SARS-CoV-2 entry requires sequential cleavage of the spike glycoprotein at the S1/S2 and the S2ʹ cleavage sites to mediate membrane fusion. SARS-CoV-2 has a polybasic insertion (PRRAR) at the S1/S2 cleavage site that can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture-adapted SARS-CoV-2 virus with an S1/S2 deletion, we show that the polybasic insertion endows SARS-CoV-2 with a selective advantage in lung cells and primary human airway epithelial cells, but impairs replication in Vero E6, a cell line used for passaging SARS-CoV-2. Using engineered spike variants and live virus competition assays and by measuring growth kinetics, we find that the selective advantage in lung and primary human airway epithelial cells depends on the expression of the cell surface protease TMPRSS2, which enables endosome-independent virus entry by a route that avoids antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin cleavage site was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals, unlike wild-type virus. Analysis of 100,000 SARS-CoV-2 sequences derived from patients and 24 human postmortem tissues showed low frequencies of naturally occurring mutants that harbour deletions at the polybasic site. Taken together, our findings reveal that the furin cleavage site is an important determinant of SARS-CoV-2 transmission.
Bibliographical noteFunding Information:
SARS-CoV-2 virus was initially provided by Public Health England and we thank M. Zambon, R. Gopal and M. Patel for their help. This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grant nos. BB/ R013071/1 (T.P.P., W.S.B.), BB/R007292/1 (L.B., W.S.B.), BB/S008292/1 (J.C.B., W.S.B.) and BB/M02542X/1 (A.D.D., D.A.M.), and Wellcome Trust grant nos. 205100 (D.H.G., R.Y.S.-D., W.S.B.) and no. 200187 (J.Z., R.F., W.S.B.). This work was also supported by MRC grant no. MR/R020566/1 (M.K.W., A.D.D.) and US FDA grant no. HHSF223201510104C (A.D.D., D.A.M.). Additional support was provided from a grant from the King’s College London King’s Together Programme and the King’s College London BHF Centre of Research Excellence grant no. RE/18/2/34213 to M.G. O.C.S. was supported by a Wellcome Trust studentship, R.K. was supported by Wellcome fellowship no. 216353/Z/19/Z, R.P. was supported by an MRC DTP studentship, J.A.H. was supported by a BBSRC DTP studentship and E.S. was supported by an Imperial College President’s Scholarship.
Human samples used in this research project were obtained from the Imperial College Healthcare Tissue Bank (ICHTB). ICHTB is supported by the National Institute for Health Research Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. ICHTB is approved by Wales REC3 to release human material for research (17/WA/0161), and the samples for this project (R20012) were issued from subcollection reference number MED_MO_20_011.
© 2021, The Author(s), under exclusive licence to Springer Nature Limited.
- viral transmission
- virus-host interaction