key: cord-0815262-eacle7rt
authors: Nguyen, Hanh T.; Zhang, Shijian; Wang, Qian; Anang, Saumya; Wang, Jia; Ding, Haitao; Kappes, John C.; Sodroski, Joseph
title: Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects
date: 2020-10-23
journal: bioRxiv
DOI: 10.1101/2020.10.22.351569
sha: d55cfff224d977047503114957c27daf6c20b9a3
doc_id: 815262
cord_uid: eacle7rt

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.

was expressed in 293T cells alone or in combination with HIV-1 Gag/protease, which 148 promotes the budding of lentivirus VLPs. The S gp precursor as well as the mature S1 149 and S2 glycoproteins could be detected in the cell lysates, on the cell surface and on 150 lentivirus VLPs (Fig. 2) . The S1 gp was shed into the medium of cells expressing the 151 wild-type S gp alone (Fig. 3A) . Very low levels of S gp were detected in the particles 152 prepared from the culture medium of cells expressing the wild-type S gp without HIV-1 153 Gag, indicating that the vast majority of the S gp pelleted in the presence of HIV-1 Gag 154 is in lentivirus VLPs and not in extracellular vesicles ( Fig. 2 and 3B ). We confirmed the 155 presence of VLPs of the expected size and morphology decorated with S glycoproteins 156 by electron microscopy with a gold-labeled S-reactive convalescent serum (data not 157 shown). A higher ratio of cleaved:uncleaved S gp was present on lentivirus VLPs than 158 in the cell lysates or on the surface of S gp-expressing cells, as seen previously (32,38) 159 ( Fig. 2 and 3B ). Selective incorporation of the cleaved S glycoproteins was also 160 observed in VSV pseudotypes (Fig. 4) . The uncleaved wild-type S gp in cell lysates and 161 on the cell surface was modified mainly by high-mannose glycans, whereas the cleaved 162 S1 gp contained complex carbohydrates (Fig. 3C ). Both the cleaved (major) fraction 163 and uncleaved (minor) fraction of the wild-type S gp incorporated into lentivirus VLPs 164 were modified by complex glycans, indicating passage through the Golgi (51). This 165 result is similar to our finding that HIV-1 envelope glycoproteins can be transported to To examine the proteolytic processing and glycosylation of the wild-type S gp in 170 the context of cells producing SARS-CoV-2 VLPs, we coexpressed the M protein or the protein was coexpressed with the S gp, faster-migrating forms of the S1 and S2 174 glycoproteins were evident in the cell lysates, on the cell surface and on VLPs (Fig. 3D) . 175 In cells treated with Brefeldin A, an inhibitor of anterograde transport through the Golgi 176 (57-59), these faster-migrating forms predominated in M-expressing cells, but were also 177 evident even in cells without M. Sensitivity to PNGase F and Endoglycosidase H 178 identified these S1/S2 species as hypoglycosylated glycoforms modified by high- The furin cleavage site reduces virus infectivity, stability and sensitivity to ACE2 212 and neutralizing antibodies, but enhances S gp syncytium-forming ability 213 At some point during its evolution from an ancestral bat coronavirus, the SARS- 214 CoV-2 S gp acquired a multibasic cleavage site for furin-like proteases at the S1/S2 215 junction (9,15,41,42). To evaluate the impact of this evolutionary change, we altered 216 the two residues immediately N-terminal to the proposed furin cleavage site (FurinMut in 217 Fig. 1) . Compared with the wild-type SARS-CoV-2 S gp, the FurinMut S gp was 218 expressed at higher levels in 293T cells and was efficiently incorporated into lentivirus 219 VLPs, but was inefficiently processed into S1 and S2 glycoproteins ( Fig. 2 and 3A) . 220 Two glycoforms of the uncleaved FurinMut, one with only high-mannose glycans and 221 the other with complex glycans, were present in cell lysates and on the cell surface; 222 however, only the glycoform modified by complex carbohydrates and presumably 223 transported through the Golgi was found in lentivirus VLPs (Fig. 3C ).

In contrast to the wild-type S gp, FurinMut did not mediate syncytium formation or 226 induce cell-cell fusion in the alpha-complementation assay ( Fig. 5B and 6 ). However, 227 the cell-fusing activity of the FurinMut S gp was dramatically increased by coexpression 228 of TMPRSS2 and ACE2 in 293T target cells (Fig. 7B ). The infectivity of the FurinMut-229 pseudotyped virus was 7-50 times higher than that of viruses pseudotyped with the wild-230 type S gp; the infectivity difference between viruses pseudotyped with FurinMut and 231 wild-type S glycoproteins was greater for HIV-1 pseudotypes than for VSV pseudotypes 232 ( Fig. 6 and 8A ). The infectivity of the FurinMut virus incubated on ice was more stable 233 than that of the wild-type S virus (Fig. 8B) . FurinMut viruses were more sensitive to 234 inhibition by soluble ACE2 and convalescent sera than wild-type viruses (Fig. 8C ).

Thus, the acquisition of the furin cleavage site by the SARS-CoV-2 S gp decreased 236 virus stability, infectivity and sensitivity to soluble ACE2 and neutralizing antibodies, but 237 greatly enhanced the ability to form syncytia.

Alteration of other potential cleavage sites reduces S gp processing and function 240 The SARS-CoV-1 S gp lacks a favorable cleavage site for furin-like proteases, 241 but during infection of a target cell is thought to be cleaved at a nearby secondary site 242 and at the S2′ site by cellular proteases (Cathepsin L, TMPRSS2) (62-64). Alteration of the secondary cleavage site near the S1/S2 junction (S1/S2Mut) in the SARS-CoV-2 S 244 gp resulted in complete lack of proteolytic processing, inefficient incorporation into 245 lentivirus VLPs, and loss of function in cell-cell fusion and infectivity assays (Fig. 2, 3C , 246 5B and 6). Alteration of the S2′ site (S2′Mut) also led to lack of S processing, although 247 low levels of VLP incorporation and infectivity were detected. The S2′Mut gp in the 248 VLPs was Endoglycosidase H-resistant, suggesting that it is modified by complex 249 carbohydrates (Fig. 3C) . The S2′Mut gp did not detectably mediate cell-cell fusion (Fig.   250 6). Thus, despite the presence of the furin cleavage site in these mutants, proteolytic 251 processing did not occur. Both mutants exhibited severe decreases in infectivity and 252 cell-cell fusion. However, coexpression of TMPRSS2 in the ACE2-expressing target 253 cells enhanced cell-cell fusion by the S1/S2Mut gp but not by S2′Mut gp (Fig. 7B ).

The D614G change in the predominant SARS-CoV-2 strain increases 256 S1-trimer association, virus infectivity and sensitivity to soluble ACE2 and 257 neutralizing antisera 258 The change in Asp 614 to a glycine residue (D614G) is found in the predominant 259 emerging SARS-CoV-2 strains worldwide (43,44). The D614G S gp was cleaved 260 slightly more efficiently than the wild-type S gp, but shed less S1 into the medium of 261 expressing cells (Fig. 2, 3A and 6) . The efficiencies of cell-cell fusion mediated by the 262 D614G and wild-type S glycoproteins were comparable ( Fig. 5B and 6 ). Both lentivirus 263 and VSV vectors pseudotyped with the D614G S gp infected cells 4-16-fold more 264 efficiently than viruses with the wild-type S gp ( Fig. 6 and 8A ). The stabilities of the 265 viruses pseudotyped with D614G and wild-type S glycoproteins on ice were comparable 266 (Fig. 8B) . Importantly, the viruses with D614G S gp were approximately 7-fold more sensitive to soluble ACE2 and 2-5-fold more sensitive to neutralizing antisera than 268 viruses with wild-type S gp (Fig. 8D ). Soluble ACE2 bound and induced the shedding of 269 S1 from D614G VLPs more efficiently than from VLPs with the wild-type S gp (Fig. 9A   270 and B). The lentivirus VLPs with D614G S gp exhibited a significantly greater 271 association of the S1 subunit with the trimer (half-life greater than 5 days at 37°C) 272 compared with viruses with wild-type S gp (half-life 2-3 days at 37°C) (Fig. 9C ). S1 273 association with detergent-solubilized S gp trimers was greater for D614G than for wild-274 type S over a range of temperatures from 4-37°C (Fig. 9D) . Thus, the D614G change 275 enhances virus infectivity, responsiveness to ACE2 and S1 association with the trimeric 276 spike.

Changes in the S2 fusion peptide decrease infectivity 279 The putative fusion peptide of the SARS-CoV-2 S2 glycoprotein (residues 816-280 834) has been identified by analogy with the SARS-CoV-1 S gp. Changes in the fusion 281 peptide and a more C-terminal S2 region in SARS-CoV-1 S gp have been suggested to 282 result in fusion-defective mutants (65-67). We introduced analogous changes into the 283 putative fusion peptide of the SARS-CoV-2 S gp (L821A and F823A), and also made a 284 change (F888R) in the downstream region implicated in SARS-CoV-1 S gp function.

The L821A and F823A mutants were processed slightly less efficiently than the wild-286 type S gp. Compared with the wild-type S gp, the F888R mutant exhibited a lower ratio 287 of the S1 gp in cell lysates relative to the S1 gp in cell supernatants, suggesting a 288 decrease in the association of S1 with the S trimer ( Fig. 3A and 6 ). Modest decreases 289 in the level of S glycoproteins on lentivirus VLPs were observed for all three mutants. 290 formed were smaller than those induced by the wild-type S gp ( Fig. 5B and 6 ). F888R 292 was severely compromised in the ability to mediate cell-cell fusion; however, this ability 293 was recovered when TMPRSS2 was coexpressed with ACE2 in the target cells ( Fig.   294 7B). The infectivity of viruses pseudotyped with these three mutants was greatly 295 decreased, relative to viruses with wild-type S gp ( Fig. 6 and 7A ). In summary, these 296 S2 ectodomain changes exert pleiotropic effects, ultimately compromising virus 297 infectivity.

Palmitoylated membrane-proximal cysteines in the S2 endodomain contribute to 300 virus infectivity 301 The S2 endodomain of SARS-CoV-1 is palmitoylated, contains an ERGIC-302 retention signal, and contributes to the interaction of S with the M protein during virus 303 assembly (45-50). We altered the highly similar endodomain of the SARS-CoV-2 S gp, 304 which contains ten cysteine residues potentially available for palmitoylation. The 305 endodomain/cytoplasmic tail was deleted in the ΔCT mutant, leaving only the two 306 membrane-proximal cysteine residues (Fig. 1 ). In the ΔERsig mutant, the putative 307 ERGIC-retention signal at the C terminus of the endodomain was deleted. In three 308 additional mutants, the N-terminal five cysteine residues (1 st 5C-to-A), the C-terminal 309 five cysteine residues (2 nd 5C-to-A), or all ten cysteine residues (10 C-to-A) were altered 310 to alanine residues. The ΔCT and ΔERsig mutants were expressed on the cell surface at higher 313 levels than that of the wild-type S gp, and the level of ΔCT on lentivirus VLPs was 314 significantly increased (Fig. 2, 3C and 6 ). Both ΔCT and ΔERsig mediated syncytium formation and virus infection at levels comparable to those of the wild-type S gp (Fig. 5B   316 and 6). Therefore, except for the two membrane-proximal cysteine residues, the SARS-317 CoV-2 S2 endodomain is not absolutely required for cell-cell fusion or virus entry.

Proteolytic processing of the 2 nd 5C-to-A and 10 C-to-A mutants was more 320 efficient than that of the wild-type S gp ( Fig. 2 and 6 ). Although all three mutants with 321 altered endodomain cysteine residues were expressed on the cell surface and To evaluate the palmitoylation of the wild-type and mutant S glycoproteins, we 329 used hydroxylamine cleavage and mPEG-maleimide alkylation to mass-tag label the 330 palmitoylated cysteine residues (68). The majority of the wild-type S2 gp was 331 palmitoylated, with different species containing one to four acylated cysteines (Fig. 10 ).

The ΔCT, 1 st 5C-to-A and 2 nd 5C-to-A mutants consisted of two species, one without 333 palmitoylation and the other with a single palmitoylated cysteine. The ratio of 334 palmitoylated:unmodified S2 glycoprotein was significantly greater for the 2 nd 5C-to-A 335 mutant than for the ΔCT and 1 st 5C-to-A mutants. The 10 C-to-A mutant was not 336 detectably palmitoylated. Thus, palmitoylation can occur on multiple different cysteine 337 residues in the SARS-CoV-2 S2 endodomain; however, palmitoylation appears to occur more efficiently on the N-terminal endodomain cysteines, which contribute to virus 339 infectivity. "Restraining residues" participate in molecular bonds that maintain Class I viral 433 envelope glycoproteins in pretriggered conformations (72-73). Alteration of these restraining residues results in increased triggerability (Fig. 13) . Asp 614 appears to fit 435 the definition of a restraining residue. The D614G change increases virus infectivity and 436 responsiveness to ACE2, but also results in moderate increases in sensitivity to 437 neutralizing antisera. The proximity of Asp 614 (in the S1 C-terminal domain (CTD2)) to 438 the S2 fusion peptide-proximal region (FPPR), which abuts the S1 CTD1 and has been 439 proposed to suppress the opening of the S1 RBD (40), could account for these effects increased triggerability. The more triggerable FurinMut and D614G mutants solve this 455 potential problem in different ways. By retaining a covalent bond between S1 and S2, 456 FurinMut maintains and even increases spike stability. D614G retains an intact furin 457 cleavage site but strengthens the association of S1 with the S trimer, possibly by improving S1 CTD2-S2 interactions. The resulting higher spike density allows FurinMut 459 and D614G to take replicative advantage of increased ACE2 triggerability. with a mouse anti-S1 antibody. Band intensity was determined as described above.

The subunit association index of each mutant was calculated as follows: In the inhibition assay, to allow for optimal contact, target cells were scraped and 718 resuspended in medium, and inhibitors were added at the indicated concentrations.

Cells were then incubated at 37 o C in 5% CO 2 for 2 hr before they were added to 720 effector cells, as described above. VLPs containing the wild-type or D614G S gp prepared as described above were used 778 to measure the binding of soluble ACE2 (sACE2) to the viral spike, and to study the 779 effect of sACE2 binding on the shedding of the S1 gp from the spike. After clarification 780 and filtration, the VLP-containing cell supernatants were incubated with different 781 concentrations of sACE2 at either 0°C or 37°C for one hour. Afterwards, the VLPs were pelleted at 14,000 x g for 1 hr at 4°C, and analyzed by Western blotting as described The protein precipitates were dissolved in 60 µl of TEA buffer with 4 mM EDTA and 4% 805 SDS. Half of the dissolved protein solution was mixed with 1 M hydroxylamine in TEA buffer (pH 7.3) + 0.2% Triton X-100, while the remaining half was mixed with TEA buffer 807 + 0.2% Triton X-100 followed by a 350-rpm rotation for 1 hr at room temperature. the wild-type SARS-CoV-2 S gp. The processing index represents the product of the S1/uncleaved S and S2/uncleaved S ratios for each S variant, relative to that of the 1215 wild-type SARS-CoV-2 S gp. The association of the S1 and S2 subunits was assessed 1216 by measuring the ratio of S1 in lysates to S1 in the supernatants of 293T cells 1217 transfected with plasmids expressing the S gp variant alone; the lysate/supernatant S1 1218 ratio for each S gp variant was then normalized to that observed for the wild-type 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 +++ 1.00 ± 0.00 

Origin and evolution of pathogenic evidence for membrane cycling from Golgi to ER

Brefeldin A's effects on endosomes, lysosomes, and the TGN 1022 suggest a general mechanism for regulating organelle structure and membrane 1023 traffic

Alpha-complementation assay for HIV envelope glycoprotein-mediated fusion

Optimized Pseudotyping 1029

Cathepsin L functionally 1031 cleaves the severe acute respiratory syndrome coronavirus class I fusion protein 1032 upstream of rather than adjacent to the fusion peptide

Protease-mediated enhancement of severe acute respiratory syndrome 1035 coronavirus infection

Host cell proteases: critical determinants 1037 of coronavirus tropism and pathogenesis

Identification and characterization of the putative fusion peptide of the 1040 severe acute respiratory syndrome-associated coronavirus spike protein

Characterization of a highly conserved domain within the severe acute 1044 respiratory syndrome coronavirus spike protein S2 domain with characteristics of 1045 a viral fusion peptide

Identification of the fusion peptide-containing region in betacoronavirus 1048 spike glycoproteins

Mass-tag labeling reveals site-specific and endogenous levels of protein 1051 S-fatty acylation

Anthrax toxin requires ZDHHC5-1053 mediated palmitoylation of its surface-processing host enzymes

Structures and distributions of SARS-1057

CoV-2 spike proteins on intact virions

The beta20-beta21 of gp120 1068 is a regulatory switch for HIV-1 Env conformational transitions

Comparison of uncleaved and mature 1072 human immunodeficiency virus membrane envelope glycoprotein trimers

Proteolytic processing of the human 1075 immunodeficiency virus envelope glycoprotein precursor decreases 1076 conformational flexibility

envelope glycoproteins adopt downstream conformations that remain responsive 1080 to conformation-preferring ligands

Selective recognition of oligomeric HIV-1 1082 primary isolate envelope glycoproteins by potently neutralizing ligands requires 1083 efficient precursor cleavage

Distinct mutation in the feline coronavirus spike protein cleavage activation site in 1086 a cat with feline infectious peritonitis-associated meningoencephalomyelitis

Structure of the hemagglutinin precursor cleavage site, a 1090 determinant of influenza pathogenicity and the origin of the labile conformation

The S2 subunit of QX-type infectious bronchitis 1094 coronavirus spike protein is an essential determinant of neurotropism

Human 1098 influenza A H5N1 virus related to a highly pathogenic avian influenza virus

Proteolytic 1101 cleavage of the E2 glycoprotein of murine coronavirus: host-dependent 1102 differences in proteolytic cleavage and cell fusion

Role of host cellular proteases in the 1105 pathogenesis of influenza and influenza-induced multiple organ failure

Modifications to 1116 the hemagglutinin cleavage site control the virulence of a neurotropic H1N1 1117 influenza virus

Receptor binding and priming of the spike 1120 protein of SARS-CoV-2 for membrane fusion

The impact of mutations in SARS-CoV-2 spike on viral 1124 Infectivity and antigenicity

Neurovirulent murine 1126 coronavirus JHM.SD uses cellular zinc metalloproteases for virus entry and cell-1127 cell fusion

Persistence of viral RNA, 1130 widespread thrombosis and abnormal cellular syncytia are hallmarks of COVID-1131 19 lung pathology. medRxiv

Matrix metalloproteinases 1133 in lung: multiple, multifarious, and multifaceted

Golgi bypass: skirting around the heart of 1135 classical secretion

Viral membrane fusion

Improvement of 1138 the reverse tetracycline transactivator by single amino acid substitutions that 1139 reduce leaky target gene expression to undetectable levels

Gene delivery by lentivirus vectors

SARS-CoV-2 S gp is shown, with the boundaries of the mature S1, S2 and S2′ 1147 glycoproteins indicated. The S gp regions include the signal peptide (Sig), the N-1148 terminal domain (NTD), receptor-binding domain (RBD), C-terminal domains (CTD1 and 1149 CTD2), fusion peptide (FP), heptad repeat regions (HR1 and HR2)

endodomain/cytoplasmic tail (CT) and endoplasmic reticulum retention signal (ERsig)

For the S2 1153 cytoplasmic tail mutants, the C-termini of the wild-type and mutant S glycoproteins are 1154 depicted, with the positions of cysteine residues indicated by vertical tick marks. 1155 1156 FIG 2. Expression and processing of the SARS-CoV-2 S gp variants. 293T cells were 1157 cotransfected with a plasmid encoding HIV-1 Gag/protease and either pcDNA3.1 or 1158 plasmids expressing SARS-CoV-1 S gp or wild-type or mutant SARS-CoV-2 S 1159 glycoproteins

Cell-surface S glycoproteins were precipitated by convalescent 1162 serum NYP01 and then Western blotted for the S1 and S2 glycoproteins (Note that 1163 NYP01 does not recognize the SARS-CoV-1 S gp)

S2 and ACE2. HIV-1 p24 and p17 Gag proteins in the VLP lysates were

Coomasie blue staining. (B) HIV-1 VLPs pseudotyped with the wild-type or D614G 1266 glycoproteins were incubated with the indicated concentrations of sACE2 for 1 hr at

The VLPs were pelleted and lysed. VLP lysates were Western blotted for S1 and 1268

The S1/S ratio as a function of sACE2 concentration is shown in the graphs in the 1270 middle and right panels

VLPs pseudotyped with the wild-type or D614G S glycoproteins were incubated at 37°C 1273 for the indicated number of days, after which the VLPs were pelleted

As negative controls, supernatants from 293T cells expressing the S 1275 glycoproteins without HIV-1 Gag were processed in parallel (lanes 1 and 2, Gag-)

Lysates from 293T cells transiently expressing the His 6 -tagged wild-type or D614G 1277 glycoproteins were incubated at the indicated temperatures for 1 hr. The S 1278 glycoproteins were then precipitated by Ni-NTA resin and Western blotted. The blots 1279 shown are representative of those obtained in two independent experiments and the 1280 graphs show the means and standard deviations from two independent experiments

Statistical significance was evaluated using Student's t-test

Palmitoylation of the SARS-CoV-2 S2 endodomain/cytoplasmic tail. Lysates 1285 prepared from 293T cells expressing the wild-type SARS-CoV-2 S gp or the indicated mutants were subjected to acyl-PEG exchange (68). When NH 2 OH is left out

palmitoylated cysteine residues are not de-acylated and therefore not available for 1288 reaction with mPEG-maleimide. The cell lysates were Western blotted with an anti-S2

The mono-PEGylated (S2*) and di-PEGylated (S2**) species are indicated

Note that the ΔCT mutant retains two membrane-proximal cysteine residues. The 1291 results shown are representative of those obtained in two independent experiments. 1292 1293 FIG 11. Metalloprotease inhibitors block S-mediated cell-cell fusion and syncytium 1294 formation. (A) The effect of marimastat and ilomastat, two matrix metalloprotease 1295 inhibitors, on two parallel alpha-complementation assays was tested. In one assay, the 1296 effector cells express the wild-type SARS-CoV-2 S gp and 293T cells expressing ACE2 1297 were used as target cells

The β-galactosidase values were normalized to those seen in the 1301 absence of inhibitors. The means and standard deviations from at least two 1302 independent experiments are shown. (B) 293T-ACE2 cells were cotransfected with 1303 plasmids expressing eGFP and the wild-type SARS-CoV-2 S gp, after which ilomastat 1304 (10 µM) or marimastat (10 µM) was added. For the HIV-1 control, 293T cells were 1305 transfected with plasmids expressing eGFP, CD4, CCR5 and the HIV-1 JR-FL envelope 1306 glycoproteins. The cells were imaged 24 hours after transfection. (C) The time course 1307 is shown for 293T-ACE2 cells transfected with an eGFP-expressing plasmid and either 1308 pcDNA3.1 or a plasmid expressing the wild-type SARS-CoV-2 S gp, in the absence (S) 1309 or presence of ilomastat (10 µM) or marimastat (10 µM). (D) 293T cells were then Western blotted for the S2 gp. Treatment with 2-BP reduces S2 palmitoylation, but 1335 also results in decreased S gp processing. The results shown in panels A, C and E are 1336 representative of those obtained in two independent experiments

Statistical significance was evaluated using Student's t-test

Model for the effects of two evolutionary changes on SARS-CoV-2 S gp 1342 function. The conformational transitions of the SARS-CoV-2 S gp during functional 1343 activation by ACE2 binding and proteolytic cleavage are depicted. The proposed 1344 effects of changes in S gp reactivity/triggerability on viral phenotypes follow from studies 1345 of other Class I viral envelope glycoproteins

D614G S glycoproteins are positioned along the pathway according to their phenotypes

variants. Forty-eight hours later, cell supernatants were filtered (0.45-µm) and