Abstract
Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.
| Original language | English |
|---|---|
| Pages (from-to) | 27637-27645 |
| Number of pages | 9 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 117 |
| Issue number | 44 |
| DOIs | |
| State | Published - Nov 3 2020 |
Keywords
- Antibody
- Chikungunya virus
- Cryo-EM
- Epitope
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In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, No. 44, 03.11.2020, p. 27637-27645.
Research output: Contribution to journal › Article › peer-review
TY - JOUR
T1 - Structural basis of Chikungunya virus inhibition by monoclonal antibodies
AU - Zhou, Qun Fei
AU - Fox, Julie M.
AU - Earnest, James T.
AU - Ng, Thiam Seng
AU - Kim, Arthur S.
AU - Fibriansah, Guntur
AU - Kostyuchenko, Victor A.
AU - Shi, Jian
AU - Shu, Bo
AU - Diamond, Michael S.
AU - Lok, Shee Mei
N1 - Funding Information: ACKNOWLEDGMENTS. This work was supported by the Duke-National University of Singapore Signature Research Programme funded by the Ministry of Health, Singapore, and awarded to S.-M.L.; and by NIH Grants R01 AI141436, R01 AI114816, R01 AI127513, U19 AI142790, and contract AI201800001 awarded to M.S.D. We thank the Defense Medical & Environmental Research Institute for the generous gifts of the Chikungunya virus Ross strain and Chikungunya virus East African strain. Funding Information: This work was supported by the Duke-National University of Singapore Signature Research Programme funded by the Ministry of Health, Singapore, and awarded to S.-M.L.; and by NIH Grants R01 AI141436, R01 AI114816, R01 AI127513, U19 AI142790, and contract AI201800001 awarded to M.S.D. We thank the Defense Medical & Environmental Research Institute for the generous gifts of the Chikungunya virus Ross strain and Chikungunya virus East African strain. Funding Information: 9. B. S. Schnierle, Cellular attachment and entry factors for chikungunya virus. Viruses 11, 1078 (2019). Materials and Methods A detailed description of all materials and methodology is included in SI Appendix, Materials and Methods. This includes the cells and virus, mAb CHK-263 and CHK-124 production and digestion, plaque reduction neutralization test, in vivo experiments, pre-and postattachment neutralization assays, dynamic light scattering assay, attachment RT-qPCR assay, DiD-labeled virus fusion assay, virus egress assay, Mxra8-blocking experiment, biolayer interferometry assay, statistical analysis, cryo-EM sample preparation, data collection, processing, and analysis. Sample Preparation for Cryo-EM. CHIKV virus samples were mixed with the CHK-263 Fab or CHK-124 Fab or CHK-263 IgG at a molar ratio of 1.5 Fab/IgG to every E2 protein, incubated at 37 °C for 30 min, and then kept at 4 °C before freezing on cryo-EM grids. About 2 μL of the sample was applied to a precooled cryo-EM grid with lacey carbon covered with a thin carbon film. The grid was then blotted with filter paper at 4 °C with 100% humidity for 1 to 2 s and flash-frozen in liquid ethane by using the Vitrobot Mark IV plunger (FEI). Data Collection and Image Processing. The images of the frozen CHIKV-Ross:CHK-124 Fab complex, CHIKV-East African:CHK-263 Fab complex, and CHIKV-East African:CHK-263 IgG complex were taken with the FEI Titan Krios electron microscope at 300 keV with a nominal magnification of 47,000× and a FEI Falcon II direct electron detector. Images were collected using Leginon (46) in movie mode, with a total exposure of 2 s and a total dose of ∼20 e−/Å2. The frames from each movie were aligned using MotionCorr (47) to produce full-dose images used for particle selection and orientation search, and the images from the first several frames amounting to the dose of about 20 e−/Å2 were used in 3D reconstruction. The images were taken at a defocus range of 0.5-to 2.5-μm. cisTEM (48) and Gctf (49) were used to estimate the astigmatic defocus parameters. Particle picking was undertaken using cisTEM. Orientation search and 3D reconstruction of the picked particles were performed using cisTEM and RELION (50, 51). The reconstruction process is detailed in SI Appendix, Materials and Methods and Table S1. Data Availability. The cryo-EM map of the CHIKV:CHK-124 Fab complex, CHIKV:CHK-263 Fab complex, CHIKV:CHK-263 IgG complex, subregion around the five-fold vertex of the CHIKV:CHK-263 Fab complex, and subregion around the two-fold vertex of the CHIKV:CHK-263 IgG complex has been deposited in the Electron Microscopy Data Bank under the accession codes EMD-30476, EMD-30477, EMD-30478, EMD-30479, and EMD-30480, respectively. Their atomic models have been deposited in the Protein Data Bank under the accession codes 7CVY, 7CVZ, 7CW0, 7CW2, and 7CW3, respectively. 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Tumkosit et al., Anti-chikungunya virus monoclonal antibody that inhibits viral fusion and release. J. Virol. 94, e00252-20 (2020). 44. J. Jin et al., Neutralizing antibodies inhibit chikungunya virus budding at the plasma membrane. Cell Host Microbe 24, 417–428.e5 (2018). 45. G. R. Klimpel, “Immune defenses” in Medical Microbiology, ed. 4, S Baron, Ed. (Uni-versity of Texas Medical Branch at Galveston, 1996). 46. B. Carragher et al., Leginon: An automated system for acquisition of images from vitreous ice specimens. J. Struct. Biol. 132, 33–45 (2000). 47. X. Li, S. Zheng, D. A. Agard, Y. Cheng, Asynchronous data acquisition and on-the-fly analysis of dose fractionated cryoEM images by UCSFImage. J. Struct. Biol. 192, 174–178 (2015). 48. T. Grant, A. Rohou, N. Grigorieff, cisTEM, user-friendly software for single-particle image processing. eLife 7, e35383 (2018). 49. K. Zhang, Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016). 50. J. 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PY - 2020/11/3
Y1 - 2020/11/3
N2 - Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.
AB - Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.
KW - Antibody
KW - Chikungunya virus
KW - Cryo-EM
KW - Epitope
UR - http://www.scopus.com/inward/record.url?scp=85095670112&partnerID=8YFLogxK
U2 - 10.1073/pnas.2008051117
DO - 10.1073/pnas.2008051117
M3 - Article
C2 - 33087569
AN - SCOPUS:85095670112
SN - 0027-8424
VL - 117
SP - 27637
EP - 27645
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 44
ER -