Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cell entry starts with membrane attachment and ends with spike (S) protein–catalyzed membrane fusion depending on two cleavage steps, namely, one usually by furin in producing cells and the second by TMPRSS2 on target cells. Endosomal cathepsins can carry out both. Using real-time three-dimensional single-virion tracking, we show that fusion and genome penetration require virion exposure to an acidic milieu of pH 6.2 to 6.8, even when furin and TMPRSS2 cleavages have occurred. We detect the sequential steps of S1-fragment dissociation, fusion, and content release from the cell surface in TMPRRS2-overexpressing cells only when exposed to acidic pH. We define a key role of an acidic environment for successful infection, found in endosomal compartments and at the surface of TMPRSS2-expressing cells in the acidic milieu of the nasal cavity.
| Original language | English |
|---|---|
| Article number | e2209514119 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 119 |
| Issue number | 38 |
| Early online date | 1 Sept 2022 |
| DOIs | |
| Publication status | Published - 20 Sept 2022 |
| Externally published | Yes |
Funding
ACKNOWLEDGMENTS. We thank Stephen C. Harrison for comments, suggestions, and extensive editorial assistance and members of our laboratories for help and encouragement; the staff at Helsinki University Hospital Diagnostic Center Virology and Immunology for providing human nasal swabs for virus isolation, Suvi Kuivanen and Teemu Smura for virus propagation, sequencing, and discussions, and Sanna M€aki for excellent technical work; E.S. (T.K. laboratory) for excellent laboratory management; Tegy John Vadakkan for maintaining the spinning-disc confocal microscope; Lena Tveriakhina (Blacklow laboratory, Harvard Medical School) for western blot analysis; and Bing Chen for providing soluble ACE2. We appreciate the following: NIH Maximizing Investigators’ Research Award (MIRA) GM130386 (T.K.); NIH Grant AI163019 (S.P.J.W. and T.K.); the Danish Technical University (T.K.); Sana Biotechnology (T.K.); Harvard Virology Program, NIH Training Grant T32 AI07245 postdoctoral fellowship (A.J.B.K.); Academy of Finland Research Grant 318434 (G.B. and R.O.); Academy of Finland Research Grant 336490 (O.V.); Helsinki University Hospital Funds TYH2021343 and Jane and Aatos Erkko Foundation (O.V.); and the University of Helsinki Graduate Program in Microbiology and Biotechnology (R.O.); NIH Grant AI030557 (V.K.). We thank Stephen C. Harrison for comments, suggestions, and extensive editorial assistance and members of our laboratories for help and encouragement; the staff at Helsinki University Hospital Diagnostic Center Virology and Immunology for providing human nasal swabs for virus isolation, Suvi Kuivanen and Teemu Smura for virus propagation, sequencing, and discussions, and Sanna M€aki for excellent technical work; E.S. (T.K. laboratory) for excellent laboratory management; Tegy John Vadakkan for maintaining the spinning-disc confocal microscope; Lena Tveriakhina (Blacklow laboratory, Harvard Medical School) for western blot analysis; and Bing Chen for providing soluble ACE2. We appreciate the following: NIH Maximizing Investigators’ Research Award (MIRA) GM130386 (T.K.); NIH Grant AI163019 (S.P.J.W. and T.K.); the Danish Technical University (T.K.); Sana Biotechnology (T.K.); Harvard Virology Program, NIH Training Grant T32 AI07245 postdoctoral fellowship (A.J.B.K.); Academy of Finland Research Grant 318434 (G.B. and R.O.); Academy of Finland Research Grant 336490 (O.V.); Helsinki University Hospital Funds TYH2021343 and Jane and Aatos Erkko Foundation (O.V.); and the University of Helsinki Graduate Program in Microbiology and Biotechnology (R.O.); NIH Grant AI030557 (V.K.).
Keywords
- 3D imaging
- infection route
- live-cell imaging
- SARS-CoV-2
- virus entry