key: cord-327808-k3jec87p
authors: Zhu, Yunkai; Feng, Fei; Hu, Gaowei; Wang, Yuyan; Yu, Yin; Zhu, Yuanfei; Xu, Wei; Cai, Xia; Sun, Zhiping; Han, Wendong; Ye, Rong; Chen, Hongjun; Ding, Qiang; Cai, Qiliang; Qu, Di; Xie, Youhua; Yuan, Zhenghong; Zhang, Rong
title: The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission
date: 2020-08-25
journal: bioRxiv
DOI: 10.1101/2020.08.25.266775
sha: 
doc_id: 327808
cord_uid: k3jec87p

The global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

SARS-CoV-2 and SARS-CoV share nearly 80% nucleotide sequence identity 43 and use the same cellular receptor, angiotensin-converting enzyme 2 (ACE2), to enter 44 target cells (Hoffmann et al., 2020b; Zhou et al., 2020) . However, the newly emerged CoV-2 spike in cells promotes cell-cell membrane fusion, which is reduced after deletion 52 of the RRAR sequence or when expressing SARS-CoV S protein lacking these 53 residues (Hoffmann et al., 2020a; Xia et al., 2020) . Pseudovirus or live virus bearing 54 SARS-CoV-2 spike deletion at the S1/S2 junction decreased the infection in Calu-3 cells 55 and attenuated infection in hamsters (Hoffmann et al., 2020a; Lau et al., 2020) . The 56 sequence at the S1/S2 boundary seems to be unstable, as deletion variants are 57 observed both in cell culture and in patient samples (Lau et al., 2020; Liu et al., 2020;  58 Ogando et al., 2020; Wong et al.) . SARS-CoV-2 entry is mediated by sequential 59 cleavage at the S1/S2 junction site and additional downstream S2' site of spike protein.

The sequence at the S1/S2 boundary contains a cleavage site for the furin protease, 61 which could preactivate the S protein for membrane fusion and potentially reduce the 62 dependence of SARS-CoV-2 on plasma membrane proteases, such as transmembrane 63 serine protease 2 (TMPRSS2), to enable efficient cell entry (Shang et al., 2020) . Here, 64 we evaluate how the deletion at the S1/S2 junction impacts virus entry and cell tropism, infection, as measured by N antigen-positive cells, was sensitive to inhibition by E-64d 104 but not camostat in Vero cells ( Figure 1F ). When TMPRSS2 was expressed, both 105 camostat and E-64d inhibited the infectivity of Sfull, indicating that expression of 106 TMPRSS2 could promote the membrane fusion entry pathway. Remarkably, E-64d and 107 camostat had no effect on Sfull virus in A549-ACE2 cells, suggesting that in this cell Sfull 108 may use other TMPRSS2 homologs or trypsin-like proteases to activate fusion at the 109 plasma membrane since TMPRSS2 expression is absent in A549 cells (Matsuyama et 110 al., 2020) . We observed a similar phenotype even when cells were treated with a high insertion of multiple basic residues at the S1/S2 cleavage site and thus resembles the 121 Sdel virus ( Figure 1A) . Indeed, E-64, but not camostat, efficiently inhibited SARS-CoV 122 pseudovirus infection in multiple cell types ( Figure S2D ). These results demonstrate that 123 the deletion at the S1/S2 junction site propels the virus to enter cells through the 124 endosomal fusion pathway, which is less efficient than the fusion pathway at the plasma 125 membrane in airway epithelial cells as indicated by the reduced infectivity in Calu-3 cells.

Both Sdel and SARS-CoV may share a similar entry pathway. 131 Richardson et al., 2018; Zhang et al., 2018; Zhang et al., 2016) . A lack of suitable 132 human physiologically relevant cell lines and the S protein-induced syncytia formation in 133 cells have made such a screen for SARS-CoV-2 very challenging. We found that Sdel 

The top candidates from the CRISPR screen were determined according to their 144 MAGeCK score ( Figure 2B ). The top hit was ACE2, the cellular receptor that confers 145 susceptibility to SARS-CoV-2, which confirmed the validity of the screen. Additionally, 146 the gene encoding cathepsin L (CTSL), a target of our earlier assay using E-64d that is and actin-related protein 2/3 (Arp2/3) complex, which have significant roles in 159 endosomal cargo sorting (Liu et al., 2016; McNally and Cullen, 2018) . We also identified 

To define the stage of viral infection that each of the 32 validated genes acted, 174 one representative sgRNA per gene was selected for study in A549-ACE2 cells. Due to 175 its known antiviral activity, ASCC3 was not targeted. We confirmed that editing of these 176 genes did not affect cell viability ( Figure S4B) . The gene-edited cells were infected with 177 pseudovirus bearing the Sdel virus S protein or, as a control, the glycoprotein of 

suggest that these genes mediate Sdel virus entry. Notably, pseudovirus bearing the 183 spike protein of SARS-CoV, which lacks the multiple basic residues at the S1/S2 184 junction as Sdel, exhibited a phenotype similar to Sdel pseudovirus and Sdel live virus 185 ( Figure 3C and 2C) . Editing of these genes, including those encoding CTSL, cholesterol 186 transporters NPC1/2, WDR81/91, and TFE3, markedly reduced infection, suggesting 187 that Sdel and SARS-CoV may utilize similar entry machinery ( Figure 3C ). Intriguingly, 188 these genes edited also significantly inhibited the infection by pseudovirus bearing the 189 spike protein of MERS-CoV in A549-ACE2-DPP4 cells ( Figure 3D ). Although the furin 190 cleavage site is present at the S1/S2 boundary of MERS-CoV{Millet, 2014 #374}, it 191 preferentially enters the A549 cell via endosomal pathway as indicated by its sensitivity 192 to E-64d inhibitor ( Figure S2E ). This is possibly due to the lack of proper protease to representative sgRNA per gene was tested ( Figure 3E ). The editing efficiency of some 198 these genes by sgRNAs was confirmed by western blotting ( Figure S3B ). As expected, 199 editing of CTSL did not reduce infection, as the Sfull virus enters A549-ACE2 cells via an 200 endosomal-independent pathway (demonstrated in Figure 1F ). In general, editing of 201 genes encoding complexes that regulate the retrieval and recycling of cargo significantly 202 reduced infection, albeit to a lesser extent than observed with the Sdel live virus. 

U18666A, a cationic sterol, binds to the NPC1 protein to inhibit cholesterol export 208 from the lysosome, resulting in impaired endosome trafficking, late endosome/lysosome 209 membrane fusion (Cenedella, 2009; Ko et al., 2001; Lu et al., 2015) . U1866A has been 210 shown to inhibit the S protein-driven entry of SARS-CoV, Middle East Respiratory 211 Syndrome coronavirus (MERS-CoV), and the human coronaviruses NL63 and 229E, 212 with the most efficient inhibition observed with SARS-CoV (Wrensch et al., 2014) . The 213 antiviral effect of U18666A on type I feline coronavirus (FCoV) has also been 214 characterized in vitro and in vivo (Doki et al., 2020; Takano et al., 2017) . We found that, and Cullen, 2018). We hypothesized that disruption of these complexes might affect the 231 binding or transit of virions. To this end, we performed binding and internalization assays 232 using Sfull virus in A549-ACE2 cells. The genes COMMD3, VPS29, and CCDC53, which 233 encode proteins that are comprise CCC, retromer, and WASH complexes, respectively, 234 were each edited; effects on expression were confirmed by western blotting (Figure   235 S3B). Notably, binding and internalization of Sfull virions to these cells was significantly 236 decreased compared to control sgRNA ( Figure 4A ).

The entry receptor ACE2 is critical for SARS-CoV-2 infection. To determine 238 whether cell surface expression of ACE2 is regulated by these complexes, gene-edited 

VPS29 and C16orf16 that were identified in our screen, also are shared functionally by 258 the retromer and CCC complexes (Norwood et al., 2011; Phillips-Krawczak et al., 2015) . 

The S1/S2 boundary of spike protein impacts infection and disease in hamsters

In cell culture, we demonstrated that the Sdel virus resulted in a switch from the 283 plasma membrane to endosomal fusion pathway for entry. In Calu-3 lung cells, which 284 model more physiologically relevant airway epithelial cells, this switch led to a less 285 efficient endosomal entry process. Since virus entry is the first step in establishing 286 infection, we hypothesized that deletion at the S1/S2 boundary might reduce virus 287 infectivity and transmissibility in vivo. Indeed, using the golden Syrian hamster model, a 288 previous study showed that a SARS-CoV-2 variant with a 30-nucleotide deletion at the 289 S1/S2 junction caused milder disease and less viral infection in the trachea and lungs 290 compared to a virus lacking the deletion(Lau et al., 2020).

We evaluated the tissue tropism of the Sfull and Sdel virus following intranasal 292 inoculation of golden Syrian hamsters. Nasal turbinates, trachea, lungs, heart, kidney, 293 spleen, duodenum, brain, serum, and feces were collected. Sfull virus replicated robustly 294 and reached peak titer at day 1 post infection, with a mean titer 31-, 126-, and 1259-fold 295 higher than Sdel in the turbinates, trachea, and lungs, respectively ( Figure 5A ). While

Sdel virus replication was delayed, no significant differences were observed by day 4 in 297 these three tissues (Figure 5B ). At days 2 and 4, five pieces of fresh feces were 298 collected from each hamster. Although no infectious virus was detected by focus-forming 299 assay (data not shown), viral RNA levels were higher in fecal samples for Sfull (20 and 300 40-fold) than Sdel at days 2 and 4, respectively ( Figure 5B ). Likely related to this, no 301 infectious virus was detected in the duodenum, and Sfull RNA was 6.3-fold higher than 302 Sdel at day 4 ( Figure S7A ). In serum, we detected no difference in viremia at day 1, but 303 Sfull RNA was 63-and 32-fold higher than Sdel at days 2 and 4, respectively ( Figure   304 S7B). In other extrapulmonary organs, infectious virus was not consistently detected 305 (data not shown). In general, brain tissue had the highest viral RNA copy number, and 306 all organs showed higher levels of Sfull RNA at day 2 or 4 compared to Sdel except for 307 the liver and kidneys (Figure S7C-G) . Weight loss was only observed in hamsters 308 inoculated with Sfull and decreased as much as ~18% at days 5 and 6 ( Figure S7H ).

The S1/S2 boundary of spike protein modulates the transmission

To determine the impact of deletion at the S1/S2 junction on transmissibility by 311 direct contact exposure, six hamsters were inoculated intranasally with Sfull or Sdel 312 virus. At 24 h post inoculation, each donor hamster was transferred to a new cage and 313 co-housed with one naïve hamster for 3 days. For donors (day 4 post-inoculation), tissue 314 samples were processed (Figure 5A and 5B, and Figure S7 ). For contact hamsters 315 (day 3 post-exposure), nasal turbinate, trachea, and lungs were collected for infectious 316 virus titration and histopathological examination. The peak titers in turbinate, trachea, 317 and lungs from Sfull-exposed hamsters reached 8, 6.6, and 7.4 logs, respectively (6.6 318 logs, 6.2 logs, and 6.1 logs on average, respectively) ( Figure 5C ). Unexpectedly, no 319 infectious virus was detected in these three tissues from Sdel-exposed hamsters ( Figure   320 5C). In lung sections from hamsters that were exposed to Sfull-infected animals, we 321 observed mononuclear cell infiltrate, protein-rich fluid exudate, hyaline membrane 322 formation, and haemorrhage ( Figure 5D ). In contrast, no or minimal histopathological 323 change was observed in the lung sections from hamsters that were exposed to Sdel-324 infected animals ( Figure 5D ). To examine viral spread in the lungs, we performed RNA 325 in situ hybridization (ISH). Viral RNA was clearly detected in bronchiolar epithelial cells in 326 hamsters exposed to Sfull-infected animals ( Figure 5E ) whereas it was rarely detected 327 in hamsters exposed to Sdel-infected animals. Similaly, abundant RNA was observed in 328 the nasal turbinate epithelium ( Figure 5F ). These results indicated that transmission of 329 Sfull from infected hamsters to co-housed naïve hamsters was efficient whereas the 330 deletion at the S1/S2 boundary in the S protein of Sdel markedly reduced transmission. 

The notion that this deletion at the S1/S2 boundary discriminates the entry 357 pathway used by the virus was supported by the large number of endosomal entry host 358 factors uncovered in our genome-wide CRISPR screen. Genes for the endosomal entry-359 specific enzyme CTSL and for regulating endolysomal trafficking and membrane fusion, 360 such as NPC1/2 and WDR81/91, were required for Sdel, but not for Sfull virus infection.

In parallel, we discovered a panel of entry factors common to both Sdel and Sfull that 

After 48 or 72 h, the luciferase activity was determined using Nano-Glo® Luciferase 466 Assay kit (Promega #N1110) according to the manufacturer's instructions. The same 467 volume of assay reagent was added to each well and shake for 2 min, After incubation at 468 room temperature for 10 min, luminescence was recorded by using a FlexStation 3 469 (Molecular Devices) with an integration time of 1 second per well. CellTiter-Glo ® reagent was added to each well and allowed to shake for 2 min. After 

Sequence alignment of spike protein encompassing the cleavage site between S1 and 637 S2 subunits. The spike proteins of SARS-CoV-2 without (Sfull strain) and with (Sdel with Dunnett's test; **P < 0.01; ***, P < 0.001; ****P < 0.0001; ns, not significant. 

and tissues from intestine, brain, heart, liver, spleen, and kidney (day 2, 4) were 818 harvested (n=6 per day). Viral RNAs were extracted for RT-qPCR analysis. The viral 819 load in the brain was also titrated by focus-forming assay. The dashed lines represent Mechanisms of Cholesterol Binding to the NPC1 and NPC2 Proteins. Advances in 866 experimental medicine and biology 1135, 139-160. 867 Doench, J.G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E.W., Donovan, K.F., 868 Smith, I., Tothova, Z., Wilen, C., Orchard, R., et al. (2016) . Optimized sgRNA design to 869 maximize activity and minimize off-target effects of CRISPR-Cas9. Nature C.A., Singla, A., Starokadomskyy, P., Deng, Z., Osborne, D.G., Li, 960 H., Dick, C.J., Gomez, T.S., Koenecke, M., Zhang, J.S., et al. (2015) . COMMD1 is 961 linked to the WASH complex and regulates endosomal trafficking of the copper 962 transporter ATP7A. Molecular biology of the cell 26, 91-103. 963 Qi, J., Zhou, Y., Hua, J., Zhang, L., Bian, J., Liu, B., Zhao, Z., and Jin, S. (2020) . The 964

scRNA-seq expression profiling of the receptor ACE2 and the cellular protease 965 TMPRSS2 reveals human organs susceptible to COVID-19 infection. bioRxiv, 966 2020 .2004 .2016 Rapiteanu 

A Unique 1005

Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia 1006

-nCoV) Potentially Related to Viral Transmissibility

TFEB regulates lysosomal positioning by modulating TMEM55B expression and 1010 JIP4 recruitment to lysosomes

Natural transmission of bat-like SARS-CoV-2DPRRA variants 1013 in COVID-19 patients

IFITM proteins inhibit entry driven 1015 by the MERS-coronavirus spike protein: evidence for cholesterol-independent 1016 mechanisms

The role of furin cleavage site in SARS-CoV-2 spike protein-mediated 1019 membrane fusion in the presence or absence of trypsin. Signal transduction and targeted 1020 therapy 5

TMPRSS2 and 1023 TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

Mxra8 is a receptor for multiple 1027 arthritogenic alphaviruses

A CRISPR screen defines a signal 1030 peptide processing pathway required by flaviviruses

A pneumonia outbreak associated with a new coronavirus 1033 of probable bat origin

Protease inhibitors 1036 targeting coronavirus and filovirus entry

biotechnology 34, 184-191. 871 Doki, T., Tarusawa, T., Hohdatsu, T., and Takano, T. (2020) . In Vivo Antiviral Effects of 872 U18666A Against Type I Feline Infectious Peritonitis Virus. Pathogens 9. 873Fedoseienko, A., Wijers, M., Wolters, J.C., Dekker, D., Smit, M., Huijkman, N., 874 Kloosterhuis, N., Klug, H., Schepers, A., Willems van Dijk, K., et al. (2018)