SARS-CoV-2 Omicron spike H655Y mutation is responsible for enhancement of the endosomal entry pathway and reduction of cell surface entry pathways

Abstract

The SARS-CoV-2 Omicron variant reportedly displays decreased usage of the cell surface entry pathway mediated by the host transmembrane protease, serine 2 (TMPRSS2) and increased usage of the endosomal entry pathway mediated by cathepsin B/L. These differences result in different cell tropisms and low fusogenicity from other SARS-CoV-2 variants. Recent studies have revealed that host metalloproteases are also involved in cell surface entry and fusogenic activity of SARS-CoV-2, independent of TMPRSS2. However, the involvement of metalloproteinase-mediated cell entry and fusogenicity in Omicron infections has not been investigated. Here, we report that Omicron infection is less sensitive to the metalloproteinase inhibitor marimastat, like the TMPRSS2 inhibitor nafamostat, and is more sensitive to the cathepsin B/L inhibitor E-64d than infections with wild-type SARS-CoV-2 and other variants. The findings indicate that Omicron preferentially utilizes the endosomal pathway rather than cell surface pathways for entry. Moreover, the Omicron variant also displays poor syncytia formation mediated by metalloproteinases, even when the S cleavage status mediated by fusion-like proteases is unchanged. Intriguingly, the pseudovirus assay showed that a single mutation, H655Y, of the Omicron spike (S) is responsible for the preferential entry pathway usage without affecting the S cleavage status. These findings suggest that the Omicron variant has altered entry properties and fusogenicity, probably through the H655Y mutation in its S protein, leading to modulations of tissue and cell tropism, and reduced pathogenicity.

Author summary

Recent studies have suggested that the SARS-CoV-2 Omicron variant displays altered cell tropism and fusogenicity, in addition to immune escape. However, comprehensive analyses of the usage of viral entry pathways in Omicron variant have not been performed. Here, we used protease inhibitors to block each viral entry pathway mediated by the three host proteases (cathepsin B/L, TMPRSS2, and metalloproteinases) in various cell types. The results clearly indicated that Omicron exhibits enhanced cathepsin B/L-dependent endosome entry and reduced metalloproteinase-dependent and TMPRSS2-dependent cell surface entry. Furthermore, the H655Y mutation of Omicron S determines the relative usage of the three entry pathways without affecting S cleavage by the host furin-like proteases. Comparative data among SARS-CoV-2 variants, including Omicron, may clarify the biological and pathological phenotypes of Omicron but increase the understanding of disease progression in infections with other SARS-CoV-2 variants.

SciScore for 10.1101/2022.03.21.485084: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The primary antibodies used were mouse anti-SARS-CoV-2 S2 domain (1:1000, GTX632604, GeneTex, Irvine, CA, USA), rabbit anti-Flag-tag (1:1000, PM020, MBL, Woburn, MA, USA), mouse anti-tubulin (1:1000, CP06, Millipore, Billerica, MA, USA), and mouse anti-VSVM (1:1000, 23H12, absolute antibody, Oxford, UK).	anti-SARS-CoV-2 S2 domain suggested: None anti-Flag-tag suggested: None anti-tubulin suggested: None CP06 suggested: (Millipore Cat# CP06, RRID:AB_2617116) anti-VSVM suggested: None
The …

SciScore for 10.1101/2022.03.21.485084: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The primary antibodies used were mouse anti-SARS-CoV-2 S2 domain (1:1000, GTX632604, GeneTex, Irvine, CA, USA), rabbit anti-Flag-tag (1:1000, PM020, MBL, Woburn, MA, USA), mouse anti-tubulin (1:1000, CP06, Millipore, Billerica, MA, USA), and mouse anti-VSVM (1:1000, 23H12, absolute antibody, Oxford, UK).	anti-SARS-CoV-2 S2 domain suggested: None anti-Flag-tag suggested: None anti-tubulin suggested: None CP06 suggested: (Millipore Cat# CP06, RRID:AB_2617116) anti-VSVM suggested: None
The secondary antibodies used were horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG (NA934; GE Healthcare) and HRP-linked donkey anti-mouse IgG (NA931V; GE Healthcare).	HRP)-linked donkey anti-rabbit IgG suggested: None HRP-linked donkey anti-mouse IgG suggested: None
After 24 h infection, fixed cells were incubated with anti-SARS-CoV-2 nucleocapsid (1:1000, GTX135357) primary antibody for 16 h at 4 °C and detected with anti-rabbit-Alexa488 (1:200, A11008, Invitrogen, Carlsbad, CA, USA) secondary antibodies for 40 min at room temperature.	anti-SARS-CoV-2 nucleocapsid (1:1000 suggested: None anti-rabbit-Alexa488 suggested: None A11008 suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)
Experimental Models: Cell Lines
Sentences	Resources
Cells and viruses: VeroE6 (CRL-1586), 293T (CRL-3216), A704 (HTB-45) and Calu-3 (HTB-55) cells were obtained from American Type Culture Collection (Rockville, MD, USA).	293T suggested: None Calu-3 suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
Caco-2 cells (RCB0988) were obtained from RIKEN BioResource Research Center (Tsukuba, Japan).	Caco-2 suggested: None
Immunofluorescence staining: Immunofluorescence analysis of HEC50B cells was performed as described previously [21].	HEC50B suggested: JCRB Cat# NIHS0391, RRID:CVCL_2929)
Experimental Models: Organisms/Strains
Sentences	Resources
OVISE (JCRB1043), HEC50B (JCRB1145), and VeroE6-TMPRSS2 (JCRB 1819) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).	VeroE6-TMPRSS2 (JCRB 1819 suggested: None
VeroE6-TMPRSS2 (JCRB 1819) cells were cultured in DMEM containing 10% FBS and 1 mg/mL G418.	VeroE6-TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
Software and Algorithms
Sentences	Resources
Caco-2 cells (RCB0988) were obtained from RIKEN BioResource Research Center (Tsukuba, Japan).	RIKEN BioResource suggested: (RIKEN BioResource Center, RRID:SCR_003250)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

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Results from scite Reference Check: We found no unreliable references.

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SARS-CoV-2 Omicron spike H655Y mutation is responsible for enhancement of the endosomal entry pathway and reduction of cell surface entry pathways

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