic tail (7, 8) . We therefore tested whether the cytoplasmic tail of ACE2 is required for SARS- We next tested whether the enzyme activity of ACE2 is required for SARS-CoV-2 infection. ACE2 is a Zinc metalloprotease with a single HEXXH zinc-binding motif (37, 38) . In addition, ACE2 structure modeling and site-directed mutagenesis analysis showed that His505 plays an essential role in the catalysis of ACE2(39-41) (Fig 5D) . To test whether enzymatic activity of ACE2 is required for SARS-CoV-2 entry, 293T cells overexpressing ACE2 that bears two zinc-binding histidine mutations (H374N&H378N, CM1) were transduced with S pseudovirus. Figure 5F showed that this mutant support viral infection equally well to wild type ACE2. Similarly, 293T cells overexpressing ACE2 catalytic site histidine mutation (H505L, CM2), or the combination of all three histidine mutations (H374N&H378N&H505L, CM3) could be transduced by S pseudovirus as efficiently (Fig.5D-F) . These results suggested that the enzymatic activity of ACE2 is not required for SARS-CoV-2 infection. As the best characterized endocytosis pathways, clathrin-mediated endocytosis has been reported to be involved in various viral infection, including SARS-CoV (14) . Here by using different chemical inhibitors and CRISPR/Cas9 targeting the major protein in clathrin coat formation, we demonstrated that SARS-CoV-2 entry also was dependent on clathrin. While we are preparing this manuscript, Bayati et al. reported that the Spike protein of SARS-CoV-2 endocytosis is dependent on clathrin and S pseudovirus can be blocked by CHC siRNA treatment (42) . Consistently, we also showed that SARS-CoV-2 pseudovirus enters cell via CME, in addition, we also showed that this process is independent of caveolaemediated endocytosis, another common endocytosis pathway. The fact that Caco2 cells, which do not express caveolin (43) , could support SARS-CoV-2 infection efficiently (44) also suggested that this virus could infect cells independent of caveolae. PI4P and PI4KB have been reported to be essential for SARS-CoV infection(23) and a recent report showed that PIKfyve inhibitors could block S pseudovirus infection (4), which all suggested that phosphoinositides pathways are important for coronavirus infection. Here we further demonstrated that PI4P and PI(4,5)P2 are essential molecules for SARS-CoV-2 infection and PI4KB is also required for SARS-CoV-2 entry. The role of cytoplasmic tail of ACE2 in SARS-CoV infection is controversial (14, 45) . We found that the cytoplasmic domain as well as the enzymatic activity of ACE2 are not required for efficient virus entry, which are consistent with the facts that the receptorbinding domain of S interacts with the N-terminal peptidase domain of ACE2 and the enzymatic sites are not in direct contacts with spike as revealed by the S-ACE2 complex structure (46, 47) . In summary, we demonstrated that SARS-CoV-2 pseudovirus infects cells via pH-dependent endocytosis, which is dependent on clathrin, but not caveolae. Phosphoinositides pathways, particularly, PI4KB is required for efficient virus infection. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 are not required for viral infection. Overall, these results provided new insights into SARS-CoV-2 cellular entry and present more precise targets for antiviral drug development. Huh 7, Vero E6 and 293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), nonessential amino acids, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. ACE2, Sac1 and INPP5E were amplified from cDNA library prepared with Huh 7 cells and each was cloned into a lentiviral expression vector pSMPUW. ACE2 truncation and mutants were obtained with overlap extension PCR amplification using primers listed in Table S1 . Codon-optimized spike of SARS-CoV-2 was purchased from GenScript and subcloned to pcDNA3.1. All constructs were confirmed by sequencing. 293T-ACE2 stable cell lines were generated by transfecting 293T cells with pSMPUM-ACE2 and selected with blasticidin. Antibiotic-resistant single clones were screened with immunoblotting and high ACE2-expressing clones were expanded for future use. Antibodies used in this study include: SARS-CoV-2 spike (GeneTex, Taiwan), HA tag, EEA1 Chloroquine, ammonium chloride, bafilomycin A1, chlorpromazine(CPZ), Filipin, MCD were purchased from Sigma. Pitstop was purchased from Abcam. PIK93, Wortmannin were purchased from MedChemExpress. Pseudovirus was prepared with a previously described protocol(4) with minor modification. Briefly, 293T cells were co-transfected with lentiviral packaging plasmid psPAX2, pcDNA3.1-Spike, pMD2.G, or empty vector, and lentiviral expression vector pSMPUW engineered with either GFP or NanoLuc luciferase. At 48h, 68h post-transfection, virus supernatants were harvested and filtered through 0.45 μm pore-size filters. Viral supernatants were then centrifuged at 30,000rpm for 2h in Himac ultracentrifuge S52ST rotor at 4 °C over a 20% sucrose solution, and virus stocks were resuspended with PBS and frozen at -80 °C. Unless otherwise indicated, Huh 7 cells were used for entry assays. For phosphatase inhibition test, 293T-ACE2 cells were first transfected with Sac1 or INPP5E. 30 hrs later, cells were split for immunoblotting and pseudovirus transduction. For ACE2 mutants assay, 293T cells were transfected with ACE2 or the indicated ACE2 mutant and 30 hrs later, cells were split for immunoblotting and pseudovirus transduction. To transduce cells with pseudovirus, cells seeded in 24-well plate were inoculated with 30ul of concentrated pseudovirus. About 36 hrs post inoculation, cells were lysed and NanoLuc luciferase activity was measured with Nano-Glo® Luciferase Assay System (Promega, Madison, WI) according to manufacturer's instructions using a Synergy Neo 2 plate reader (BioTek, Winooski, VT). For cells transduced with GFP pseudovirus, photos were taken at 56 hrs post inoculation. For virus entry inhibition studies, cells were pretreated with indicated concentrations of inhibitors for 1 hr at 37 °C before pseudovirus inoculation. NanoLuc luciferase activity was measured as described above. Immunofluorescence staining was performed as described in (48, 49) . Images were taken with a Nikon C2 laser scanning confocal microscope in sequential scanning mode to limit crosstalk between fluorochromes. shRNA knockdown or CRISPR knockout cell lines construction VSV-G pseudotyped shRNA or CRISPR lentiviruses were produced as previously reported (48, 50) . Briefly, 293T cells were co-transfected with psPAX2, pMD2.G and pLKO.1-Puro based shRNA or pLentiCRISPR V2 based sgRNA lentiviral vectors and supernatants were harvested and used for transduction. sgRNA sequences and shRNA sequences used were listed in Table S2 . Cells transduced with lentiviral particles were then selected with puromycin for stable cell lines. All values represent means ± standard deviations and represent the results of a minimum of three independent experiments. Where applicable, the two-tailed Student's t test was used to compare the means of control and experimental groups. The authors declare that they have no conflict of interest. Organelle. J Virol 93:e00742-19. SARS and MERS: recent insights into emerging coronaviruses Bat origin of human coronaviruses Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein The Cell Biology of the SARS Coronavirus Receptor, Angiotensin-Converting Enzyme 2 The emerging role of ACE2 in physiology and disease Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Opinion: Cell entry machines: a common theme in nature? Endocytosis unplugged: multiple ways to enter the cell Coronavirus membrane fusion mechanism offers a potential target for antiviral development Early events of SARS coronavirus infection in vero cells Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic tail deleted SARS coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway Mechanisms of clathrin-mediated endocytosis Phosphoinositide regulation of clathrin-mediated endocytosis Phosphoinositides in endocytosis Regulation of endocytosis by phosphatidylinositol 4,5-bisphosphate and ENTH proteins Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate Molecular mechanism and physiological functions of clathrin-mediated endocytosis Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic Phosphatidylinositol 4-kinase IIIbeta is required for severe acute respiratory syndrome coronavirus spikemediated cell entry Fusion of Enveloped Viruses in Endosomes Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition Structure of caveolae Disruption of Lipid Raft Function Increases Expression and Secretion of Monocyte Chemoattractant Protein-1 in 3T3-L1 Adipocytes Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains Pathophysiological Role of Caveolae in Hypertension Protein sorting by tyrosine-based signals: adapting to the Ys and wherefores Signaling on the endocytic pathway Endocytosis and signalling: intertwining molecular networks Fc receptor endocytosis is controlled by a cytoplasmic domain determinant that actively prevents coated pit localization Integrin cytoplasmic tail interactions ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captoprilinsensitive carboxypeptidase Angiotensinconverting enzyme-2 (ACE2): comparative modeling of the active site, specificity requirements, and chloride dependence Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis ACE2 X-ray structures reveal a large hingebending motion important for inhibitor binding and catalysis SARS-CoV-2 infects cells following viral entry via clathrin-mediated endocytosis Detergent-resistant membrane microdomains from Caco-2 cells do not contain caveolin Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 Oxysterolbinding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking The Role of the Phosphatidylinositol 4-Kinase PI4KA in Hepatitis C Virus-Induced Host Membrane Rearrangement Nir2 Is an Effector of VAPs Necessary for Efficient Hepatitis C Virus This work was supported by the National Natural Science Foundation of China (81871662), Xi'an Jiaotong University Fund (xzy012019066 and xzy032020037) and Xi'an Jiaotong University Health Science Center-Qinnong Bank Fund (QNXJTU-04& QNXJTU-07).