Question

SARS-CoV2 is a coronavirus that infects the cells of the mammalian respiratory epithelium. To replicate it needs to take over the host cells secretory pathway to generate essential viral proteins. You are working to try and find a treatment for this virus. Answer all the following questions related to the virus and the secretory system:

a) You discover that the virus needs to insert a protein called ‘Spike’ into the plasma membrane for newly formed viral particles to be infectious. The RNA for Spike is in the cytosol. Describe briefly SIX key steps for how the Spike protein gets into the plasma membrane.

b) You determine the sequence of the Spike protein and identify the following amino acid sequence motifs. What is the function of each sequence (i) Asn–X–Ser/Thr (ii) A long stretch of hydrophobic residues near the centre of the sequence. (iii) Asp-X-Glu (Di-acidic sequence)

c) The Spike protein also contains a sequence you recognise as a cleavage site for an enzyme called Furin. You know Furin cleavages can activate other pro-proteins. What does this tell you about Spike?

d) To infect epithelial cells the SARS-CoV Spike protein binds to a receptor on the plasma membrane and is endocytosed. Describe THREE key steps that are essential in this process.

e) Reading back over Questions a-d, name a protein you could target in a treatment for COVID-19 and explain why you chose it.

Answer 1

a. Enveloped viruses enter cells by viral glycoprotein-mediated binding to host cells and subsequent fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. Palmitate adducts are generally required for protein-mediated fusions, but their precise roles in the process are unclear. To obtain additional insights into the S-mediated membrane fusion process, we focused on these acylated carboxyl-terminal intravirion tails. Substituting alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S-mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral heptad repeat peptides that bind only to folding intermediates in the S-mediated fusion process and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly 10 times longer than native S proteins. This slower refolding was also reflected in the paucity of postfusion six-helix bundle configurations among the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. These studies reveal a previously unappreciated role for covalently attached lipids on the endodomains of viral proteins eliciting membrane fusion reactions

Biological membranes are configured in large part by protein-mediated fission and fusion reactions. Enveloped viruses can reveal the principles of these processes because their assembly and budding from infected cells requires membrane fissions, and their entry into susceptible cells depends on membrane fusions. Glycoproteins extending from virion surfaces mediate the fusion process. These specialized integral membrane proteins are in metastable high energy configurations on virus surfaces, and they drive coalescence of opposing virus and cell membranes by undergoing a series of energy-releasing unfolding and refolding events (1). The structural rearrangements are triggered by virus binding to cellular receptors (2) and by the acidic, proteolytic environments encountered after viruses are endocytosed (3,–,5). These reactions begin with an unfolding process that reveals hydrophobic fusion peptides (FPs)2 that dagger into cellular membranes. This is then followed by a refolding process that, in analogy to a closing hairpin, brings FPs and associated cellular membranes toward the virion membranes, driving formation of a lipid stalk connecting the opposing outer membrane leaflets (6) and culminating in complete cell-virion membrane coalescence (7, 8). For viral fusion proteins in the so-called “class I” category, the arms of the prehairpin intermediates are each trihelical bundles designated as heptad repeats 1 and 2 (HR1 and HR2), and closure to the postfusion state therefore creates six-helix bundles (6-HBs) of antiparallel HR1 and -2 segments, with FPs abutted next to transmembrane (TM) spans in the coalesced membrane (see Fig. 6 for a depiction of this process). Viral fusion proteins in other classes go through related refoldings to effect membrane coalescence, but the hairpin arms are not necessarily α-helical (1).

Although this view of viral protein-mediated membrane fusion is satisfying in many ways, important details are missing. For example, the importance of the TM and endodomain (ENDO) portions of the surface proteins demand more prominent attention in the membrane fusion models. Because these TM and ENDO regions are not structurally resolved, it can be difficult to accurately add them into the models. However, abundant literature indicates that TM-ENDO portions of many different virus fusion proteins do operate to control virus-cell and cell-cell fusion (9,–,12). An influenza hemagglutinin fusion protein with a glycosylphosphatidylinositol anchor replacing its TM-ENDO domains was able to mediate outer membrane leaflet fusions (i.e. hemifusion) but could not create full membrane fusions (13). The animal retrovirus envelope proteins contain long ENDO domains that include the “R peptides” that, once removed by proteolysis, facilitate the fusion reaction (14, 15). Truncation of the human immunodeficiency virus (HIV) envelope ENDO tail modulates its fusogenicity (16). Finally, it is notable that many viral fusion protein ectodomain fragments lacking TM and ENDO domains fold into postfusion states (17, 18), suggesting that membrane-anchoring parts help maintain functional metastable high energy conformations.

It is not entirely clear how the intravirion parts of the fusion protein influence reactions that are carried out by the much larger exterior portion of the protein. We and others consider it plausible that changes in the fusion protein endodomain impact refolding rates, which in turn control the route and timing of virus entry. This is because the transitions from prehairpin intermediate to postfusion states require large scale transit of TM-ENDO domains across lipid stalks (19), which may be a rate-limiting step in the process.

We investigate the cell entry of coronaviruses (CoVs). The CoVs are enveloped, plus-strand RNA viruses causing respiratory and gastrointestinal diseases in animals and humans. The prototype human pathogenic CoV is severe acute respiratory syndrome (SARS)-CoV (20). We have found that the CoVs provide a good model in which one can study the relationship between endodomain changes and fusion reaction kinetics. CoV spike (S) proteins are solely sufficient to mediate virus-cell fusion and cell entry. The S protein ectodomains are trimers (21) with classical “class I” fusion protein characteristics (22). The relative positions of fusion peptides (23), HR regions (24, 25), and TM span are known, and condensed six-helix bundles of antiparallel HR1 and HR2 have been crystallographically resolved (26, 27) (see Fig. 1). The S protein endodomains comprising the carboxyl termini are set apart by their abundance of cysteine residues. Many if not all of these cysteines are well known to be post-translationally acylated with palmitate and/or stearate adducts (28,–,31); these post-translational modifications add considerable lipophilicity to the endodomains and probably position the ENDO tails against the inner face of virion membranes. Indeed, the S proteins are set apart from other enveloped virus glycoproteins in having very richly acylated endodomains. There are nine acylated cysteines in coronavirus S, whereas there are only three in influenza HA (32) and two in HIV gp160 (33). Interference with S endodomain palmitoylation, either by engineered mutations or pharmacologic agents, diminishes or eliminates S-mediated membrane fusion activities (28, 29, 31, 34), but the mechanisms by which these endodomain alterations influence membrane fusion activities are unknown.

Here we explore the mechanistic basis for these observations. Our findings indicate that spikes harboring endodomain cysteine mutations can fold into prefusion forms, can reach infected cell surfaces, and can mediate cell-cell fusions. However, the endodomain mutant spikes that we evaluate here cannot efficiently incorporate into secreted virions, and those few that do incorporate into virions cannot efficiently support virus-cell entry and fusion because they are slow at refolding into postfusion forms. We interpret these findings in the context of the class I protein-mediated membrane fusion pathway and suggest that endodomain palmitates serve to anchor spike protein trimers onto virion membranes such that metastable prefusion spike conformations can be maintained and also progress through conformational intermediates in a timely fashion.

EXPERIMENTAL PROCEDURES

Cells

Murine 17cl1 fibroblasts (35) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 5% tryptose phosphate broth (Difco) and 5% heat-inactivated fetal bovine serum (FBS). 293T, FCWF (36), and HeLa-CEACAM (carcinoembryonic antigen cell adhesion molecule isoform 1a; cell line number 3) cells (37) were grown in DMEM supplemented with 10% FBS. All growth media were buffered with 0.01 M sodium HEPES (pH 7.4).

Plasmid DNAs

MHV-A59 S and M cDNAs were PCR-amplified using template pMH54-A59 (38, 39) and cloned into pCAGGS.MCS (40) between SacI and XmaI restriction sites. Mutations in the pCAGGS-S construct were created using mutagenic primers and a site-directed mutagenesis protocol. (QuikChange® XL; catalogue number 200519-5; Stratagene). All plasmid constructs were sequenced to confirm the presence of desired mutations. Primers and primer sequences are available upon request.

Recombinant Viruses

Recombinant MHVs were created via targeted RNA recombination (39). Mutations in the pMH54-E-FL-M construct (41) were created using site-directed mutagenesis, as described above. The plasmid DNAs were linearized by digestion with PacI and used as templates for in vitro transcription reactions using T7 RNA polymerase and reagents from Ambion (mMESSAGE mMACHINE®; catalogue number AM1344). Transcripts were electroporated into ∼107 feline FCWF cells that were infected 4 h earlier with recombinant coronavirus feline MHV-A59 (39), using a Bio-Rad Gene Pulser II. The electroporated FCWF cells were added to a monolayer of ∼106 17cl1 cells. Recombinant viruses, identified by syncytia development on 17cl1 cells, were then collected from media and isolated by three cycles of plaque purification on 17cl1 cells. Mutations fixed into the recombinant MHVs were confirmed by reverse transcription PCR and sequencing. Sequence determinations included ∼300 nucleotides spanning the intended site-directed mutations.

Radiolabeling and Virus Purification

Viruses were adsorbed to 17cl1 cells at a multiplicity of infection of 1 for 1 h at 37 °C in serum-free DMEM and then aspirated and replaced with DMEM supplemented with 5% FBS. At 12 h postinfection, media were removed, and cells were rinsed extensively with saline. For radiolabeling with 35S-labeled amino acids, cells were first incubated for 30 min at 37 °C in labeling medium (methionine- and cysteine-free DMEM containing 1% dialyzed FBS). The cells were then replenished with labeling medium containing 60 μCi/ml Tran 35S-label (MP Biomedicals, Irvine, CA), and incubated for 4 h at 37 °C. Media collected from infected cell cultures were centrifuged for 10 min at 2,000 × g and then for 20 min at 20,000 × g and then overlaid on top of discontinuous sucrose gradients consisting of 5 ml of 30% and 2 ml of 50% (w/w) sucrose in HNB buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.01% bovine serum albumin). Virions were equilibrated at the 30–50% sucrose interface, using a Beckman Spinco SW41 rotor at 40,000 rpm for 2 h at 4 °C and recovered by fractionation from air-gradient interfaces.

Immunoprecipitations and Immunoblotting

293T cells were co-transfected via calcium phosphate (42, 43) with pCAGGS-M and pCAGGS-S constructs. At 40 h post-transfection, the cell monolayers were lysed in HNB buffer containing 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% protease inhibitor (Sigma P2714). Cell lysates were first clarified by centrifugation at 2,000 × g for 5 min, and then 160,000 cell equivalents were mixed with 0.01 ml of 1 mg/ml N-CEACAM-Fc (44) and 0.06 ml of protein G magnetic beads (NEB Corp., Inc.) for 2 h at 25 °C. Beads were rinsed three times with HNB buffer containing 0.5% Nonidet P-40, 0.5% sodium deoxycholate. Proteins were eluted from beads by the addition of electrophoresis sample buffer (0.125 M Tris (pH 6.8), 10% dithiothreitol, 2% SDS, 10% sucrose, 0.004% bromphenol blue) and heating to 95 °C for 5 min and subsequently subjected to SDS-PAGE. SDS gels were transferred to polyvinylidene difluoride membranes that were subsequently blocked for 1 h with 5% nonfat milk powder in TBS-T (25 mM Tris-HCl (pH 7.5), 140 mM NaCl, 2.7 mM KCl, 0.05% Tween 20). S proteins were detected with murine mAb 10G (45) (1:2000 in TBS-T). M proteins were detected with murine mAb J.3.1 (46) (1:500 in TBS-T).

Pseudotyped Virions and Transductions

To generate pseudotyped HIV particles, 293T cells were co-transfected via calcium phosphate (42, 43) with pNL4.3-Luc R-E- (National Institutes of Health AIDS Research and Reference Program number 3418) and the various pCAGGS-S constructs. After 2 days, media were collected, clarified for 10 min at 2,000 × g, and then overlaid on top of a 30% sucrose cushion in HNB buffer and centrifuged at 40,000 rpm for 2 h at 4 °C using a Beckman SW41 rotor. Pelleted particles were resuspended in HNB buffer and stored at −80 °C. For biochemical analysis, pelleted HIV pseudoparticles were resuspended in electrophoresis sample buffer and processed by immunoblotting as described above. S proteins were detected with murine mAb 10G (45) (1:2,000 in TBS-T). HIV capsid protein (p24) was detected with murine mAb αp24 (National Institutes of Health AIDS Research and Reference Program) (1:5000 in TBS-T). For transductions, HIV particles, normalized to p24 levels, were adsorbed to HeLa-CEACAM cells in serum-free DMEM for 2 h. Subsequently, the inoculum was removed and replaced with DMEM supplemented with 10% FBS.

At 2 days post-transduction, the cells were rinsed with saline and dissolved in luciferase lysis buffer (Promega E397A). Luminescence was measured upon the addition of luciferase substrate (Promega E1501) using a Veritas microplate luminometer (Turner BioSystems). In some experiments, HR2 peptide (25 μM) was added as indicated under “Results.”

Protease Digestion Assay

For the protease digestion assay (47), 104 plaque-forming units of recombinant A59 (rA59) coronavirus in 20 μl of DMEM supplemented with 5% FBS or HIV pseudoparticles in 20 μl of HNB buffer were incubated with N-CEACAM-Fc (2 μM) for various times at 37 °C. After samples were placed on ice for 10 min, proteinase K (Sigma) was added at a final concentration of 10 μg/ml, and digestion was carried out at 4 °C for 20 min. Reactions were terminated by the addition of electrophoresis sample buffer and subjected to SDS-PAGE and immunoblotting as described above.

Cell-Cell Fusion Assay

Cell-cell fusion was performed as described previously (48). Briefly, effector cells (HeLa) were transiently transfected with a pCAGGS vector encoding T7 polymerase and the various pCAGGS-S constructs using Lipofectamine 2000 reagent (Invitrogen). Target cells were generated by Lipofectamine transfection of HeLa-CEACAM cells with pT7pro-EMC-luc, which encodes firefly luciferase under T7 promoter control (49). At ∼6 h post-transfection, the target cells were quickly trypsinized and added to adherent effector cells in a 1:1 effector/target cell ratio. After a ∼4-h co-cultivation period, luciferase levels were quantified as described above.

RESULTS

Effect of Endodomain Mutations on S Incorporation into Virions

The MHV strain A59 S protein has nine cytoplasmic (endodomain) cysteines, most or all of which are known to be stably thioacylated with palmitic acids (28,–,31) We mutated those most distal from the transmembrane span, the carboxyl-terminal Cys1300, Cys1303, and Cys1304, to alanines, with the expectation that these changes would prevent S palmitoylation at these positions and thus untether the ends of the S tails from cytosolic membrane faces (Fig. 1A). Our goal was to discern the functional consequences of these changes. To this end, we used targeted RNA recombination to direct mutations into the MHV genome, thus creating a series of recombinant MHV viruses harboring cysteine-to-alanine substitutions. The parent virus we used is a recombinant MHV-A59 strain engineered to produce firefly luciferase, identical to that developed by de Haan et al. (50).

Biochemical evaluation of the newly generated recombinant viruses involved 35S radiolabeling of infected 17cl1 cell monolayers. 35S-virions were harvested from culture media, purified by density gradient ultracentrifugation, and evaluated for radioactive protein content by SDS-PAGE and autoradiography. The single mutant C1304A recombinant virions were indistinguishable from WT rA59 in these electrophoretic analyses (data not shown). Fig. 2A depicts the virion proteins associated with wild type (WT) rA59 in comparison with C1303A/C1304A rA59. The C1303A/C1304A mutant virions were noticeably depleted in S protein content (11-fold relative to WT). The triple mutant C1300A/C1303A/C1304A recombinant viruses were never isolated despite several attempts, suggesting that a threshold of spike density is required for virus viability.

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FIGURE 2.

Effect of S endodomain cysteine mutations on virion incorporation and association with M proteins. A, recombinant virions were metabolically radiolabeled with 35S-amino acids and purified by sucrose density gradient ultracentrifugation. Equal 35S radioactivities were collected from each purified virion preparation, electrophoresed on SDS gels, and detected by autoradiography. S agg, S aggregates; S unc, uncleaved S; N, nucleocapsid protein; M, membrane protein. Molecular masses are shown in kilodaltons. B, 293T cells co-expressing the indicated S constructs with M proteins were dissolved in Nonidet P-40/deoxycholate buffer, and S·M complexes were captured using an MHV-soluble receptor immunoadhesin (nCEACAM-Fc) bound to magnetic protein G beads. Eluted proteins were detected by Western immunoblotting using S- and M-specific mAbs. 1C-A, C1304A; 2C-A, C1303A/C1304A; 3C-A, C1300A/C1303A/C1304A.

An explanation for the reduced incorporation of endodomain mutant spikes into virions appeals to disruption of S protein interaction with M proteins, the M proteins being the key orchestrating agents in the virion assembly process (51, 52). Thus, we co-expressed the various spike constructs individually with M protein in 293T cells and subsequently dissolved cell monolayers in a buffer containing both Nonidet P-40 and sodium deoxycholate, a detergent formulation known to preserve association between S and M proteins (53). S·M complexes were captured using the S-binding immunoadhesin N-CEACAM-Fc (44) and magnetic protein G beads. Eluted proteins were detected by Western blot using anti-S and anti-M antibodies, and the results (Fig. 2B) revealed that the poor incorporation of endodomain mutant spikes into recombinant virions correlated with their failure to efficiently associate with M proteins.

Effect of Endodomain Mutations on Spike-mediated Membrane Fusion

To investigate the role of endodomain cysteines on the membrane fusion reaction, we first performed cell-cell fusion assays. To this end, cells transfected with various pCAGGS-spike constructs were co-cultivated with target cells containing murine CEACAMs, the primary MHV receptors. Spike-bearing cells contained phage T7 polymerase, and CEACAM cells harbored luciferase genes whose transcription required the T7 polymerases, making it so that luciferase enzyme activities increased in response to spike-induced cell-cell fusions. From these assays, we found that all spikes induced similar luciferase accumulations (Fig. 3A). Thus, at least within a 4-h cell co-cultivation period, the various endodomain mutant spikes were equivalent in cell-cell fusion activities.

An inference from the results of cell-cell fusion assays is that the various spike proteins accumulate equivalently on cell surfaces. If so, then the spike proteins might incorporate equivalently onto HIV virus cores budding from plasma membrane sites, making HIV-coronavirus S pseudoparticles appropriate for virus-cell fusion assays. Such HIV-S pseudoviruses could replace authentic rA59 coronaviruses for use in virus-cell fusion assays, the rA59 viruses being unsuitable for correlating endodomain changes with virus-cell fusion because of the confounding effect of these endodomain changes on the assembly of spikes into virions (Fig. 2A).

HIV-CoV S pseudotype virions were produced by co-transfecting 293 cells with an envelope-deficient HIV vector (pNL4-3-Luc-R-E) along with pCAGGS-S constructs. Released pseudoparticles were harvested from culture media, purified by sucrose gradient ultracentrifugation, and subjected to SDS-PAGE. The data (Fig. 3B) revealed that WT and endodomain mutant spikes did indeed incorporate onto HIV particles with equal efficiencies. However, when the HIV-S particles were used to transduce CEACAM receptor-bearing target cells, the single (C1304A) double (C1303A/C1304A), and triple (C1300A/C1303A/C1304A) cysteine mutants were about 2, 20, and 40 times less efficient at delivering the HIV cores into cells, as measured by a luciferase reporter that is part of the recombinant HIV genome (Fig. 3C). These data indicate that endodomain cysteines and most likely their palmitate adducts are specifically needed to facilitate effective virus-cell fusion.

We wanted to investigate the mechanism by which these endodomain mutations suppressed virus entry. One possibility is that S-mediated entry was impaired because endodomain mutations reduced the affinity of S ectodomains for CEACAM receptors. To address this speculation, we produced highly purified 35S-labeled WT and C1303A/C1304A rA59 virions and assessed their immunoprecipitation with N-CEACAM-Fc. In 1-h, 4 °C incubation periods, the 35S radioactivities that were captured varied by <10% between WT and C1303A/C1304A virions. Furthermore, we observed no significant differences in the association of 35S-labeled WT and Cys → Ala pseudovirions with CEACAM-bearing host cells (data not shown).

Given that the endodomain mutations had no obvious effect on receptor interactions, their suppression of virus entry was probably at the level of membrane fusion. To address this possibility and evaluate S-mediated fusion in detail, we monitored S protein refolding events with an HR2 peptide that was previously shown to be a potent fusion inhibitor (22). The HR2 peptide used (NVTFLDLTYEMNRIQDAIKKLNESYINLKE) corresponds to residues 1225–1254 of the MHV strain A59 spike. The view is that HR2 peptides bind exposed HR1 trimers, thereby occluding the cis refolding of endogenous HR2 helices onto HR1, preventing 6-HB formation, membrane fusion, and virus entry (54, 55). These exposed HR1 trimers are present only in transitional S protein folding states; in support of this statement, we found that HR2 peptides could be incubated indefinitely with virions at 50 μM (50 × EC50) (56) at 37 °C, and after diluting to 0.5 nM (0.0005 x EC50), they effect no inhibition of plaque development.

In our experiments, we used the HR2 peptide as a tool to monitor the exposure of HR1 (reflecting S unfolding) and subsequent disappearance of HR1 (reflecting S refolding into postfusion 6-HBs) during virus entry into cells. In this experimental design, we applied HIV-S pseudoparticles to CEACAM-bearing HeLa cells at 4 °C and incubated to equilibrium. Unbound particles were aspirated and replaced with prewarmed 37 °C media, because the 37 °C temperature is required for fusion and for S protein conformational changes (57, 58). Then the HR2 peptide (25 μM) was added at the 37 °C temperature shift and subsequently removed at 0-, 2-, 4-, 8-, 16-, 32-, and 64-min time intervals (Fig. 4A) or added at early 0-, 2-, 4-, 8-, 16-, 32-, and 64-min time intervals after the temperature shift (Fig. 4B). At the 64 min time point, all cells were rinsed, replenished with complete media, and then assayed 40 h later for accumulated luciferase, which served as the readout for S-mediated psuedovirus entry.

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FIGURE 4.

Time course of entrance into and exit from HR2-sensitive folding states. HIV particles normalized to p24 content were prebound to HeLa-CEACAM cells at 4 °C for 1 h. Unbound particles were then aspirated, and 37 °C serum-free DMEM with or without HR2 peptide (25 μM) was added to the cells. The HR2 peptides were subsequently removed at 0-, 2-, 4-, 8-, 16-, and 32-min time intervals (A) or added at 0-, 2-, 4-, 8-, 16-, and 32-min time intervals after the temperature shift (B). At the 64 min time point, all cells were rinsed and replenished with DMEM supplemented with 10% FBS, and luciferase accumulations were assayed 2 days post-transduction. 1C-A, C1304A; 2C-A, C1303A/C1304A; 3C-A, C1300A/C1303A/C1304A.

When HR2 peptide was present from 0 to 64 min after the 37 °C temperature shift, WT S-mediated infection was blocked by more than 1,000-fold (Fig. 4B). However, when HR2 peptide was present from 2 to 64 min, blockade was only about 20-fold, suggesting that ∼5% of the entry-related WT S protein refolding events took place within the first 2 min after 37 °C temperature shift. When HR2 was added after 16 min at 37 °C, blockade was only 2–3-fold, again suggesting that ∼30–50% of entry was completed within 16 min. Quite strikingly, and in sharp contrast to the rapid refolding of the wild type S proteins, the single (C1304A), double (C1303A/C1304A), and triple (C1300A/C1303A/C1304A) endodomain mutant pseudoviruses were more sensitive to inhibition by HR2 peptides added late after 37 °C temperature shift, with the extent of this sensitivity to HR2 inhibition correlating directly with the degree of Cys → Ala substitution. Entry mediated by the triple mutant S proteins was completely inhibited by HR2 peptides added as late as 16 min after the 37 °C shift, suggesting that the HR1 trihelix exposed itself in delayed fashion and/or remained exposed for remarkably prolonged periods in relation to the wild type protein. A reasonable speculation is that this slower fusion kinetics accounted for the general inefficiencies of the endodomain-mutant S proteins in mediating virus entry (Fig. 3C). This same degree of slower fusion kinetics is not revealed by the much longer 4-h cell-cell fusion assay (Fig. 3A).

The kinetics of S protein refolding was further examined using a biochemical approach. A distinct experimental advantage of the coronaviruses is that their S proteins can be triggered to refold into 6-HBs in reductionist in vitro assays by relatively simple exposure to soluble receptors at 37 °C temperature (47, 57). The resulting 6-HBs, being extraordinarily stable (59), can be visualized in Western blots as ∼58 kDa protease-resistant bands (47). We incubated wild-type and double cysteine mutant (C1303A/C1304A) virions with soluble receptor (N-CEACAM-Fc) at 4 °C and, once at equilibrium, shifted to 37 °C for various time periods. Increased levels of 6-HBs were observed with 37 °C incubation time (Fig. 5A). Far more striking was the finding that the endodomain mutant C1303A/C1304A S proteins were less prone to advancing into 6-HB configurations (Fig. 5A). Similar experiments performed with HIV-S pseudoviruses generated corroborating findings of diminished 6-HBs in C1303A/C1304A and C1300A/C1303A/C1304A S proteins (Fig. 5B). The distal carboxyl-terminal cysteines and/or their palmitate adducts increase the facility of S-mediated refolding into postfusion forms.

DISCUSSION

Viral fusion proteins have distinctive, sequence-specific TM and ENDO domains. Deleting or replacing these regions with similar hydrophobic sequences can eliminate fusion function (60,–,64). Sequence specificity indicates that the TM and ENDO domains have functions beyond mere anchoring of their respective ECTO domains. During membrane fusion, the TM spans transit through hemifusion “lipid stalk” structures, and in postfusion states, the TM spans link stably onto FPs (6, 65) (also see Fig. 6). In addition to amino acid sequence specificities, the TM spans of viral fusion proteins appear to have unusual length requirements as well. Whereas a 20-residue α helix can vertically span a lipid bilayer, viral fusion proteins have hydrophobic, putative TM spans ranging from ∼25 to ∼50 residues. There are several proposed operating mechanisms for these lengthy hydrophobic helices. One view is that the long hydrophobic stretches, if positioned during prefusion states at oblique angles relative to effector membrane planes, might create membrane deformations or dimples that facilitate transitions into lipid stalk conformations (19, 66). Another superior viewpoint is that long hydrophobic anchoring helices are required so that they can be accommodated at various orientations within the curved membrane architectures arising during bilayer fusions (9) (also see Fig. 6). Last, anchoring motifs may operate at the latest fusion stages to ensure that the transit of TM spans through lipid stalks comes concomitant with complete bilayer fusions (19).

We performed this research out of our understanding that coronavirus S proteins have distinctive TM-ENDO domain features that might further reveal fusion operating mechanisms. The portion of the coronavirus TM-ENDO region that is highly hydrophobic and probably α-helical includes ∼42 amino acids, from Lys1263 to Asp1305, in MHV A59 (see Fig. 1B). The COOH-terminal part of this region comprises the cysteine-rich motif, and if all cysteines are palmitoylated, as is strongly suggested by [3H]palmitate labeling (28, 30), then this region would be extraordinarily lipophilic. Indeed, each S trimer would add 27 16-carbon acyl chain lipids to the intravirion membrane leaflet. Several reports evaluating truncated coronavirus S proteins missing part or all of these acylated tails have provided valuable data on the minimal tail lengths required to preserve biological function (28, 30, 67, 68). We used a more subtle approach to evaluate tail activities by substituting one or more of the nine cysteines in the palmitoylation motif with alanines. We expected that the reduced palmitoylation in the Cys → Ala mutants would have deleterious effects on membrane fusion, in accordance with earlier reports (28), but would not entirely eliminate fusion activities in the way that the truncation mutants do, making it so that we could gain some insights into the specific points in the fusion reaction where the palmitates might be operating.

One of our findings was that the distal cysteine-to-alanine substitutions in the endodomain reduced spike protein incorporation into virions. Hydrophobic palmitates may determine assembly of spike into virus particles by helping position the endodomain along the cytoplasmic face of lipid bilayers, thereby facilitating interaction with the assembly-orchestrating M protein. It has already been established that the S-M interaction is generally dependent on S protein palmitoylation, since the addition of a pharmacologic inhibitor of palmitoylation (2-bromopalmitate) inhibits efficient S·M complex formation (29). This report indicates that the most distal carboxyl-terminal cysteines/palmitates are crucial elements for S incorporation. Notably, for other class I fusion proteins, such as HIV-1 Env and influenza HA, palmitoylation of endodomain cysteines is also required for assembly (69, 70), although these requirements vary with influenza virus strains. For HIV and influenza, assembly and budding take place at or near the plasma membrane in lipid raft microdomains (71, 72), and the requirements for glycoprotein incorporation into virions might be explained by the biophysical partitioning of palmitoylated proteins into lipid rafts (73). Coronaviruses bud into the ER (74, 75), where raft-defining lipids are relatively rare (76). Thus, the palmitate requirements for S assembly are less clear, but it is possible that the extraordinary degree of S palmitoylation organizes adjacent ER lipids into rigid arrays that are akin to raftlike environments. Indeed, if the endodomains are α-helical, as predicted by bioinformatics, then palmitates extending from cysteines spaced 3–4 residues apart would be within ∼5 Å of each other, forming nanoarrays of adjacent saturated fatty acids underneath each S trimer. This hypothetical lipid organization around S proteins might create ER membrane environments that are crucial to coronavirus assembly. The next step in understanding assembly may come in dissecting viral protein-lipid and lipid-lipid interactions.

There were direct relationships between S assembly and S-mediated membrane fusion competence. For example, relative to wild-type S, the C1303A/C1304A mutant was poorly incorporated into virions (Fig. 2A) and was a compromised membrane fusogen (Figs. 3C and 4). These relationships argue for a sorting process at the budding sites, with inclusion of S proteins into virions according to palmitoylation status. This sorting process insures that only the most palmitoylated, fastest fusing S proteins are integrated into secreted virions. S proteins with less palmitoylation sort to cell surfaces as free proteins and perform related cell-cell fusions. This cell-cell fusion activity appears to be far less dependent on quick fusion reactions because the wild type and Cys → Ala mutants were indistinguishable in our assays of syncytial formation (Fig. 3A).

On cell surfaces, the wild type and Cys → Ala mutant S proteins probably occupy similar raftlike environments, because all S forms were equally incorporated into the HIV-based pseudoviruses that are known to bud from lipid raft microdomains (77) (Fig. 3B). Using these HIV-S pseudoviruses, we found that the stepwise substitution of one, two, and then three COOH-terminal cysteines caused progressively declining transduction. This result could not be explained by any obvious defects in S protein structure or density on pseudoviruses because uncleaved and cleaved S forms were equally abundant in all viruses (Fig. 3B). Therefore, we sought out more subtle effects of the endodomain mutations on the virus entry process by using HR2 peptides, potent inhibitors of virus entry, as probes for the intermediate folded S protein conformations (Fig. 4). By adding HR2 peptides into media at various times before and after initiating the S refolding reaction, we could assess the time required for S proteins to enter into and out of the intermediate prehairpin state (see Fig. 6). These experiments yielded enlightening results, allowing us to conclude that the endodomain mutants remained HR2-sensitive for prolonged periods, in essence slowing the kinetics of refolding relative to wild type S proteins.

Endodomain mutant S proteins transition from native to unfolded prehairpin states at the same rate as wild type spikes, because HR2 peptides added 0–2 min after initiating S refolding resulted in a ∼10-fold reduction for all S-mediated transductions (Fig. 4A). Similarly, equivalent inhibitions were observed when HR2 peptides were added 0–4 min after initiation. In contrast, when HR2s were introduced at various times after initiating S refoldings, the Cys → Ala mutants were preferentially blocked (Fig. 4B). These data support a view in which the duration of the prehairpin state is regulated by the palmitoylated endodomains. We consider it likely that virus S proteins are triggered to unfold into the prehairpin (HR2-sensitive) state at cell surfaces, immediately after binding cell surface CEACAM receptors. Particularly for those viruses with underpalmitoylated S proteins, we suggest that this prephairpin architecture remains as viruses enter endosomes, whereupon all but the core HR1-HR2 fusion machinery is cleaved away by endosomal proteases (4, 78). Following this proteolysis, hairpin closure might then ensue, effecting the membrane fusion event. The timely completion of this hairpin closure appears to be correlated with virus infectivity.

As we expected, the kinetics of S protein refolding was also reflected by the relative abundances of proteinase K-resistant 6-HB hairpin forms in the various virus preparations. Our experiments here were modeled after those of Taguchi et al. (47), who found that coronavirus S proteins can be triggered to refold into 6-HBs by exposure to soluble receptors. Indeed, soluble receptors created increasing 6-HB levels with increasing incubation time (Fig. 5A), and the endodomain mutations impeded this 6-HB formation in accordance with the number of endodomain mutations (Fig. 5B).

All of these findings solicit speculations on the way in which the endodomains, specifically the cysteines and/or their palmitate adducts, change the rate-limiting step of the membrane fusion reaction. Given that the endodomain Cys → Ala mutations progressively extend the HR2-sensitive stage, we suggest that the absence of these cysteines-palmitates raises an activation energy barrier between the HR2-sensitive and 6-HB stage. It was recently discovered that the SARS-CoV HR2 regions exist in a monomer-trimer equilibrium (79). The idea is that the equilibrium has to be shifted toward monomers, so that separated HR2 helices can each invert relative to HR1 and attach in antiparallel fashion onto the HR1 trimers (see Fig. 6). Given that the HR2 regions in isolation can stick together into trimers (79), the role of the endodomain cysteines-palmitates could be to anchor the transmembrane spans such that a separation of HR2 monomers is maintained in the native S structure. This prevention of HR2 trimerization in the native structure would then allow membrane fusion to occur in a timely fashion. We take our cues here from the cryoelectron microscopy reconstructions of HIV that reveal a tripod-like arrangement for virus spikes coming out of the virion membrane (80, 81). Class I protein-mediated membrane fusion may depend on prefusion spikes with separated HR2 domains. Palmitoylation of juxtamembranous cysteines may induce the transmembrane domain to tilt relative to the lipid bilayer plane, as suggested by Abrami et al. (82), who found that unusually long transmembrane spans could be accommodated within membrane interiors if palmitoylated endodomain cysteines were nearby to presumably keep the spans from adopting a perpendicular orientation relative to the membrane. If this concept applies to the S proteins, then extracellular extension from the membrane bilayer might be progressively more oblique with increasing endodomain palmitoylation, and in turn, the degree to which HR2 regions remain separated and poised for the membrane fusion reaction would relate to the extent of endodomain palmitoylation.

One final and obvious point about our study is that the workings of viral fusion proteins can only be partially understood by analyzing the structure and function of soluble protein ectodomains. The way that viral fusion proteins are embedded into virion and infected cell membranes is crucial to our understanding. For the coronaviruses, extensive palmitoylation of fusion protein endodomains may set up a metastable membrane embedment that is both preferentially selected for assembly into virions and set up for rapid membrane fusion-related refolding.

B.The Amino Acids and Their Properties Proteins are constructed of a series of amino acids in one continuous string synthesized together in the cellular protein synthetic machinery of polyribosomes. The front end first to be synthesized is called the Nterminus, because the nitrogen at one end of each amino acid remains exposed, while the other end is called the C-terminus, because the C at the other end of each amino acid in the growing chain is exposed. Synthesis always goes N to C terminal, generally shown as left to right. In the example above from SARS, ELDKY would be a five-amino acid peptide, with the E N-terminal and the Y C-terminal. The letters are from the single letter code for the 20 different amino acids that commonly occur in human and animal proteins. The single-letter codes for the amino acids are shown in Figure 3, grouped into 8 separate clusters of amino acids with similar properties.

All amino acids are built on the same backbone that forms the protein chain itself. They are differentiated on the basis of the very different side chains that impart specific molecular character to each one. The first group is composed of Glycine (Gly, G) and Alanine (Ala, A) that both have very short side chains. In the case of glycine, none in fact, just a hydrogen. In the case of alanine, a single methyl group of only three atoms. Glycine is effectively a spacer, allowing free rotation for that spot in the protein sequence. Alanine provides very little bulk, and fits almost anywhere. The second amino group to the right comprises the aliphatic series of amino acids, imparting hydrophobicity (greasiness) to their position in the protein chain. Valine (Val, V) is the smallest, just three more atoms than Alanine, and Leucine (Leu, L) three more. Leucine is one of the most common amino acids in proteins, providing basic hydrophobic bulk wherever its place in the protein chain. Both V and L are symmetrical in shape. Isoleucine (Ile, I) is similar in size to Leucine, but asymmetrical. Methionine (Met, M) differs from the others in having a sulfur atom near its outer end, rather than a carbon. It is distinguished by always being the first amino acid in any protein chain, because gene expression always begins with RNA that codes for it. At the lower left are two amino acids grouped for their uniqueness, while at the same time being hydrophobic. In Proline (Pro, P) the backbone atoms are cyclized into a ring structure. Instead of free rotation around each backbone bond, the two ends of Proline are locked into a 130 degree angle to one another. Proline creates kinks in the protein chain at critical locations. Cysteine (Cys, C) is unique in that it has a terminal sulfhydryl group (-SH). Two cysteines can become covalently bound to one another, forming an S-S or disulfide bond between different regions of the protein chain, locking them together in a fixed configuration to one another. Cysteines are often highly conserved landmarks in proteins very important for stabilizing secondary structure. To the right of P and C are Phenylalanine (Phe, F), Tryptophan (Trp, W), and Tyrosine (Tyr, Y), the aromatic amino acids with either a planar benzene ring or an indole double-ring for highly hydrophobic side chains. In this regard, W might well stand for whopper. It is much larger than any other hydrophobic side chain. Wherever it is found constitutes a veritable center of hydrophobicity. It has a high natural affinity for cholesterol found in cellular target membranes (Yau et al. 1998). The four groups to the right of the above groups in Figure 3 are more hydrophilic, and tend to be found on the outside of proteins. Next at the top are Serine (Ser, S) and Threonine (Thr, T). These are hydroxylated amino acids (-OH). Apart from readily interacting with water, they may also be the site where polysaccharide adducts can be added to the protein chain in what is called an O-glycosidic linkage, effectively sugar coating to that region of protein. Below S and T are Glutamine (Gln, Q), Asparagine (Asn, N) and Histidine (His, H). These are mostly neutral amino acids with secondary amines. Q is notable because it has a strong propensity to be part of an alpha helix; N, because it can serve as a site for N-linked polysaccharide adducts, another type of sugar coating. H is relatively rare, with an imidazole ring for a side chain that can impart a slight charge. H is frequently found where proteins interact with some sort of substrate, with H as the reactive group on the protein. On the top right are the two basic amino acids, Lysine (Lys, K) and Arginine (Arg, R) for which the side chains end in a free amino groups, imparting a positive charge to that part of the protein chain. Since cell surfaces are negatively charged, K and R have a natural affinity. They are also sites for endoproteolytic cleavage of proteins by cellular proteases similar to trypsin and furin. As such, they have a key role in maturation of viral fusion/entry proteins when in certain critical locations. Arginine is very large, comparable to tryptophan in size, but on the hydrophilic side Finally, on the lower right are Glutamate (Glu, E), and Aspartate (Asp, D), the acidic amino acids that terminate in a carboxylic group (- COOH) and impart a negative charge to the protein chain. In terms of protein structure, they differ significantly in that E has a strong propensity (like its neutral homologue Q) to reside in alpha helices, whereas D, only shorter by a three atom methylene group, much less so. When modeling for alpha helices (see Lupas 1996), such as those shown in Figure 1, my basic rule has always in fact been simple, “watch your Es and Qs”, especially when clustered with A, L, F, W and K. G, P, S, and T are often found in turns, especially when clustered together. The other amino acids are more malleable in terms of protein structure, but clusters of I, V, Y, and M are common in beta-pleated sheet regions. To those who study protein sequence and structure, the amino acids are not just beads on a long string, in the case of S1/S2 over 1200 amino acids long. The sequences are interpretable in terms of character, structure and function of each particular stretch of protein, as we are about to examine in comparing the S1/S2 sequences of nCoV2019 and SARS.

c.

The spike glycoprotein of the newly emerged SARS-CoV-2 contains a potential cleavage site for furin proteases. This observation has implications for the zoonotic origin of the virus and its epidemic spread in China.

The membrane of coronaviruses harbors a trimeric transmembrane spike (S) glycoprotein (pictured) which is essential for entry of virus particles into the cell. The S protein contains two functional domains: a receptor binding domain, and a second domain which contains sequences that mediate fusion of the viral and cell membranes. The S glycoprotein must be cleaved by cell proteases to enable exposure of the fusion sequences and hence is needed for cell entry.

The nature of the cell protease that cleaves the S glycoprotein varies according to the coronavirus. The MERS-CoV S glycoprotein contains a furin cleavage site and is probably processed by these intracellular proteases during exit from the cell. The virus particles are therefore ready for entry into the next cell. In contrast, the SARS-CoV S glycoprotein is uncleaved upon virus release from cells; it is likely cleaved during virus entry into a cell.

Proteolytic cleavage of the S glycoprotein can determine whether the virus can cross species, e.g. from bats to humans. For example, the S glycoprotein from a MERS-like CoV from Ugandan bats can bind to human cells but cannot mediate virus entry. However, if the protease trypsin is included during infection, the S glycoprotein is cleaved and virus entry takes place. This observation demonstrates that cleavage of the S glycoprotein is a barrier to zoonotic coronavirus transmission.

Examination of the protein sequence of the S glycoprotein of SARS-CoV-2 reveals the presence of a furin cleavage sequence (PRRARS|V). The CoV with the highest nucleotide sequence homology, isolated from a bat in Yunnan in 2013 (RaTG-13), does not have the furin cleavage sequence. Because furin proteases are abundant in the respiratory tract, it is possible that SARS-CoV-2 S glycoprotein is cleaved upon exit from epithelial cells and consequently can efficiently infect other cells. In contrast, the highly related bat CoV RaTG-13 does not have the furin cleavage site.

Whether or not the furin cleavage site within the S glycoprotein of SARS-CoV-2 is actually cleaved remains to be determined. Meanwhile, it is possible that the insertion of a furin cleavage site allowed a bat CoV to gain the ability to infect humans. The furin cleavage site might have been acquired by recombination with another virus possessing that site. This event could have happened thousands of years ago, or weeks ago. Upon introduction into a human – likely in an outdoor meat market – the virus began its epidemic spread.

Furins are also known to control infection by avian influenza A viruses, in which cleavage of the HA glycoprotein is needed for entry into the cell. Low-pathogenic avian influenza viruses contain a single basic amino acid at the cleavage site in the HA protein which is cleaved by proteases that are restricted to the respiratory tract. Insertion of a furin cleavage site in the HA of highly pathogenic avian H5N1 influenza viruses leads to replication in multiple tissues and higher pathogenicity, due to the distribution of furins in multiple tissues.

Acquisition of the furin cleavage site might be viewed as a ‘gain of function’ that enabled a bat CoV to jump into humans and begin its current epidemic spread.

D. In order to examine the entry of SARS-CoV into cells, cell lines were first generated which stably expressed ACE2 fused to either a Myc tag or GFP. ACE2 expression in the 293E-ACE2-Myc cell lines was confirmed by western blotting (Supplementary information Figure S1), while ACE2 receptor expression in the 293E-ACE2-GFP cell lines was directly visualized by fluorescence. Figure 1A shows that ACE2-GFP is localized primarily at the cell surface, suggesting that expression and localization of ACE2 in this cell line is similar to that in Vero E6 cells. The 293E-ACE2-GFP cell line allowed for direct tracking of the movement of the receptor under a fluorescence microscope. In agreement with a previous report, which showed that ACE2 expression in some nonpermissive cells renders them permissive to infection 15, both of these stable cell lines can be infected by spike-bearing pseudoviruses.

We first tested whether spike protein alone could enter cells using the 293E-ACE2-GFP cell line, since spike protein was believed to mediate virus entry. Purified recombinant spike protein fused to the human IgG Fc fragment (S1190-Fc) was added to 293E-ACE2-GFP cells. After incubation at 37 °C for 3 h, a number of GFP-containing vesicles were observed in the perinuclear area (Figure 1B), whereas vesicles were not observed in the control cells treated with Fc protein alone (Figure 1C), suggesting that spike protein specifically induced translocation of ACE2-GFP from the cell surface to the interior of the cells. Statistical analysis of the percentage of cells in which vesicles accumulated showed that there was a significant difference between the effects of these treatments on virus entry (Supplementary information Figure S2). A dual staining assay for spike protein and ACE2-GFP showed colocalization of these two proteins (Figure 1D), which suggests that spike protein was bound to ACE2 in the vesicles and that spike protein was taken up by the cell, most likely by endocytosis. The fact that spike protein induced translocation of ACE2 from the cell surface to intracellular compartments is in agreement with our previous finding that surface expression of ACE2 in Vero E6 cells decreased after incubation with spike protein at 37 °C for 3 h 16.

After an additional incubation at 37 °C, receptor-containing vesicles were no longer visible within the cells, and green signals were instead seen clustered near the cell surface, suggesting that the receptors were recycled (Figure 1E). This process can be blocked by lysosomotropic agents, which are reported to function by elevating the pH of acidic compartments, thereby inhibiting dissociation of the ligand from the receptor and trapping the receptor in the endosome 17. We found that after treatment with ammonium chloride, bafilomycin A1, or chloroquine, the viral receptor was trapped within perinuclear vacuoles, even after a 14-h incubation, a period that would allow the receptor to be recycled to the cell surface under normal conditions . These results reflect the normal traffic of membrane flow; that is, cargos are first internalized by a besieged membrane, which fuses with the early endosome and then the late endosome, where cargos are dissociated from the receptors. Receptors are usually recycled back to the cell membrane, while cargos are targeted to lysosomes.

SARS-CoV spike-bearing pseudoviruses enter cells via endocytosis

Due to the highly contagious nature of SARS-CoV, we used spike protein-bearing pseudoviruses to study the virus entry route. Pseudoviruses are often used to mimic the entry of real viruses, such as hepatitis C virus 18, 19, Ebola virus, and Marburg virus 20, into host cells. This strategy is a powerful tool for studying early events in the life cycle of a virus. Therefore, we used retroviral pseudoviruses bearing the SARS-CoV spike protein to infect ACE2-GFP-expressing HEK293E cells. After a 3-h incubation at 37 °C, GFP-containing vesicles were detected within the cells (Figure 2A), while there were few intracellular vesicles when the same cell lines were treated with a control pseudovirus bearing VSV-G protein on the surface (Figure 2B). Dual labeling of spike protein and ACE2 also showed colocalization (Figure 2C), which indicates that the pseudovirus may be contained in the vesicles. After an additional 10 h incubation, few vesicles were observed (Figure 2D) for both treatments. This result was similar to that observed following treatment with spike protein alone. Moreover, after treatment with ammonium chloride, bafilomycin A1, or chloroquine, GFP-containing vesicles were detected, even after a 12-h incubation (Figure 2E and unpublished data), suggesting that these reagents inhibited receptor recycling. These results were nearly identical to those obtained following spike protein treatment, which indicated that SARS-CoV may enter cells via endocytosis. Although the pseudovirus can express GFP, the green vesicles observed in these cells at this time point resulted from cellular ACE2-GFP rather than from virally expressed GFP, as expression from the viral GFP gene was not observed at 12 h after infection. This notion was verified with ACE2-Myc-expressing HEK293E cells, in which no GFP expression was observed at 12 h postinfection with the pseudovirus

The endocytic pathway is usually thought to be pH-dependent. Therefore, if SARS-CoV can enter cells via endocytosis, lysosomotropic agents should inhibit virus infection. Vero E6 cells have been reported to be naturally permissive for SARS-CoV. We infected Vero E6 cells with spike-bearing pseudovirus in the presence or absence of lysosomotropic agents. Since the pseudovirus expresses GFP, infected cells can be distinguished from uninfected cells by viral GFP expression. Figure 2F shows that pseudovirus infection of Vero E6 cells leads to viral GFP expression, while the lysosomotropic agents inhibited GFP expression. This result suggests that successful virus entry is pH-dependent.

To provide further confirmation that SARS-CoV enters host cells via endocytic pathways, dual immunofluorescence labeling with antibodies specific for the SARS-CoV spike protein and the early endosome marker protein early endosome antigen 1 (EEA1) was performed, followed by confocal microscopy analysis. After 1 h of infection, a punctate pattern characterized by strong colocalization of the spike protein and EEA1 was observed, confirming that SARS-CoV was targeted to early endosomes (Figure 2J-L). Alexa-labeled transferrin was used as a positive control (Figure 2G-I), since the endocytosis of transferrin has been well characterized. After binding of transferrin to its receptor, the complex was shown to be internalized in coated vesicles, followed by fusion with endosomes 21, 22, 23.

Thus, the spike-bearing pseudovirus, like spike protein, can cause ACE2 receptor translocation in a pH-dependent manner, and the pseudovirus infects Vero E6 cells in a pH-dependent manner. In addition, the pseudovirus is targeted to the early endosome after it enters the cell. Taken together, these results indicate that the spike-bearing pseudovirus can enter cells via endocytosis.

SARS-CoV can enter cells in the absence of clathrin-mediated endocytosis

After determining that SARS-CoV can enter cells via endocytosis, we attempted to identify the specific endocytic pathway exploited by this virus. The endocytic pathways exploited by animal viruses to enter host cells include macropinocytosis, the clathrin-dependent pathway, and the caveolae-dependent pathway, as well as routes that are not as well characterized, such as clathrin- and caveolae-independent pathways. The clathrin-dependent pathway is the most common of these pathways. Because drugs can produce pleiotropic effects, we employed several complementary approaches to determine the role of clathrin-mediated endocytosis in the entry of SARS-CoV into cells 4.

Chlorpromazine (CPZ), a drug commonly used to inhibit clathrin-mediated endocytosis, causes clathrin lattices to assemble on endosomal membranes and prevents the assembly of coated pits at the cell surface 24. The effect of CPZ on clathrin-mediated endocytosis was first tested with transferrin labeled with Alexa 594 (Alexa 594 Tf) using Vero E6 cells. In mock-treated cells, transferrin appeared to cluster in the perinuclear region, while in the CPZ-treated cells, transferrin uptake was blocked, leaving transferrin at the cell surface (Figure 3A and ​and3B).3B). This result confirms the effectiveness of CPZ. We then tested the effect of CPZ on spike-pseudovirus entry using two different methods: confocal microscopy and spectrofluorometer measurement. After Vero E6 cells were treated with CPZ and incubated with spike-bearing pseudoviruses for 1 h, the ability of spike-bearing pseudovirus to enter Vero E6 cells was examined using confocal microscopy. Figure 3C and ​and3D3D showed that the spike-bearing pseudovirus can enter Vero E6 cells despite CPZ treatment. In order to quantify the effect of CPZ on viral entry, Vero E6 cells were treated with the indicated amounts of CPZ and were then infected with spike-bearing pseudoviruses. The relative infectivity of the viruses was determined by measuring the level of GFP expression at 60 h postinfection using a spectrofluorometer. Our results demonstrated that CPZ did not significantly inhibit virus entry

We then used the more specific siRNA approach to further explore the role of clathrin-mediated endocytosis in SARS-CoV entry. HEK293E-ACE2-Myc cells were used because of their high transfection efficiency. Cells were transfected with siRNA specific for clathrin heavy chain (HC) or control siRNA. Seventy-two hours post-transfection, the effects of the siRNA were assayed using western blotting and the cells were infected with pseudoviruses. After another 48 h, the cells were lysed and viral GFP expression was quantified using a spectrofluorometer. Although the expression of clathrin HC was knocked-down in the cells transfected with the specific siRNA compared to cells transfected with control siRNA, the infectivity of the virus was not significantly affected (Figure 3F).

We further employed a dominant-negative variant of Eps15 to explore the role of clathrin-mediated endocytosis in SARS-CoV entry. Eps15 plays an important role in clathrin-mediated endocytosis, and expression of the dominant-negative Eps15 construct GFP-EΔ95/295 has been reported to inhibit clathrin-mediated endocytosis, while the construct GFP-D3Δ2 can be used as a control 25, 26. To confirm disruption of the clathrin-dependent endocytic pathway, the transfected cells were incubated with Alexa-594 transferrin. As expected, the EΔ95/295 construct, but not the D3Δ2 construct, successfully inhibited transferrin uptake . However, we found that SARS-CoV was able to enter cells expressing GFP-EΔ95/295 as efficiently as it entered cells expressing GFP-D3Δ2 ( demonstrating that expression of the dominant-negative variant Eps15 did not block SARS-CoV entry. All of these results indicate that SARS-CoV is able to enter cells that lack a functional clathrin-dependent endocytic pathway.

SARS-CoV enters cells independent of caveolae-mediated endocytosis

Caveolae-dependent endocytosis is a newly characterized endocytosis pathway. Caveolae are small, flask-shaped invaginations in the plasma membrane composed of high levels of cholesterol and glycosphingolipids as well as the integral membrane protein caveolin 27. Because cholesterol is a prominent component of lipid rafts, which are involved in caveolae formation, sequestration of cholesterol with the sterol-binding drugs filipin and nystatin impairs caveolae-mediated endocytosis 4. To determine whether SARS-CoV enters cells through a caveolae-mediated pathway, we treated cells with filipin and nystatin. As a control, the effects of these drugs on caveolae-mediated endocytosis were examined by measuring the uptake of cholera toxin subunit B (CTB), since it is targeted to caveolae and its uptake can be blocked by these kinds of drugs 28, 29. Data presented in Figure 5A-C indicate that these drugs block CTB uptake, since Alexa-594 CTB clustered at the cell surface following drug treatment, compared to its concentration near the nucleus in mock-treated cells (Supplementary information Figure S4). When treated cells were infected with pseudovirus, virus entry was not inhibited by treatment with filipin or nystatin (Figure 5D and ​and5F),5F), but was inhibited by another drug, methyl-β-cyclodextrin (MβCD) (Figure 5E), an oligosaccharide used to deplete cholesterol from cell membranes. The effects of these drugs on virus entry were measured quantitatively. Treatment of Vero E6 cells with MβCD was shown to inhibit pseudovirus entry in a dose-dependent manner (Figure. 5H), while filipin and nystatin had no inhibitory effect on virus entry

To further study whether the caveolae-dependent pathway was involved in the endocytosis of this virus, a colocalization assay of spike protein and caveolin-1 was performed. The infected cells were immunolabeled for SARS-CoV spike protein as a marker of the pseudoviruses and for caveolin-1 as a marker of the caveolae. No significant colocalization of the pseudovirus and caveolin-1 specific signals was observed . Taken together, these results indicate that SARS-CoV is able to enter cells in a caveolin-independent manner.

E.

According to the above , this process is followed by a piece of the spike protein, called the fusion peptide, interacting directly with the host cell membrane and facilitating merging to form a fusion pore, or opening.

They said the virus then transfers its genome into the host cell through this pore, and eventually hijacking the host cell's machinery to produce more viruses.

In the study, found that charged atoms of calcium interacting with the fusion .