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Cell entry mechanisms of SARS-CoV-2

Cell entry mechanisms of SARS-CoV-2 a,1 a,1 a,1 a a a a,2 Jian Shang , Yushun Wan , Chuming Luo , Gang Ye , Qibin Geng , Ashley Auerbach , and Fang Li  Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108 Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved April 27, 2020 (received for review February 18, 2020) A novel severe acute respiratory syndrome (SARS)-like coronavirus spike protein is present as a trimer, with three receptor-binding (SARS-CoV-2) is causing the global coronavirus disease 2019 S1 heads sitting on top of a trimeric membrane fusion S2 stalk (COVID-19) pandemic. Understanding how SARS-CoV-2 enters hu- (Fig. 1B). The cell entry mechanism of SARS-CoV has been man cells is a high priority for deciphering its mystery and curbing extensively studied. SARS-CoV S1 contains a receptor-binding its spread. A virus surface spike protein mediates SARS-CoV-2 en- domain (RBD) that specifically recognizes angiotensin-converting try into cells. To fulfill its function, SARS-CoV-2 spike binds to its enzyme 2 (ACE2) as its receptor (17–19). The RBD constantly receptor human ACE2 (hACE2) through its receptor-binding do- switches between a standing-up position for receptor binding and main (RBD) and is proteolytically activated by human proteases. a lying-down position for immune evasion (20, 21) (Fig. 1B). Here we investigated receptor binding and protease activation of Moreover, to fuse membranes, SARS-CoV spike needs to be SARS-CoV-2 spike using biochemical and pseudovirus entry assays. proteolytically activated at the S1/S2 boundary, such that S1 Our findings have identified key cell entry mechanisms of dissociates and S2 undergoes a dramatic structural change (22, SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding af- 23). These SARS-CoV entry-activating proteases include cell finity than SARS-CoV RBD, supporting efficient cell entry. Second, surface protease TMPRSS2 and lysosomal proteases cathepsins paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 (22, 23) (Fig. 1A). These features of SARS-CoV entry contribute spike is comparable to or lower than that of SARS-CoV spike, suggest- to its rapid spread and severe symptoms and high fatality rates of ing that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is infected patients (24–26). preactivated by proprotein convertase furin, reducing its dependence The past several months saw an explosion of studies on the cell on target cell proteases for entry. The high hACE2 binding affinity of entry mechanisms of SARS-CoV-2, sometimes with conflicting the RBD, furin preactivation of the spike, and hidden RBD in the spike findings. Like SARS-CoV, SARS-CoV-2 also recognizes human potentially allow SARS-CoV-2 to maintain efficient cell entry while ACE2 (hACE2) as its receptor (27–29). We recently determined evading immune surveillance. These features may contribute to the the crystal structure of SARS-CoV-2 RBD complexed with wide spread of the virus. Successful intervention strategies must tar- hACE2, which revealed subtle but functionally important dif- get both the potency of SARS-CoV-2 and its evasiveness. ferences between SARS-CoV-2 and SARS-CoV in receptor recognition (30). These differences enable SARS-CoV-2 RBD to COVID-19 SARS-CoV-2 SARS-CoV ACE2 receptor proprotein | | | | have a significantly higher hACE2 binding affinity than SARS- convertase furin CoV RBD does (30). However, the cryo-electron microscopy (cryo-EM) structure of SARS-CoV-2 spike revealed that its he emergence and rapid spread of a novel severe acute re- RBD is mostly in the lying-down state (31, 32), a state associated Tspiratory syndrome (SARS)-like coronavirus SARS-CoV-2 is with ineffective receptor binding. In addition, there have been destroying global health and economy (1, 2). To date, conflicting reports on the hACE2-binding affinities of SARS-CoV-2 SARS-CoV-2 has infected over 3 million people and caused and SARS-CoV spikes (32–34). more than 200,000 deaths. It forces much of the world to adopt a lockdown mode, causing staggering economic fallout and human Significance suffering (https://www.cdc.gov/coronavirus/novel-coronavirus-2019. html). These numbers dwarf the impact of the related SARS A key to curbing SARS-CoV-2 is to understand how it enters coronavirus (SARS-CoV), which caused about 8,000 infections and cells. SARS-CoV-2 and SARS-CoV both use human ACE2 as entry 800 deaths (3, 4). Compared to SARS-CoV, many SARS-CoV-2 receptor and human proteases as entry activators. Using bio- patients develop low levels of neutralizing antibodies and suffer chemical and pseudovirus entry assays and SARS-CoV as a prolonged illness (5–7). These clinical features indicate that comparison, we have identified key cell entry mechanisms of SARS-CoV-2 evades the human immune surveillance more effec- SARS-CoV-2 that potentially contribute to the immune evasion, tively than SARS-CoV does. When viruses evolve to escape im- cell infectivity, and wide spread of the virus. This study also mune surveillance, they often suffer reduced fitness and become clarifies conflicting reports from recent studies on cell entry of less infectious (8–10). Yet SARS-CoV-2 remains highly infectious SARS-CoV-2. Finally, by highlighting the potency and the eva- (11, 12). The combination of immune evasion and high infectivity siveness of SARS-CoV-2, the study provides insight into in- may contribute to the wide spreadofSARS-CoV-2.Tocurb tervention strategies that target its cell entry mechanisms. SARS-CoV-2, it is important to uncover the molecular mechanisms that enable it to both evade immune surveillance and maintain high Author contributions: J.S., Y.W., and F.L. designed research; J.S., Y.W., C.L., G.Y., Q.G., and infectivity. Here, using biochemical and pseudovirus entry assays A.A. performed research; J.S., Y.W., C.L., G.Y., Q.G., A.A., and F.L. analyzed data; and F.L. wrote the paper. and SARS-CoV as a comparison, we investigate these mechanisms at an essential step of viral infection: the cell entry of SARS-CoV-2. The authors declare no competing interest. Coronavirus entry into host cells is an important determinant This article is a PNAS Direct Submission. of viral infectivity and pathogenesis (13, 14). It is also a major This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). target for host immune surveillance and human intervention strategies (15, 16). To enter host cells, coronaviruses first bind to J.S., Y.W., and C.L. contributed equally to this work. a cell surface receptor for viral attachment, subsequently enter To whom correspondence may be addressed. Email: [email protected]. endosomes, and eventually fuse viral and lysosomal membranes This article contains supporting information online at https://www.pnas.org/lookup/suppl/ doi:10.1073/pnas.2003138117/-/DCSupplemental. (13, 14) (Fig. 1A). A virus surface-anchored spike protein me- diates coronavirus entry (Fig. 1 B and C). On mature viruses, the First published May 6, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2003138117 PNAS | May 26, 2020 | vol. 117 | no. 21 | 11727–11734 MICROBIOLOGY Fig. 1. PPC motif in SARS-CoV-2 spike protein. (A) Different stages of coronavirus entry where host cellular proteases may activate coronavirus spikes. (B) Schematic drawing of the three-dimensional (3D) structure of coronavirus spike. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, trans- membrane anchor; IC, intracellular tail. (C) Schematic drawing of the 1D structure of coronavirus spike. NTD, N-terminal domain. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are structural units in coronavirus S2 that function in membrane fusion. (D) Sequence comparison of the spike proteins from SARS-CoV-2, SARS-CoV, and two bat SARS-like coronaviruses in a region at the S1/S2 boundary. Only SARS-CoV-2 spike contains a putative PPC motif—RRAR (residues in the box). The assumed PPC cleavage site is in front of the arginine residue labeled in red. The spike region mutated from SARS-CoV-2 sequence (TNSPRRA) to SARS-CoV sequence (SLL) is labeled in blue. GenBank accession numbers are QHD43416.1 for SARS-CoV-2 spike, AFR58740.1 for SARS- CoV spike, MG916901.1 for bat Rs3367 spike, and QHR63300.2 for bat RaTG13 spike. In addition to receptor binding, protease activators for mediated by SARS-CoV-2 spike. Three types of target cells were SARS-CoV-2 entry have been examined. It has been shown that used: HeLa cells (human cervical cells) exogenously expressing TMPRSS2 and lysosomal proteases are both important for hACE2, Calu-3 cells (human lung epithelial cells) endogenously SARS-CoV-2 entry (33, 34). In avian influenza viruses, propro- expressing hACE2, and MRC-5 cells (human lung fibroblast tein convertase (PPC) motif in the surface glycoprotein is a cells) endogenously expressing hACE2. hallmark of high pathogenesis (35). However, although SARS- To detect the cleavage state of SARS-CoV-2 spike on the CoV-2 spike contains a PPC motif at the S1/S2 boundary, it was surface of pseudoviruses, we packaged SARS-CoV-2 pseudovi- reported that PPC cleavage of the spike protein did not enhance ruses in HEK293T cells (human embryonic kidney cells) and SARS-CoV-2 entry into cells (31), challenging the well-established performed Western blot on the pseudoviruses. The result concept on the role of PPC motif. This raised questions about the showed that SARS-CoV-2 spike had been cleaved during viral role of PPC motif in SARS-CoV-2 entry. packaging (Fig. 2A). We then mutated the putative PPC site in Here we investigate the receptor binding and protease acti- SARS-CoV-2 spike to the corresponding sequence in SARS- vations of SARS-CoV-2 spike, using SARS-CoV spike as a CoV spike; the mutant SARS-CoV-2 spike was no longer comparison. Our results identify important cell entry mecha- cleaved during viral packaging (Fig. 2A). Further, we performed nisms of SARS-CoV-2 that potentially contribute to the immune pseudovirus entry assay using both wild-type SARS-CoV-2 evasion, cell infectivity, and wide spread of the virus. The find- pseudoviruses and PPC site mutant SARS-CoV-2 pseudoviruses. ings reconcile conflicting recent reports on cell entry of The result showed that SARS-CoV-2 pseudoviruses efficiently SARS-CoV-2. By revealing the surprising strategies that SARS- entered all three types of target cells (Fig. 2B). In contrast, the CoV-2 adopts to infect humans while evading immune surveil- mutant SARS-CoV-2 pseudoviruses demonstrated significantly lance, the findings provide insight into possible intervention reduced efficiency in entering the same cells (Fig. 2B). The strategies targeting cell entry of the virus. remaining cell entry of the mutant SARS-CoV-2 pseudoviruses Results was likely due to the activation from other host proteases that play partially overlapping and cumulative roles with PPCs (see Through examining the sequence of SARS-CoV-2 spike, we below). Therefore, we have identified and confirmed the PPC identified a putative cleavage site for PPCs at the S1/S2 cleavage site in SARS-CoV-2 spike, and shown that PPC cleav- boundary (Fig. 1 C and D). Curiously, this putative PPC site is age of SARS-CoV-2 spike during viral packaging is critical for absent in the spikes of SARS-CoV and SARS-like bat corona- SARS-CoV-2 to enter three different types of target cells. viruses. In this study, we investigated the role of PPC, along with To provide further evidence for the role of prior PPC cleavage other proteases, in SARS-CoV-2 entry. To this end, we estab- in SARS-CoV-2 entry, we treated HEK293T cells with PPC in- lished a pseudovirus entry assay for SARS-CoV-2. More specif- hibitor (PPCi) during packaging of wild-type SARS-CoV-2 pseu- ically, replication-deficient lentiviruses were pseudotyped with SARS-CoV-2 spike (i.e., SARS-CoV-2 pseudoviruses) and doviruses, and then subjected the PPCi-treated SARS-CoV-2 used to enter target cells. This type of pseudovirus assay sepa- pseudoviruses to entry into the aforementioned three types of target cells. The result showed that PPCi treatment inhibited PPC rates viral entry from other steps of the viral infection cycle (e.g., replication), enabling us to focus on the viral entry step that is cleavage of SARS-CoV-2 spike on pseudoviruses, and that the 11728 | www.pnas.org/cgi/doi/10.1073/pnas.2003138117 Shang et al. cathepsins activate SARS-CoV-2 entry. Similarly, SARS-CoV entry can also be activated by TMPRSS2 and lysosomal ca- thepsin (Fig. 4B). Moreover, prior treatment of pseudovirus- packaging cells with PPCi, combined with treatment of pseudovirus- targeted cells with either camostat or E64d, further reduced the efficiency of SARS-CoV-2 pseudovirus entry into HeLa cells (Fig. 4A). Thus, TMPRSS2 and lysosomal cathepsins both have cumulative effects with furin on activating SARS-CoV-2 entry. In contrast, neither camostat nor E64d has cumulative effects with PPCi on activating SARS-CoV entry (Fig. 4B). Overall, these results demonstrate that cell surface proteases and lysosomal proteases can both activate SARS-CoV-2 entry; in addition, furin and these other proteases have cumulative effects on activating Fig. 2. Role of PPC motif in SARS-CoV-2 spike-mediated cell entry. (A) SARS-CoV-2 entry. Cleavage state of SARS-CoV-2 spike on the surface of pseudoviruses. Pack- Having examined the role of furin in cleaving SARS-CoV-2 aged SARS-CoV-2 pseudoviruses were subjected to Western blot analysis for spike and preactivating SARS-CoV-2 entry, we next compared detection of the cleavage state of SARS-CoV-2 spike. SARS-CoV-2 spike the hACE2-binding affinities of SARS-CoV-2 and SARS-CoV fragments were detected using anti-C9 antibody targeting the C-terminal C9 spikes. To this end, we performed a protein pull-down assay, tag of the spike protein. (Left) Wild-type (WT) SARS-CoV-2 pseudoviruses. (Right) SARS-CoV-2 pseudoviruses where the PPC motif in the spike protein using recombinant hACE2 as the bait and cell surface-expressed had been mutated to the corresponding sequence in SARS-CoV spike (see SARS-CoV-2 and SARS-CoV spikes as the targets. To eliminate Fig. 1D for details). (B) SARS-CoV-2 pseudovirus entry into three types of any potential effect of furin cleavage on SARS-CoV-2 spike’s target cells. The two types of pseudoviruses correspond to the pseudoviruses binding of hACE2, we also included SARS-CoV-2 spike with its in A. Pseudovirus entry efficiency was characterized as luciferase signal ac- furin site mutated. For cross-validation, we used hACE2 with companying entry. The entry efficiency of wild-type SARS-CoV-2 pseudovi- two different tags, His tag and Fc tag. The result showed that, ruses was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; compared to SARS-CoV spike, SARS-CoV-2 spike binds to *P < 0.05. hACE2 with lower affinity (Fig. 5A). This result is different from our recent report that SARS-CoV-2 RBD binds to hACE2 with PPCi-treated SARS-CoV-2 pseudoviruses demonstrated signifi- significantly higher affinity than SARS-CoV RBD does, which cantly reduced cell entry efficiency (Fig. 3A). In comparison, was detected using surface plasmon resonance (SPR) (30). To SARS-CoV spike was not cleaved during packaging of SARS-CoV ensure that the above discrepancy was not due to different de- pseudoviruses, and PPCi treatment during virus packaging had no tection methods, we performed protein pull-down assay using effect on the subsequent cell entry process (Fig. 3B). These results recombinant hACE2 as the bait and soluble SARS-CoV-2 and further confirm that the efficiency of SARS-CoV-2 entry into SARS-CoV RBDs as the targets. The result showed that target cells can be enhanced by the prior PPC cleavage of the SARS-CoV-2 RBD binds to hACE2 with significantly higher SARS-CoV-2 spike during viral packaging, a contrast to SARS- affinity than SARS-CoV RBD does (Fig. 5B), confirming our CoV whose cell entry does not depend on PPC preactivation. recent SPR result. Therefore, whereas SARS-CoV-2 RBD has Since the PPCi used above is a broad-spectrum PPCi, we higher hACE2 binding affinity than SARS-CoV RBD, further investigated which specific PPC activates SARS-CoV-2 SARS-CoV-2 spike has lower hACE2 binding affinity than spike using small interfering RNA (siRNA) assay. To this end, SARS-CoV spike. we packaged SARS-CoV-2 pseudoviruses in HEK293T cells that Finally, we directly compared the cell entry efficiency of were treated with furin-targeting siRNA. Furin was selected in SARS-CoV-2 and SARS-CoV pseudoviruses. Similar to recent our study because it is the prototypic PPC and it preactivates the studies (31, 34), we calibrated pseudovirus entry efficiency entry of many other viruses, including some coronaviruses (22, against expression levels of spikes. Moreover, taking into account 23). The result showed that, after furin-targeting siRNA treat- that part of SARS-CoV-2 spike molecules had been cleaved ment, the spike molecules on the packaged SARS-CoV-2 pseu- during pseudovirus packaging, we used the total amount of doviruses were intact (Fig. 3C), revealing that furin is the PPC uncleaved and cleaved spike molecules to calibrate SARS-CoV-2 that preactivates SARS-CoV-2 spike. To rule out the possibility pseudovirus entry, while using the uncleaved spike molecules to that furin-dependent activation of matrix metalloproteinases calibrate SARS-CoV pseudovirus entry. The result showed that (MMPs) led to indirect activation of SARS-CoV-2 spike, we SARS-CoV-2 and SARS-CoV pseudoviruses entered all three treated HEK293T cells with MMP inhibitor during packaging of types of target cells with similar efficiency (Fig. 5C), which is SARS-CoV-2 pseudoviruses. The result showed that, after MMP consistent with two recent studies (31, 34). inhibitor treatment, the spike molecules on the packaged Discussion SARS-CoV-2 pseudoviruses were still cleaved (Fig. 3D), dem- onstrating that MMP is not involved in the activation of With mounting infections, fatalities, and economic losses caused SARS-CoV-2 spike. Taken together, these findings show that by SARS-CoV-2, it is imperative that we understand the cell furin is the PPC that preactivates SARS-CoV-2 spike (1, 2). entry mechanisms of SARS-CoV-2. However, recent studies To investigate the role of other proteases in SARS-CoV-2 have presented puzzling and sometimes conflicting findings on entry, we performed pseudovirus entry assay in the presence of how SARS-CoV-2 enters cells, raising pressing scientific ques- inhibitors that specifically target these other proteases. First, tions (30–32, 34). For example, which virus binds to hACE2 SARS-CoV-2 pseudovirus entry into all three types of target cells more tightly, SARS-CoV-2 or SARS-CoV? What is the role of was reduced in the presence of TMPRSS2 inhibitor camostat furin in SARS-CoV-2 entry? How does SARS-CoV-2 success- (Fig. 4A), suggesting that these cells endogenously express fully evade human immune surveillance while maintaining its TMPRSS2 and that these TMPRSS2 molecules activate high cell infectivity? The current study addresses these questions SARS-CoV-2 entry. Second, SARS-CoV-2 pseudovirus entry by detailing the cell entry mechanisms of SARS-CoV-2. into all three types of target cells was reduced in the presence of Receptor recognition is an important determinant of corona- lysosomal cathepsin inhibitor E64d (Fig. 4A). Hence, lysosomal virus infection and pathogenesis. It is also one of the most Shang et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11729 MICROBIOLOGY Fig. 3. Effect of PPCs on SARS-CoV-2 spike-mediated cell entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of PPCi. The pseudoviruses were packaged in the presence of different concentrations of PPCi before they were subjected to cell entry; (-) control: no pseudovirus was added. Also shown is the Western blot result of the corresponding pseudoviruses (packaged in the presence of different concentrations of PPCi). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; **P 0.01; *P < 0.05. (B) SARS- CoV pseudovirus entry into three types of target cells in the presence of PPCi. The experiments were performed in the same way as in A, except that SARS-CoV spike replaced SARS-CoV-2 spike in pseudoviruses. The entry efficiency of SARS-CoV pseudoviruses without any treatment was taken as 100%. (C) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with siRNA. (Left) Pseudoviruses packaged in cells treated with siRNA-negative control. (Right) Pseudoviruses packaged in cells treated with furin-targeting siRNA. (D) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with MMP inhibitor. (Left) Pseudoviruses packaged in cells not treated with MMP inhibitor. (Right) Pseudoviruses packaged in cells treated with MMP inhibitor. important targets for host immune surveillance and human in- analyses (30). In addition, using protein pull-down assay, the tervention strategies. The current study and other recent studies current study confirmed that SARS-CoV-2 RBD has higher have revealed two patterns of results on the hACE2 binding hACE2 binding affinity than SARS-CoV RBD does. Second, affinity of SARS-CoV-2. First, with regard to the RBD, despite the potency of its RBD’s binding to hACE2, the entire SARS-CoV-2 RBD has significantly higher hACE2 binding af- SARS-CoV-2 spike does not bind to hACE2 any more strongly finity than SARS-CoV RBD does. This was shown in our recent than SARS-CoV spike does. Using protein pull-down assay, the study using SPR assay as well as structural and mutagenesis current study showed that SARS-CoV-2 spike binds to hACE2 Fig. 4. Effect of other protease inhibitors on SARS-CoV-2 entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of protease inhibitors. For pseudoviruses treated with PPCi, the pseudoviruses were packaged in the presence of PPCi (5 μM) before they were subjected to cell entry. For pseudoviruses treated with TMPRSS2 inhibitor camostat or lysosomal protease inhibitor E64d, pseudovirus entry was performed in the presence of camostat (50 μM) or E64d (50 μM). The cleavage state of SARS-CoV-2 spike was the same as in Fig. 3A (5 μM PPCi condition). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; *P < 0.05. (B) SARS-CoV pseudovirus entry into three types of target cells. The treatments were done in the same way as in A. 11730 | www.pnas.org/cgi/doi/10.1073/pnas.2003138117 Shang et al. To maintain its high infectivity while keeping its RBD less accessible, SARS-CoV-2 relies on a second strategy—host pro- tease activation. Host protease activation is a significant de- terminant of coronavirus infection and pathogenesis, and a significant target for host immune surveillance and human in- tervention strategies. Using a combination of mutagenesis, pro- tease inhibitors, and siRNA approaches, here we showed that furin preactivation enhances SARS-CoV-2 pseudovirus entry into different types of hACE2-expressing cell lines, including lung epithelial and lung fibroblast cell lines. We also showed that cell surface protease TMPRSS2 and lysosomal cathepsins acti- vate SARS-CoV-2 pseudovirus entry and that both TMPRSS2 and cathepsins have cumulative effects with furin on SARS-CoV-2 entry. In comparison, SARS-CoV pseudovirus entry is activated by TMPRSS2 and cathepsins, but not furin. Furin preactivation allows SARS-CoV-2 to be less dependent on target cells, enhancing its entry into some target cells, particu- larly cells with relatively low expressions of TMPRSS2 and/or lysosomal cathepsins. This has also been observed with furin- preactivated avian influenza viruses (32). However, a recent study showed that furin preactivation enhances SARS-CoV-2 pseudovirus entry into BHK cells (baby hamster kidney fibro- blast cells), but reduces SARS-CoV-2 pseudovirus entry into Vero cells (African green monkey kidney epithelial cells) (31). These seemingly conflicting results can be explained by how coronavirus entry is regulated by proteases. Protease activation of coronavirus spikes potentially leads to the final structural Fig. 5. Comparison of receptor binding affinity and cell entry efficiency of change of coronavirus S2 needed for membrane fusion; this SARS-CoV-2 and SARS-CoV. (A) Spike pull-down assay using hACE2 as the process is irreversible and needs to be tightly regulated (13). bait and cell-associated coronavirus spike molecules as the targets. (Top) Indeed, it has been shown that, on SARS-CoV-2 virus particles, Cell-expressed coronavirus spike molecules including SARS-CoV-2 spike, many spike molecules have already undergone the final struc- SARS-CoV-2 spike containing a mutant furin site as in Fig. 2A, SARS-CoV tural change (36). Hence, in principle, virus particles pre- spike, and MERS-CoV spike. These spike molecules all contain a C-terminal activated by furin may have unchanged or reduced entry C9 tag. (Middle) Pull-down result using His -tagged hACE2. (Bottom) Pull- efficiency in some types of cells with high expressions of down result using Fc-tagged hACE2. (B) RBD pull-down assay using Fc- tagged hACE2 as the bait and soluble coronavirus RBDs as the targets. TMPRSS2 and/or lysosomal proteases; this may particularly be These RBD molecules all contain a C-terminal His tag. (C)(Left) Entry of the case in vitro for virus particles that are not fresh, as the final SARS-CoV-2 and SARS-CoV pseudoviruses into three types of target cells. conformational change of spike molecules may occur slowly (Right) Western blot of SARS-CoV-2 and SARS-CoV pseudoviruses used in the spontaneously or be facilitated by environmental factors (e.g., cell entry assay. high temperature, physical force, or some chemicals) (37). Overall, furin preactivation can facilitate SARS-CoV-2 to enter some types of cells (particularly those with low expressions of less strongly than SARS-CoV spike does. Another study using TMPRSS2 and/or lysosomal cathepsins) (Fig. 6A). flow cytometry assay yielded similar results (34). A third study The cell entry mechanisms of SARS-CoV-2 have implications using Blitz assay showed that SARS-CoV-2 and SARS-CoV for understanding clinical features of coronavirus disease 2019 spikes have similar hACE2 binding affinities (31). Note that (COVID-19) (Fig. 6B). The hidden RBD can evade immune the hACE2 binding affinities of SARS-CoV RBD and surveillance, potentially leading to insufficient immune re- SARS-CoV-2 spike should not be compared directly with each sponses and prolonged recovery time. Granted, there are other other (32). These findings therefore present a paradoxical pat- immune evasion strategies for coronaviruses. For example, some tern of results: Although SARS-CoV-2 RBD has higher hACE2 coronavirus nonstructural proteins can help evade the host in- binding affinity than SARS-CoV RBD, its spike has hACE2 nate immune responses (38, 39). Importantly, viruses commonly hide their RBD or other critical parts of their spike proteins binding affinity comparable to or lower than SARS-CoV spike. from host adaptive immune responses using two main strategies These contrasting patterns between the RBD and the entire (40). The first is conformational masking, where viruses conceal spike are particularly compelling in the current study because their RBDs in locations like canyons (as in the case of picorna- they were observed using the same method and under the same viruses) (41) or recessed pockets (as in the case of HIV) (42). testing conditions. The dynamic state of the RBD in coronavirus The second is glycan shielding, where viruses conceal critical spikes may explain this paradox. The RBD in coronaviruses can parts of their spike proteins behind glycan clusters (as in the case be in either a standing-up state, which enables receptor binding, of HIV, Ebola virus, and hepatitis C virus) (43). Our finding or a lying-down state, which does not bind to the host receptors about the discrepancy in hACE2 binding affinity between (20, 21). Cryo-EM studies have shown that, in SARS-CoV spike, SARS-CoV-2 RBD and spike, combined with other groups’ the RBD is mostly in the standing-up state (20, 21); however, in observation of the lying-down RBD in SARS-CoV-2 spike, SARS-CoV-2 spike, the RBD is mostly in the lying-down state suggests that the hidden RBD contributes to the immune evasion (31, 32). Therefore, compared to SARS-CoV, although of SARS-CoV-2 as one of the conformational masking strategies. SARS-CoV-2 RBD has higher hACE2 binding affinity, it is less Indeed, a recent study showed that SARS-CoV RBD-induced accessible, resulting in comparable or lower hACE2 binding af- mouse sera bind SARS-CoV-2 RBD with high affinity, but finity for SARS-CoV-2 spike (Fig. 6A). poorly neutralize SARS-CoV-2 pseudovirus entry into host cells; Shang et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11731 MICROBIOLOGY Fig. 6. Summary of cell entry mechanisms of SARS-CoV-2. (A) A schematic view of three unique features of SARS-CoV-2 entry: hidden RBD in the spike for immune evasion, RBD’s high hACE2 binding affinity for efficient entry, and furin preactivation of the spike for enhanced entry into some cells. (B) Implications of the cell entry mechanisms of SARS-CoV-2. in contrast, the same sera bind SARS-CoV RBD with high af- multiple protease activators would be needed to achieve satis- finity and neutralize SARS-CoV pseudovirus entry potently (44). factory outcome. This approach will need to consider side effects This result shows that immune surveillance recognizes hidden when these drugs target host proteins. The sophisticated cell entry mechanisms of SARS-CoV-2 pose significant challenges, RBD less well than exposed RBD. However, hidden RBD may lead to poor recognition of the host receptor and inefficient but also illuminate multiple intervention strategies that target entry into host cells. SARS-CoV-2 overcomes this problem by cell entry of the virus. evolving an RBD with high hACE2 binding affinity and a furin Materials and Methods motif that allows its spike to be preactivated. The end result is Cell Line and Plasmids. HEK293T, HeLa, Calu-3, and MRC-5 cells were obtained that the overall entry efficiencies of SARS-CoV-2 and SARS- from the American Type Culture Collection and cultured in Dulbecco’s CoV pseudoviruses are comparable. modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM Understanding the cell entry mechanism of SARS-CoV-2 can L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Life inform intervention strategies. The RBD is the most immuno- Technologies). genic region of the whole spike (15, 45). Hence, the hidden RBD Full-length SARS-CoV-2 spike (GenBank accession number QHD43416.1), of SARS-CoV-2 presents a major challenge to both vaccination SARS-CoV Spike (GenBank accession number AFR58740.1), MERS-CoV spike and antibody drug therapy due to the limited access of neutral- (GenBank accession number AFS88936.1), and human ACE2 (GenBank ac- cession number NM_021804) were synthesized (GenScript Biotech) and izing antibodies to the target. Correspondingly, there are several subcloned into the pcDNA3.1(+) vector (Life Technologies) with a C-terminal approaches for intervention strategies, with some caveats. First, C9 tag. SARS-CoV-2 RBD (residues 319 to 535), SARS-CoV RBD (residues 306 antibody drugs can be developed to bind to the RBD very tightly, to 521), MERS-CoV RBD (residues 367 to 588), and human ACE2 peptidase preferably with both a high k rate and a low k rate, such that, on off domain (residues 1 to 615) were subcloned into pFastBac vector (Life Tech- during the limited exposure of RBD, the drugs can latch onto the nologies) with an N-terminal honey bee melittin signal peptide and a RBD quickly and keep a strong hold on it. It was recently shown C-terminal His tag. For human ACE2 peptidase domain, a construct was also that recombinant ACE2 can inhibit SARS-CoV-2 infection in made containing a C-terminal Fc tag instead of the C-terminal His tag. artificial human tissues (46), suggesting that blocking the RBD is feasible. Thus, an antibody drug with significantly higher RBD Protein Expression and Purification. All of the proteins were expressed in sf9 binding affinity than ACE2 can dominate over cell surface ACE2 insect cells using the Bac-to-Bac system (Life Technologies). Briefly, His - in latching onto the RBD, blocking viral attachment. Second, tagged proteins were harvested from cell culture medium, and were puri- fied sequentially on Ni-NTA column and Superdex200 gel filtration column RBD vaccines can be developed. Because neutralizing antibodies (GE Healthcare) as described previously (30). The Fc-tagged protein was elicited by RBD vaccines may have limited access to the RBD, purified in the same way, except that protein A column replaced Ni-NTA structure-guided engineering will be needed to significantly en- column (30). Purified proteins were stored in a buffer containing 20 mM Tris hance the efficacy of RBD vaccines (45). Third, vaccines and pH7.2 and 200 mM NaCl for later use. drugs can be developed to target the membrane fusion S2 sub- unit. The success of this approach for vaccine development, Coronavirus Spike-Mediated Pseudovirus Entry Assay. Retroviruses pseudo- however, may be limited because the S2 subunit is less immu- typed with SARS-CoV-2 spike or SARS-CoV spike were generated in nogenic than the RBD (15). Last, the cell entry process of HEK293T cells, and pseudovirus entry assay was performed as previously SARS-CoV-2 can be blocked using inhibitors that target the described (48). Briefly, HEK293T cells were cotransfected with a plasmid protease activators (47). Because SARS-CoV-2 uses several carrying an Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.lu- cellular proteases as entry activators, inhibitor mixtures against c.R-E-) and pcDNA3.1(+) plasmid encoding one of the indicated spikes. 11732 | www.pnas.org/cgi/doi/10.1073/pnas.2003138117 Shang et al. Pseudoviruses were harvested 72 h after transfection, and were used to C-terminal His tag) or 5 μg hACE2-Fc (human ACE2 with a C-terminal Fc tag), enter target cells. Six hours after incubation with pseudoviruses, cells were respectively, on a roller at room temperature for 30 min. Subsequently, transferred to fresh medium. After another 66 h, cells were washed and hACE2-bound beads were washed three times with 1 mL of PBS buffer plus lysed for detection of luciferase signal (relative luciferase units or RLU). 0.05% Tween-20 (PBST) on a roller for 10 min and then were aliquoted into Target cells for pseudovirus entry assay included HeLa cells exogenously different tubes for later use. To prepare cell-associated coronavirus spike expressing human ACE2, and Calu-3 and MRC-5 cells endogenously protein, HEK293T cells were transfected with pcDNA3.1(+) plasmid encoding expressing human ACE2. coronavirus spike (containing a C-terminal C9 tag); 48 h after transfection, For pseudoviruses treated with PPCi or matrix MMP inhibitor, PPCi the spike-expressing cells were lysed using a sonicator in assay buffer and chloromethylketone (Enzo Life Sciences) or MMP inhibitor batimastat centrifuged at 12,000 × g for 2 min. The supernatants containing solubilized (Sigma-Aldrich) was added to the medium at indicated concentrations 6 h SARS-CoV-2 spike (for spike pull-down assay) or purified recombinant after transfection for pseudovirus packaging began. Pseudoviruses were coronavirus RBDs (for RBD pull-down assay) were incubated with the hACE2- harvested after an additional incubation time of 66 h. Pseudoviruses were bound beads in 2-mL tubes (spike or RBD was in excess of hACE2) on a roller then used to enter target cells. at room temperature for 1 h. Then beads were washed three times with For pseudoviruses treated with siRNA, siRNA furin and siRNA negative PBST buffer, and the bound proteins were eluted using elution buffer. The control (Thermo Fisher Scientific) were transfected separately into samples were then subjected to Western blot analysis and detected using an HEK293T cells 6 h after transfection for pseudovirus packaging began. anti-C9 tag antibody or anti-His tag antibody. Pseudoviruses were harvested after an additional incubation time of 66 h. Pseudoviruses were then subjected to Western blot analysis. For pseudoviruses treated with other protease inhibitors, target cells were Statistic Analysis. All experiments were repeated at least four times. Statistical pretreated with camostat (50 μM) (Sigma-Aldrich) or E64d (50 μM) analyses were performed using t tests. 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Cell entry mechanisms of SARS-CoV-2

Shang, Jian; Wan, Yushun; Luo, Chuming; Ye, Gang; Geng, Qibin; Auerbach, Ashley; Li, Fang
Proceedings of the National Academy of Sciences of the United States of America , Volume 117 (21) – May 6, 2020
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Abstract

a,1 a,1 a,1 a a a a,2 Jian Shang , Yushun Wan , Chuming Luo , Gang Ye , Qibin Geng , Ashley Auerbach , and Fang Li  Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108 Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved April 27, 2020 (received for review February 18, 2020) A novel severe acute respiratory syndrome (SARS)-like coronavirus spike protein is present as a trimer, with three receptor-binding (SARS-CoV-2) is causing the global coronavirus disease 2019 S1 heads sitting on top of a trimeric membrane fusion S2 stalk (COVID-19) pandemic. Understanding how SARS-CoV-2 enters hu- (Fig. 1B). The cell entry mechanism of SARS-CoV has been man cells is a high priority for deciphering its mystery and curbing extensively studied. SARS-CoV S1 contains a receptor-binding its spread. A virus surface spike protein mediates SARS-CoV-2 en- domain (RBD) that specifically recognizes angiotensin-converting try into cells. To fulfill its function, SARS-CoV-2 spike binds to its enzyme 2 (ACE2) as its receptor (17–19). The RBD constantly receptor human ACE2 (hACE2) through its receptor-binding do- switches between a standing-up position for receptor binding and main (RBD) and is proteolytically activated by human proteases. a lying-down position for immune evasion (20, 21) (Fig. 1B). Here we investigated receptor binding and protease activation of Moreover, to fuse membranes, SARS-CoV spike needs to be SARS-CoV-2 spike using biochemical and pseudovirus entry assays. proteolytically activated at the S1/S2 boundary, such that S1 Our findings have identified key cell entry mechanisms of dissociates and S2 undergoes a dramatic structural change (22, SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding af- 23). These SARS-CoV entry-activating proteases include cell finity than SARS-CoV RBD, supporting efficient cell entry. Second, surface protease TMPRSS2 and lysosomal proteases cathepsins paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 (22, 23) (Fig. 1A). These features of SARS-CoV entry contribute spike is comparable to or lower than that of SARS-CoV spike, suggest- to its rapid spread and severe symptoms and high fatality rates of ing that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is infected patients (24–26). preactivated by proprotein convertase furin, reducing its dependence The past several months saw an explosion of studies on the cell on target cell proteases for entry. The high hACE2 binding affinity of entry mechanisms of SARS-CoV-2, sometimes with conflicting the RBD, furin preactivation of the spike, and hidden RBD in the spike findings. Like SARS-CoV, SARS-CoV-2 also recognizes human potentially allow SARS-CoV-2 to maintain efficient cell entry while ACE2 (hACE2) as its receptor (27–29). We recently determined evading immune surveillance. These features may contribute to the the crystal structure of SARS-CoV-2 RBD complexed with wide spread of the virus. Successful intervention strategies must tar- hACE2, which revealed subtle but functionally important dif- get both the potency of SARS-CoV-2 and its evasiveness. ferences between SARS-CoV-2 and SARS-CoV in receptor recognition (30). These differences enable SARS-CoV-2 RBD to COVID-19 SARS-CoV-2 SARS-CoV ACE2 receptor proprotein | | | | have a significantly higher hACE2 binding affinity than SARS- convertase furin CoV RBD does (30). However, the cryo-electron microscopy (cryo-EM) structure of SARS-CoV-2 spike revealed that its he emergence and rapid spread of a novel severe acute re- RBD is mostly in the lying-down state (31, 32), a state associated Tspiratory syndrome (SARS)-like coronavirus SARS-CoV-2 is with ineffective receptor binding. In addition, there have been destroying global health and economy (1, 2). To date, conflicting reports on the hACE2-binding affinities of SARS-CoV-2 SARS-CoV-2 has infected over 3 million people and caused and SARS-CoV spikes (32–34). more than 200,000 deaths. It forces much of the world to adopt a lockdown mode, causing staggering economic fallout and human Significance suffering (https://www.cdc.gov/coronavirus/novel-coronavirus-2019. html). These numbers dwarf the impact of the related SARS A key to curbing SARS-CoV-2 is to understand how it enters coronavirus (SARS-CoV), which caused about 8,000 infections and cells. SARS-CoV-2 and SARS-CoV both use human ACE2 as entry 800 deaths (3, 4). Compared to SARS-CoV, many SARS-CoV-2 receptor and human proteases as entry activators. Using bio- patients develop low levels of neutralizing antibodies and suffer chemical and pseudovirus entry assays and SARS-CoV as a prolonged illness (5–7). These clinical features indicate that comparison, we have identified key cell entry mechanisms of SARS-CoV-2 evades the human immune surveillance more effec- SARS-CoV-2 that potentially contribute to the immune evasion, tively than SARS-CoV does. When viruses evolve to escape im- cell infectivity, and wide spread of the virus. This study also mune surveillance, they often suffer reduced fitness and become clarifies conflicting reports from recent studies on cell entry of less infectious (8–10). Yet SARS-CoV-2 remains highly infectious SARS-CoV-2. Finally, by highlighting the potency and the eva- (11, 12). The combination of immune evasion and high infectivity siveness of SARS-CoV-2, the study provides insight into in- may contribute to the wide spreadofSARS-CoV-2.Tocurb tervention strategies that target its cell entry mechanisms. SARS-CoV-2, it is important to uncover the molecular mechanisms that enable it to both evade immune surveillance and maintain high Author contributions: J.S., Y.W., and F.L. designed research; J.S., Y.W., C.L., G.Y., Q.G., and infectivity. Here, using biochemical and pseudovirus entry assays A.A. performed research; J.S., Y.W., C.L., G.Y., Q.G., A.A., and F.L. analyzed data; and F.L. wrote the paper. and SARS-CoV as a comparison, we investigate these mechanisms at an essential step of viral infection: the cell entry of SARS-CoV-2. The authors declare no competing interest. Coronavirus entry into host cells is an important determinant This article is a PNAS Direct Submission. of viral infectivity and pathogenesis (13, 14). It is also a major This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY). target for host immune surveillance and human intervention strategies (15, 16). To enter host cells, coronaviruses first bind to J.S., Y.W., and C.L. contributed equally to this work. a cell surface receptor for viral attachment, subsequently enter To whom correspondence may be addressed. Email: [email protected]. endosomes, and eventually fuse viral and lysosomal membranes This article contains supporting information online at https://www.pnas.org/lookup/suppl/ doi:10.1073/pnas.2003138117/-/DCSupplemental. (13, 14) (Fig. 1A). A virus surface-anchored spike protein me- diates coronavirus entry (Fig. 1 B and C). On mature viruses, the First published May 6, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2003138117 PNAS | May 26, 2020 | vol. 117 | no. 21 | 11727–11734 MICROBIOLOGY Fig. 1. PPC motif in SARS-CoV-2 spike protein. (A) Different stages of coronavirus entry where host cellular proteases may activate coronavirus spikes. (B) Schematic drawing of the three-dimensional (3D) structure of coronavirus spike. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, trans- membrane anchor; IC, intracellular tail. (C) Schematic drawing of the 1D structure of coronavirus spike. NTD, N-terminal domain. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are structural units in coronavirus S2 that function in membrane fusion. (D) Sequence comparison of the spike proteins from SARS-CoV-2, SARS-CoV, and two bat SARS-like coronaviruses in a region at the S1/S2 boundary. Only SARS-CoV-2 spike contains a putative PPC motif—RRAR (residues in the box). The assumed PPC cleavage site is in front of the arginine residue labeled in red. The spike region mutated from SARS-CoV-2 sequence (TNSPRRA) to SARS-CoV sequence (SLL) is labeled in blue. GenBank accession numbers are QHD43416.1 for SARS-CoV-2 spike, AFR58740.1 for SARS- CoV spike, MG916901.1 for bat Rs3367 spike, and QHR63300.2 for bat RaTG13 spike. In addition to receptor binding, protease activators for mediated by SARS-CoV-2 spike. Three types of target cells were SARS-CoV-2 entry have been examined. It has been shown that used: HeLa cells (human cervical cells) exogenously expressing TMPRSS2 and lysosomal proteases are both important for hACE2, Calu-3 cells (human lung epithelial cells) endogenously SARS-CoV-2 entry (33, 34). In avian influenza viruses, propro- expressing hACE2, and MRC-5 cells (human lung fibroblast tein convertase (PPC) motif in the surface glycoprotein is a cells) endogenously expressing hACE2. hallmark of high pathogenesis (35). However, although SARS- To detect the cleavage state of SARS-CoV-2 spike on the CoV-2 spike contains a PPC motif at the S1/S2 boundary, it was surface of pseudoviruses, we packaged SARS-CoV-2 pseudovi- reported that PPC cleavage of the spike protein did not enhance ruses in HEK293T cells (human embryonic kidney cells) and SARS-CoV-2 entry into cells (31), challenging the well-established performed Western blot on the pseudoviruses. The result concept on the role of PPC motif. This raised questions about the showed that SARS-CoV-2 spike had been cleaved during viral role of PPC motif in SARS-CoV-2 entry. packaging (Fig. 2A). We then mutated the putative PPC site in Here we investigate the receptor binding and protease acti- SARS-CoV-2 spike to the corresponding sequence in SARS- vations of SARS-CoV-2 spike, using SARS-CoV spike as a CoV spike; the mutant SARS-CoV-2 spike was no longer comparison. Our results identify important cell entry mecha- cleaved during viral packaging (Fig. 2A). Further, we performed nisms of SARS-CoV-2 that potentially contribute to the immune pseudovirus entry assay using both wild-type SARS-CoV-2 evasion, cell infectivity, and wide spread of the virus. The find- pseudoviruses and PPC site mutant SARS-CoV-2 pseudoviruses. ings reconcile conflicting recent reports on cell entry of The result showed that SARS-CoV-2 pseudoviruses efficiently SARS-CoV-2. By revealing the surprising strategies that SARS- entered all three types of target cells (Fig. 2B). In contrast, the CoV-2 adopts to infect humans while evading immune surveil- mutant SARS-CoV-2 pseudoviruses demonstrated significantly lance, the findings provide insight into possible intervention reduced efficiency in entering the same cells (Fig. 2B). The strategies targeting cell entry of the virus. remaining cell entry of the mutant SARS-CoV-2 pseudoviruses Results was likely due to the activation from other host proteases that play partially overlapping and cumulative roles with PPCs (see Through examining the sequence of SARS-CoV-2 spike, we below). Therefore, we have identified and confirmed the PPC identified a putative cleavage site for PPCs at the S1/S2 cleavage site in SARS-CoV-2 spike, and shown that PPC cleav- boundary (Fig. 1 C and D). Curiously, this putative PPC site is age of SARS-CoV-2 spike during viral packaging is critical for absent in the spikes of SARS-CoV and SARS-like bat corona- SARS-CoV-2 to enter three different types of target cells. viruses. In this study, we investigated the role of PPC, along with To provide further evidence for the role of prior PPC cleavage other proteases, in SARS-CoV-2 entry. To this end, we estab- in SARS-CoV-2 entry, we treated HEK293T cells with PPC in- lished a pseudovirus entry assay for SARS-CoV-2. More specif- hibitor (PPCi) during packaging of wild-type SARS-CoV-2 pseu- ically, replication-deficient lentiviruses were pseudotyped with SARS-CoV-2 spike (i.e., SARS-CoV-2 pseudoviruses) and doviruses, and then subjected the PPCi-treated SARS-CoV-2 used to enter target cells. This type of pseudovirus assay sepa- pseudoviruses to entry into the aforementioned three types of target cells. The result showed that PPCi treatment inhibited PPC rates viral entry from other steps of the viral infection cycle (e.g., replication), enabling us to focus on the viral entry step that is cleavage of SARS-CoV-2 spike on pseudoviruses, and that the 11728 | www.pnas.org/cgi/doi/10.1073/pnas.2003138117 Shang et al. cathepsins activate SARS-CoV-2 entry. Similarly, SARS-CoV entry can also be activated by TMPRSS2 and lysosomal ca- thepsin (Fig. 4B). Moreover, prior treatment of pseudovirus- packaging cells with PPCi, combined with treatment of pseudovirus- targeted cells with either camostat or E64d, further reduced the efficiency of SARS-CoV-2 pseudovirus entry into HeLa cells (Fig. 4A). Thus, TMPRSS2 and lysosomal cathepsins both have cumulative effects with furin on activating SARS-CoV-2 entry. In contrast, neither camostat nor E64d has cumulative effects with PPCi on activating SARS-CoV entry (Fig. 4B). Overall, these results demonstrate that cell surface proteases and lysosomal proteases can both activate SARS-CoV-2 entry; in addition, furin and these other proteases have cumulative effects on activating Fig. 2. Role of PPC motif in SARS-CoV-2 spike-mediated cell entry. (A) SARS-CoV-2 entry. Cleavage state of SARS-CoV-2 spike on the surface of pseudoviruses. Pack- Having examined the role of furin in cleaving SARS-CoV-2 aged SARS-CoV-2 pseudoviruses were subjected to Western blot analysis for spike and preactivating SARS-CoV-2 entry, we next compared detection of the cleavage state of SARS-CoV-2 spike. SARS-CoV-2 spike the hACE2-binding affinities of SARS-CoV-2 and SARS-CoV fragments were detected using anti-C9 antibody targeting the C-terminal C9 spikes. To this end, we performed a protein pull-down assay, tag of the spike protein. (Left) Wild-type (WT) SARS-CoV-2 pseudoviruses. (Right) SARS-CoV-2 pseudoviruses where the PPC motif in the spike protein using recombinant hACE2 as the bait and cell surface-expressed had been mutated to the corresponding sequence in SARS-CoV spike (see SARS-CoV-2 and SARS-CoV spikes as the targets. To eliminate Fig. 1D for details). (B) SARS-CoV-2 pseudovirus entry into three types of any potential effect of furin cleavage on SARS-CoV-2 spike’s target cells. The two types of pseudoviruses correspond to the pseudoviruses binding of hACE2, we also included SARS-CoV-2 spike with its in A. Pseudovirus entry efficiency was characterized as luciferase signal ac- furin site mutated. For cross-validation, we used hACE2 with companying entry. The entry efficiency of wild-type SARS-CoV-2 pseudovi- two different tags, His tag and Fc tag. The result showed that, ruses was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; compared to SARS-CoV spike, SARS-CoV-2 spike binds to *P < 0.05. hACE2 with lower affinity (Fig. 5A). This result is different from our recent report that SARS-CoV-2 RBD binds to hACE2 with PPCi-treated SARS-CoV-2 pseudoviruses demonstrated signifi- significantly higher affinity than SARS-CoV RBD does, which cantly reduced cell entry efficiency (Fig. 3A). In comparison, was detected using surface plasmon resonance (SPR) (30). To SARS-CoV spike was not cleaved during packaging of SARS-CoV ensure that the above discrepancy was not due to different de- pseudoviruses, and PPCi treatment during virus packaging had no tection methods, we performed protein pull-down assay using effect on the subsequent cell entry process (Fig. 3B). These results recombinant hACE2 as the bait and soluble SARS-CoV-2 and further confirm that the efficiency of SARS-CoV-2 entry into SARS-CoV RBDs as the targets. The result showed that target cells can be enhanced by the prior PPC cleavage of the SARS-CoV-2 RBD binds to hACE2 with significantly higher SARS-CoV-2 spike during viral packaging, a contrast to SARS- affinity than SARS-CoV RBD does (Fig. 5B), confirming our CoV whose cell entry does not depend on PPC preactivation. recent SPR result. Therefore, whereas SARS-CoV-2 RBD has Since the PPCi used above is a broad-spectrum PPCi, we higher hACE2 binding affinity than SARS-CoV RBD, further investigated which specific PPC activates SARS-CoV-2 SARS-CoV-2 spike has lower hACE2 binding affinity than spike using small interfering RNA (siRNA) assay. To this end, SARS-CoV spike. we packaged SARS-CoV-2 pseudoviruses in HEK293T cells that Finally, we directly compared the cell entry efficiency of were treated with furin-targeting siRNA. Furin was selected in SARS-CoV-2 and SARS-CoV pseudoviruses. Similar to recent our study because it is the prototypic PPC and it preactivates the studies (31, 34), we calibrated pseudovirus entry efficiency entry of many other viruses, including some coronaviruses (22, against expression levels of spikes. Moreover, taking into account 23). The result showed that, after furin-targeting siRNA treat- that part of SARS-CoV-2 spike molecules had been cleaved ment, the spike molecules on the packaged SARS-CoV-2 pseu- during pseudovirus packaging, we used the total amount of doviruses were intact (Fig. 3C), revealing that furin is the PPC uncleaved and cleaved spike molecules to calibrate SARS-CoV-2 that preactivates SARS-CoV-2 spike. To rule out the possibility pseudovirus entry, while using the uncleaved spike molecules to that furin-dependent activation of matrix metalloproteinases calibrate SARS-CoV pseudovirus entry. The result showed that (MMPs) led to indirect activation of SARS-CoV-2 spike, we SARS-CoV-2 and SARS-CoV pseudoviruses entered all three treated HEK293T cells with MMP inhibitor during packaging of types of target cells with similar efficiency (Fig. 5C), which is SARS-CoV-2 pseudoviruses. The result showed that, after MMP consistent with two recent studies (31, 34). inhibitor treatment, the spike molecules on the packaged Discussion SARS-CoV-2 pseudoviruses were still cleaved (Fig. 3D), dem- onstrating that MMP is not involved in the activation of With mounting infections, fatalities, and economic losses caused SARS-CoV-2 spike. Taken together, these findings show that by SARS-CoV-2, it is imperative that we understand the cell furin is the PPC that preactivates SARS-CoV-2 spike (1, 2). entry mechanisms of SARS-CoV-2. However, recent studies To investigate the role of other proteases in SARS-CoV-2 have presented puzzling and sometimes conflicting findings on entry, we performed pseudovirus entry assay in the presence of how SARS-CoV-2 enters cells, raising pressing scientific ques- inhibitors that specifically target these other proteases. First, tions (30–32, 34). For example, which virus binds to hACE2 SARS-CoV-2 pseudovirus entry into all three types of target cells more tightly, SARS-CoV-2 or SARS-CoV? What is the role of was reduced in the presence of TMPRSS2 inhibitor camostat furin in SARS-CoV-2 entry? How does SARS-CoV-2 success- (Fig. 4A), suggesting that these cells endogenously express fully evade human immune surveillance while maintaining its TMPRSS2 and that these TMPRSS2 molecules activate high cell infectivity? The current study addresses these questions SARS-CoV-2 entry. Second, SARS-CoV-2 pseudovirus entry by detailing the cell entry mechanisms of SARS-CoV-2. into all three types of target cells was reduced in the presence of Receptor recognition is an important determinant of corona- lysosomal cathepsin inhibitor E64d (Fig. 4A). Hence, lysosomal virus infection and pathogenesis. It is also one of the most Shang et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11729 MICROBIOLOGY Fig. 3. Effect of PPCs on SARS-CoV-2 spike-mediated cell entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of PPCi. The pseudoviruses were packaged in the presence of different concentrations of PPCi before they were subjected to cell entry; (-) control: no pseudovirus was added. Also shown is the Western blot result of the corresponding pseudoviruses (packaged in the presence of different concentrations of PPCi). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; **P 0.01; *P < 0.05. (B) SARS- CoV pseudovirus entry into three types of target cells in the presence of PPCi. The experiments were performed in the same way as in A, except that SARS-CoV spike replaced SARS-CoV-2 spike in pseudoviruses. The entry efficiency of SARS-CoV pseudoviruses without any treatment was taken as 100%. (C) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with siRNA. (Left) Pseudoviruses packaged in cells treated with siRNA-negative control. (Right) Pseudoviruses packaged in cells treated with furin-targeting siRNA. (D) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with MMP inhibitor. (Left) Pseudoviruses packaged in cells not treated with MMP inhibitor. (Right) Pseudoviruses packaged in cells treated with MMP inhibitor. important targets for host immune surveillance and human in- analyses (30). In addition, using protein pull-down assay, the tervention strategies. The current study and other recent studies current study confirmed that SARS-CoV-2 RBD has higher have revealed two patterns of results on the hACE2 binding hACE2 binding affinity than SARS-CoV RBD does. Second, affinity of SARS-CoV-2. First, with regard to the RBD, despite the potency of its RBD’s binding to hACE2, the entire SARS-CoV-2 RBD has significantly higher hACE2 binding af- SARS-CoV-2 spike does not bind to hACE2 any more strongly finity than SARS-CoV RBD does. This was shown in our recent than SARS-CoV spike does. Using protein pull-down assay, the study using SPR assay as well as structural and mutagenesis current study showed that SARS-CoV-2 spike binds to hACE2 Fig. 4. Effect of other protease inhibitors on SARS-CoV-2 entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of protease inhibitors. For pseudoviruses treated with PPCi, the pseudoviruses were packaged in the presence of PPCi (5 μM) before they were subjected to cell entry. For pseudoviruses treated with TMPRSS2 inhibitor camostat or lysosomal protease inhibitor E64d, pseudovirus entry was performed in the presence of camostat (50 μM) or E64d (50 μM). The cleavage state of SARS-CoV-2 spike was the same as in Fig. 3A (5 μM PPCi condition). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; *P < 0.05. (B) SARS-CoV pseudovirus entry into three types of target cells. The treatments were done in the same way as in A. 11730 | www.pnas.org/cgi/doi/10.1073/pnas.2003138117 Shang et al. To maintain its high infectivity while keeping its RBD less accessible, SARS-CoV-2 relies on a second strategy—host pro- tease activation. Host protease activation is a significant de- terminant of coronavirus infection and pathogenesis, and a significant target for host immune surveillance and human in- tervention strategies. Using a combination of mutagenesis, pro- tease inhibitors, and siRNA approaches, here we showed that furin preactivation enhances SARS-CoV-2 pseudovirus entry into different types of hACE2-expressing cell lines, including lung epithelial and lung fibroblast cell lines. We also showed that cell surface protease TMPRSS2 and lysosomal cathepsins acti- vate SARS-CoV-2 pseudovirus entry and that both TMPRSS2 and cathepsins have cumulative effects with furin on SARS-CoV-2 entry. In comparison, SARS-CoV pseudovirus entry is activated by TMPRSS2 and cathepsins, but not furin. Furin preactivation allows SARS-CoV-2 to be less dependent on target cells, enhancing its entry into some target cells, particu- larly cells with relatively low expressions of TMPRSS2 and/or lysosomal cathepsins. This has also been observed with furin- preactivated avian influenza viruses (32). However, a recent study showed that furin preactivation enhances SARS-CoV-2 pseudovirus entry into BHK cells (baby hamster kidney fibro- blast cells), but reduces SARS-CoV-2 pseudovirus entry into Vero cells (African green monkey kidney epithelial cells) (31). These seemingly conflicting results can be explained by how coronavirus entry is regulated by proteases. Protease activation of coronavirus spikes potentially leads to the final structural Fig. 5. Comparison of receptor binding affinity and cell entry efficiency of change of coronavirus S2 needed for membrane fusion; this SARS-CoV-2 and SARS-CoV. (A) Spike pull-down assay using hACE2 as the process is irreversible and needs to be tightly regulated (13). bait and cell-associated coronavirus spike molecules as the targets. (Top) Indeed, it has been shown that, on SARS-CoV-2 virus particles, Cell-expressed coronavirus spike molecules including SARS-CoV-2 spike, many spike molecules have already undergone the final struc- SARS-CoV-2 spike containing a mutant furin site as in Fig. 2A, SARS-CoV tural change (36). Hence, in principle, virus particles pre- spike, and MERS-CoV spike. These spike molecules all contain a C-terminal activated by furin may have unchanged or reduced entry C9 tag. (Middle) Pull-down result using His -tagged hACE2. (Bottom) Pull- efficiency in some types of cells with high expressions of down result using Fc-tagged hACE2. (B) RBD pull-down assay using Fc- tagged hACE2 as the bait and soluble coronavirus RBDs as the targets. TMPRSS2 and/or lysosomal proteases; this may particularly be These RBD molecules all contain a C-terminal His tag. (C)(Left) Entry of the case in vitro for virus particles that are not fresh, as the final SARS-CoV-2 and SARS-CoV pseudoviruses into three types of target cells. conformational change of spike molecules may occur slowly (Right) Western blot of SARS-CoV-2 and SARS-CoV pseudoviruses used in the spontaneously or be facilitated by environmental factors (e.g., cell entry assay. high temperature, physical force, or some chemicals) (37). Overall, furin preactivation can facilitate SARS-CoV-2 to enter some types of cells (particularly those with low expressions of less strongly than SARS-CoV spike does. Another study using TMPRSS2 and/or lysosomal cathepsins) (Fig. 6A). flow cytometry assay yielded similar results (34). A third study The cell entry mechanisms of SARS-CoV-2 have implications using Blitz assay showed that SARS-CoV-2 and SARS-CoV for understanding clinical features of coronavirus disease 2019 spikes have similar hACE2 binding affinities (31). Note that (COVID-19) (Fig. 6B). The hidden RBD can evade immune the hACE2 binding affinities of SARS-CoV RBD and surveillance, potentially leading to insufficient immune re- SARS-CoV-2 spike should not be compared directly with each sponses and prolonged recovery time. Granted, there are other other (32). These findings therefore present a paradoxical pat- immune evasion strategies for coronaviruses. For example, some tern of results: Although SARS-CoV-2 RBD has higher hACE2 coronavirus nonstructural proteins can help evade the host in- binding affinity than SARS-CoV RBD, its spike has hACE2 nate immune responses (38, 39). Importantly, viruses commonly hide their RBD or other critical parts of their spike proteins binding affinity comparable to or lower than SARS-CoV spike. from host adaptive immune responses using two main strategies These contrasting patterns between the RBD and the entire (40). The first is conformational masking, where viruses conceal spike are particularly compelling in the current study because their RBDs in locations like canyons (as in the case of picorna- they were observed using the same method and under the same viruses) (41) or recessed pockets (as in the case of HIV) (42). testing conditions. The dynamic state of the RBD in coronavirus The second is glycan shielding, where viruses conceal critical spikes may explain this paradox. The RBD in coronaviruses can parts of their spike proteins behind glycan clusters (as in the case be in either a standing-up state, which enables receptor binding, of HIV, Ebola virus, and hepatitis C virus) (43). Our finding or a lying-down state, which does not bind to the host receptors about the discrepancy in hACE2 binding affinity between (20, 21). Cryo-EM studies have shown that, in SARS-CoV spike, SARS-CoV-2 RBD and spike, combined with other groups’ the RBD is mostly in the standing-up state (20, 21); however, in observation of the lying-down RBD in SARS-CoV-2 spike, SARS-CoV-2 spike, the RBD is mostly in the lying-down state suggests that the hidden RBD contributes to the immune evasion (31, 32). Therefore, compared to SARS-CoV, although of SARS-CoV-2 as one of the conformational masking strategies. SARS-CoV-2 RBD has higher hACE2 binding affinity, it is less Indeed, a recent study showed that SARS-CoV RBD-induced accessible, resulting in comparable or lower hACE2 binding af- mouse sera bind SARS-CoV-2 RBD with high affinity, but finity for SARS-CoV-2 spike (Fig. 6A). poorly neutralize SARS-CoV-2 pseudovirus entry into host cells; Shang et al. PNAS | May 26, 2020 | vol. 117 | no. 21 | 11731 MICROBIOLOGY Fig. 6. Summary of cell entry mechanisms of SARS-CoV-2. (A) A schematic view of three unique features of SARS-CoV-2 entry: hidden RBD in the spike for immune evasion, RBD’s high hACE2 binding affinity for efficient entry, and furin preactivation of the spike for enhanced entry into some cells. (B) Implications of the cell entry mechanisms of SARS-CoV-2. in contrast, the same sera bind SARS-CoV RBD with high af- multiple protease activators would be needed to achieve satis- finity and neutralize SARS-CoV pseudovirus entry potently (44). factory outcome. This approach will need to consider side effects This result shows that immune surveillance recognizes hidden when these drugs target host proteins. The sophisticated cell entry mechanisms of SARS-CoV-2 pose significant challenges, RBD less well than exposed RBD. However, hidden RBD may lead to poor recognition of the host receptor and inefficient but also illuminate multiple intervention strategies that target entry into host cells. SARS-CoV-2 overcomes this problem by cell entry of the virus. evolving an RBD with high hACE2 binding affinity and a furin Materials and Methods motif that allows its spike to be preactivated. The end result is Cell Line and Plasmids. HEK293T, HeLa, Calu-3, and MRC-5 cells were obtained that the overall entry efficiencies of SARS-CoV-2 and SARS- from the American Type Culture Collection and cultured in Dulbecco’s CoV pseudoviruses are comparable. modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM Understanding the cell entry mechanism of SARS-CoV-2 can L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (Life inform intervention strategies. The RBD is the most immuno- Technologies). genic region of the whole spike (15, 45). Hence, the hidden RBD Full-length SARS-CoV-2 spike (GenBank accession number QHD43416.1), of SARS-CoV-2 presents a major challenge to both vaccination SARS-CoV Spike (GenBank accession number AFR58740.1), MERS-CoV spike and antibody drug therapy due to the limited access of neutral- (GenBank accession number AFS88936.1), and human ACE2 (GenBank ac- cession number NM_021804) were synthesized (GenScript Biotech) and izing antibodies to the target. Correspondingly, there are several subcloned into the pcDNA3.1(+) vector (Life Technologies) with a C-terminal approaches for intervention strategies, with some caveats. First, C9 tag. SARS-CoV-2 RBD (residues 319 to 535), SARS-CoV RBD (residues 306 antibody drugs can be developed to bind to the RBD very tightly, to 521), MERS-CoV RBD (residues 367 to 588), and human ACE2 peptidase preferably with both a high k rate and a low k rate, such that, on off domain (residues 1 to 615) were subcloned into pFastBac vector (Life Tech- during the limited exposure of RBD, the drugs can latch onto the nologies) with an N-terminal honey bee melittin signal peptide and a RBD quickly and keep a strong hold on it. It was recently shown C-terminal His tag. For human ACE2 peptidase domain, a construct was also that recombinant ACE2 can inhibit SARS-CoV-2 infection in made containing a C-terminal Fc tag instead of the C-terminal His tag. artificial human tissues (46), suggesting that blocking the RBD is feasible. Thus, an antibody drug with significantly higher RBD Protein Expression and Purification. All of the proteins were expressed in sf9 binding affinity than ACE2 can dominate over cell surface ACE2 insect cells using the Bac-to-Bac system (Life Technologies). Briefly, His - in latching onto the RBD, blocking viral attachment. Second, tagged proteins were harvested from cell culture medium, and were puri- fied sequentially on Ni-NTA column and Superdex200 gel filtration column RBD vaccines can be developed. Because neutralizing antibodies (GE Healthcare) as described previously (30). The Fc-tagged protein was elicited by RBD vaccines may have limited access to the RBD, purified in the same way, except that protein A column replaced Ni-NTA structure-guided engineering will be needed to significantly en- column (30). Purified proteins were stored in a buffer containing 20 mM Tris hance the efficacy of RBD vaccines (45). Third, vaccines and pH7.2 and 200 mM NaCl for later use. drugs can be developed to target the membrane fusion S2 sub- unit. The success of this approach for vaccine development, Coronavirus Spike-Mediated Pseudovirus Entry Assay. Retroviruses pseudo- however, may be limited because the S2 subunit is less immu- typed with SARS-CoV-2 spike or SARS-CoV spike were generated in nogenic than the RBD (15). Last, the cell entry process of HEK293T cells, and pseudovirus entry assay was performed as previously SARS-CoV-2 can be blocked using inhibitors that target the described (48). Briefly, HEK293T cells were cotransfected with a plasmid protease activators (47). Because SARS-CoV-2 uses several carrying an Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.lu- cellular proteases as entry activators, inhibitor mixtures against c.R-E-) and pcDNA3.1(+) plasmid encoding one of the indicated spikes. 11732 | www.pnas.org/cgi/doi/10.1073/pnas.2003138117 Shang et al. Pseudoviruses were harvested 72 h after transfection, and were used to C-terminal His tag) or 5 μg hACE2-Fc (human ACE2 with a C-terminal Fc tag), enter target cells. Six hours after incubation with pseudoviruses, cells were respectively, on a roller at room temperature for 30 min. Subsequently, transferred to fresh medium. After another 66 h, cells were washed and hACE2-bound beads were washed three times with 1 mL of PBS buffer plus lysed for detection of luciferase signal (relative luciferase units or RLU). 0.05% Tween-20 (PBST) on a roller for 10 min and then were aliquoted into Target cells for pseudovirus entry assay included HeLa cells exogenously different tubes for later use. To prepare cell-associated coronavirus spike expressing human ACE2, and Calu-3 and MRC-5 cells endogenously protein, HEK293T cells were transfected with pcDNA3.1(+) plasmid encoding expressing human ACE2. coronavirus spike (containing a C-terminal C9 tag); 48 h after transfection, For pseudoviruses treated with PPCi or matrix MMP inhibitor, PPCi the spike-expressing cells were lysed using a sonicator in assay buffer and chloromethylketone (Enzo Life Sciences) or MMP inhibitor batimastat centrifuged at 12,000 × g for 2 min. The supernatants containing solubilized (Sigma-Aldrich) was added to the medium at indicated concentrations 6 h SARS-CoV-2 spike (for spike pull-down assay) or purified recombinant after transfection for pseudovirus packaging began. Pseudoviruses were coronavirus RBDs (for RBD pull-down assay) were incubated with the hACE2- harvested after an additional incubation time of 66 h. Pseudoviruses were bound beads in 2-mL tubes (spike or RBD was in excess of hACE2) on a roller then used to enter target cells. at room temperature for 1 h. Then beads were washed three times with For pseudoviruses treated with siRNA, siRNA furin and siRNA negative PBST buffer, and the bound proteins were eluted using elution buffer. The control (Thermo Fisher Scientific) were transfected separately into samples were then subjected to Western blot analysis and detected using an HEK293T cells 6 h after transfection for pseudovirus packaging began. anti-C9 tag antibody or anti-His tag antibody. Pseudoviruses were harvested after an additional incubation time of 66 h. Pseudoviruses were then subjected to Western blot analysis. For pseudoviruses treated with other protease inhibitors, target cells were Statistic Analysis. All experiments were repeated at least four times. Statistical pretreated with camostat (50 μM) (Sigma-Aldrich) or E64d (50 μM) analyses were performed using t tests. 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Published: May 6, 2020

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