SARS CoV-2 SPIKE GLYCOPROTEIN MUTATIONS AND CHANGES IN PROTEIN STRUCTURE

Abstract

Severe Acute Respiratory Syndrome Corona Virus-2 (SARS CoV-2) is a single-stranded positive polarity RNA virus with a high virulence effect. Spike (S) glycoprotein is the outermost component of the SARS CoV-2 virion and is important in the entry of the virus into the cell via the angiotensin converting enzyme 2 (ACE2) receptor. ACE2 plays an important role in the regulation of human blood pressure by converting the vasoconstrictor angiotensin 2 to the vasodilator angiotensin 1-7. In this study, the changes that mutations in Asian isolates may cause in S glycoprotein structure were analyzed and modeled to contribute to drug and vaccine targeting studies. Genome, proteome and mutation analyses were done using bioinformatics tools (MAFFT, MegaX, PSIPRED, MolProbity, PyMoL). Protein modelling was performed using ProMod3. We detected 26 mutations in the S glycoprotein. The changes that these mutations reveal in the general topological and conformational structure of the S glycoprotein may affect the virulence features of SARS CoV-2. It was determined that mutations converted the receptor binding domain (RBD) from down-formation to like-up formation. It is thought that conformational change occurring after mutation in RBD may cause an increase in receptor affinity. These findings could be beneficial for disease prevention of and drug/vaccine development for SARS CoV-2.

Keywords

• SARS CoV-2
• COVID19
• spike protein
• mutational changes
• protein structure

Öz

Şiddetli akut solunum yolu sendromu koronavirüsü-2 (SARS CoV-2) yüksek virülans etkiye sahip tek zincirli pozitif polariteli RNA virüsüdür. Spike (S) glikoprotein SARS CoV-2 virionunun en dıştaki bileşenidir ve anjiyotensin dönüştürücü enzim 2 (ACE2) reseptörü aracılığı ile virüsün hücreye girişinde önemlidir. ACE2, vazokonstriktör anjiyotensin 2'yi vazodilatör anjiyotensin 1-7'ye dönüştürerek insanda kan basıncının düzenlenmesinde önemli roller üstlenir. Bu çalışmada, Asya izolatlarındaki mutasyonların S glikoprotein yapısında neden olabileceği değişiklikler analiz edilmiş ve ilaç ve aşı hedefleme çalışmalarına katkıda bulunmak üzere modellenmiştir. Genom, proteom ve mutasyon analizleri biyoinformatik araçları (MAFFT, MegaX, PSIPRED, MolProbity, PyMoL) kullanılarak yapıldı. Protein modellemesi ProMod3 kullanılarak yapıldı. S glikoproteinde 26 mutasyon tespit edilmiştir. Bu mutasyonların S glikoproteininin genel topolojik ve konformasyonel yapısında ortaya çıkardığı değişiklikler, SARS CoV-2’nin virülans özelliklerini etkileyebilir. Mutasyonların reseptör bağlanma bölgesini (RBB) kapalı formasyondan açık formasyon benzeri bir yapıya dönüştürdüğü belirlenmiştir. RBB'de mutasyondan sonra meydana gelen konformasyonel değişimin reseptör afinitesinde bir artışa neden olabileceği düşünülmektedir. Bu bulgular hastalığın önlenmesi ve SARS CoV-2 ilaç ve aşı geliştirme çalışmaları için faydalı olabilir.

References

1. 1. Agor, J.K. & Özaltın, O.Y. 2018. Models for predicting the evolution of influenza to inform vaccine strain selection. Human Vaccines Immunotherapeutics, 14(3): 678-683.
2. 2. Baltimore, D. 1971. Expression of animal virus genomes. Bacteriological Reviews, 35(3): 235-241.
3. 3. Benkert, P., Biasini, M. & Schwede, T. 2011. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics, 27(3): 343-350.
4. 4. Bertoni, M., Kiefer, F., Biasini, M., Bordoli, L. & Schwede, T. 2017. Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology. Scientific Reports, 7(1): 1-15.
5. 5. Bogoch, I.I., Watts, A., Thomas-Bachli, A., Huber, C., Kraemer, M.U.G. & Khan, K. 2020. Pneumonia of unknown aetiology in Wuhan, China: Potential for international spread via commercial air travel. Journal of Travel Medicine, 27(2): 1-3.
6. 6. Bosso, M., Thanaraj, T.A., Abu-Farha, M., Alanbaei, M., Abubaker, J.& Al-Mulla, F. 2020. The two faces of ACE2: The role of ACE2 receptor and its polymorphisms in hypertension and COVID-19. Molecular Therapy Methods&Clinical Development, 18: 321-327.
7. 7. Bourgonje, A.R., Abdulle, A.E., Timens, W., Hillebrands, J.L., Navis, G.J., Gordijn, S.J., Bolling, M.C., Dijkstra, G., Voors, A.A., Osterhaus, A.D., van der Voort, P.H., Mulder, D.J. & van Goor, H. 2020. Angiotensin‐converting enzyme‐2 (ACE2), SARS‐CoV‐2 and pathophysiology of coronavirus disease 2019 (COVID‐19). The Journal of Pathology, 251(3): 228-248.
8. 8. Buchan, D.W.A., Minneci, F., Nugent, T.C.O., Bryson, K. & Jones, D.T. 2013. Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Research, 41(Web Server issue): W349-W357.

9. 9. Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K. & Madden, T.L. 2009. BLAST+: Architecture and applications. BMC Bioinformatics, 10(1): 421.
10. 10. Carroll, H., Beckstead, W., O’Connor, T., Ebbert, M., Clement, M., Snell, Q. & Mcclellan, D. 2007. DNA reference alignment benchmarks based on tertiary structure of encoded proteins. Bioinformatics, 23(19): 2648-2649.
11. 11. Cavallo, L. & Oliva, R. 2020. D936Y and other mutations in the fusion core of the SARS-Cov-2 spike protein heptad repeat 1 undermine the post-fusion assembly. bioRxiv, https://doi.org/10.1101/2020.06.08.140152
12. 12. CDC-Centers for Disease Control and Prevention, National Center for Immunization and Respiratory Diseases (NCIRD). 2019. How the flu virus can change: “drift” and “shift” https://www.cdc.gov/flu/about/viruses/change.htm (Data accessed: 01.05.2020).
13. 13. Chan, W.E., Chuang, C.K., Yeh, S.H., Chang, M.S. & Chen, S.S.L. 2006. Functional characterization of heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome Coronavirus. Journal of Virology, 80(7): 3225-3237.
14. 14. Chen, N., Zhou, M., Dong, X., Qu, J., Gong, F., Han, Y., Qiu, Y., Wang, J., Liu, Y., Wei, Y., Xia, J., Yu, T., Zhang, X. & Zhang, L. 2020. Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet, 395(10223): 507-513.
15. 15. Chen, V.B., Arendall, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S. & Richardson, D.C. 2010. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallographica Section D: Biological Crystallography, 66(1): 12-21.
16. 16. Conenello, G.M., Zamarin, D., Perrone, L.A., Tumpey, T. & Palese, P. 2007. A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS Pathogens, 3(10): 1414-1421.
17. 17. Daly, J.L., Simonetti, B., Klein, K., Chen, K., Williamson, M.K., Antón-Plágaro, C., Shoemark, D.K., Gracia, L.S., Bauer, M., Hollandi, R., Greber, U.F., Horvath, P., Sessions, R.B., Helenius, A., Hiscox, J.A., Teesalu, T., Matthews, D.A., Davidson, A.D., Collins, B.M., Cullen, P.J. & Yamauchi, Y. 2020. Neuropilin-1 is a host factor for SARS-CoV-2 infection. Science, eabd3072: 1-8.
18. 18. Davidson, A.M., Wysocki, J. & Batlle, D. 2020. Interaction of SARS-CoV-2 and other coronavirus with ACE (Angiotensin-Converting Enzyme)-2 as their main receptor- therapeutic implications. Hypertension, 76(5): 1339-1349.
19. 19. Davis, I.W., Leaver-Fay, A., Chen, V.B., Block, J.N., Kapral, G.J., Wang, X., Murray, L.W., Arendall, W.B., Snoeyink, J., Richardson, J.S. & Richardson, D.C. 2007. MolProbity: All-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Research, 35(2): 375-383.
20. 20. Ding, X., Zhang, X., Chong, H., Zhu, Y., Wei, H., Wu, X., He, J., Wang, X. & He, Y. 2017. Enfuvirtide (T20)-based lipopeptide is a potent HIV-1 cell fusion Inhibitor: implications for viral entry and inhibition. Journal of Virology, 91(18): 1-20.
21. 21. Drake, J.W. 1993. Rates of spontaneous mutation among RNA viruses. Proceedings of the National Academy of Sciences of the United States of America, 90(9): 4171-4175.
22. 22. Duffus, W.A., Levy-Mintz, P., Klimjack, M.R. & Kielian, M. 1995. Mutations in the putative fusion peptide of Semliki Forest virus affect spike protein oligomerization and virus assembly. Journal of Virology, 69(4): 2471-2479.
23. 23. Gallagher, T.M. & Buchmeier, M.J. 2001. Coronavirus spike proteins in viral entry and pathogenesis. Virology, 279(2): 371-374.
24. 24. Garcia, P.D., Ou, J.H., Rutter, W.J. & Walter, P. 1988. Targeting of the hepatitis B virus precore protein to the endorplasmic reticulum membrane: After signal peptide cleavage translocation can be aborted and the product released into the cytoplasm. Journal of Cell Biology, 106(4): 1093-1104.
25. 25. Guex, N., Peitsch, M.C. & Schwede, T. 2009. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis, 30(SUPPL. 1): S162-S173.
26. 26. Gui, M., Song, W., Zhou, H., Xu, J., Chen, S., Xiang, Y. & Wang, X. 2017. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell Research, 27(1): 119-129.
27. 27. Gustine, J.N. & Jones, D. 2020. Immunopathology of yyperinflammation in COVID-19. The American Journal of Pathology, https://doi.org/10.1016/j.ajpath.2020.08.009
28. 28. He, J., Tao, H., Yan, Y., Huang, S.Y. & Xiao, Y. 2020. Molecular mechanism of evolution and human infection with SARS-CoV-2. Viruses, 12(4): 428.
29. 29. Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., Schiergens, T.S., Herrler, G., Wu, N.H., Nitsche, A., Müller, M.A., Drosten, C. & Pöhlmann, S. 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell, 181: 271-280.
30. 30. Hsu, H.Y., Chang, M.H., Ni, Y.H. & Chen, H.L. 2004. Survey of hepatitis B surface variant infection in children 15 years after a nationwide vaccination programme in Taiwan. Gut, 53(10): 1499-1503.
31. 31. Huang, C., Wang, Y., Li, X., Ren, L., Zhao, J., Hu, Y., Zhang, L., Fan, G., Xu, J., Gu, X., Cheng, Z., Yu, T., Xia, J., Wei, Y., Wu, W., Xie, X., Yin, W., Li, H., Liu, M., Xiao, Y., Gao, H., Guo, L., Xie, J., Wang, G., Jiang, R., Gao, Z., Jin, Q., Wang, J. & Cao, B. 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The Lancet, 395(10223): 497-506.
32. 32. Ignatova, Z., Hörnle, C., Kasche, V. & Nurk, A. 2002. Unusual signal peptide directs penicillin amidase from Escherichia coli to the tat translocation machinery. Biochemical and Biophysical Research Communications, 291(1): 146-149.
33. 33. Jia, Y., Shen, G., Zhang, Y., Huang, K.-S., Ho, H.-Y., Hor, W.-S., Yang, C.H., Li, C. & Wang, W.L. 2020. Analysis of the mutation dynamics of SARS-CoV-2 reveals the spread history and emergence of RBD mutant with lower ACE2 binding affinity. BioRxiv, 2020.04.09.034942. https://doi.org/10.1101/2020.04.09.034942
34. 34. Kan, B., Wang, M., Jing, H., Xu, H., Jiang, X., Yan, M., Liang, W., Zheng, H., Wan, K., Liu, Q., Cui, B., Xu, Y., Zhang, E., Wang, H., Ye, J., Li, G., Li, M., Cui, Z., Qi, X., Chen, K., Du, L., Gao, K., Zhao, Y., Zou, X., Feng, Y., Gao, Y., Hai, R., Yu, D., Guan, Y. & Xu, J. 2005. Molecular Evolution Analysis and Geographic Investigation of Severe Acute Respiratory Syndrome Coronavirus-Like Virus in Palm Civets at an Animal Market and on Farms. Journal of Virology, 79(18): 11892-11900.
35. 35. Kadam, R.U. & Wilson, I.A. 2017. Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol. PNAS, 114(2): 206-214.
36. 36. Kamili, S. 2010. Infectivity and vaccination efficacy studies in animal models of HBV S and pol gene mutants. Antiviral Therapy, 15(3): 477-485.
37. 37. Katoh, K. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30(14): 3059-3066.
38. 38. Katoh, Kazutaka, Rozewicki, J. & Yamada, K.D. 2018. MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics, 20(4): 1160-1166.
39. 39. Khan, A., Benthin, C., Zeno, B., Albertson, T.E., Boyd, J., Christie, J.D., Hall, R., Poirier, G., Ronco, J.J., Tidswell, M., Hardes, K., Powley, W.M., Wright, T.J., Siederer, S.K., Fairman, D.A., Lipson, D.A., Bayliffe, A.I. & Lazaar, A.L. 2017. A pilot clinical trial of recombi-nant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome. Critical Care, 21(234): 1-9.
40. 40. Kim, Y., Cheon, S., Min, C.K., Sohn, K.M., Kang, Y.J., Cha, Y.J., Kang, J. Il, Han, S.K., Ha, N.Y., Kim, G., Aigerim, A., Shin, H.M., Choi, M.S., Kim, S., Cho, H.S., Kim, Y.S. & Choa, N.H. 2016. Spread of mutant middle east respiratory syndrome coronavirus with reduced affinity to human CD26 during the south Korean outbreak. MBio, 7(2): e00019-16.
41. 41. Kim, Y.S., Aigerim, A., Park, U., Kim, Y., Rhee, J.Y., Choi, J.P., Park, W.B., Park, S.W., Kim, Y., Lim, D.G., Inn, K.S., Hwang, E.S., Choi, M.S., Shin, H.S. & Cho, N.H. 2019. Sequential emergence and wide spread of neutralization escape middle east respiratory syndrome coronavirus mutants, South Korea, 2015. Emerging Infectious Diseases, 25(6): 1161-1168.
42. 42. Kruse, R.L. 2020. Therapeutic strategies in an outbreak scenario to treat the novel coronavirus originating in Wuhan, China. F1000Research, 9(72): 1-15.
43. 43. Kuljić-Kapulica, N. & Budisin, A. 1992. Coronaviruses. In Srpski arhiv za celokupno lekarstvo, 120(7-8): 215-218.
44. 44. Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6): 1547-1549.
45. 45. Li, D., Wu, J., Chen, J., Zhang, D., Zhang, Y., Qiao, X., Yu, X., Zheng, Q. & Hou, J. 2020. Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris. Protein Expression and Purification, 167(105527): 1-7.
46. 46. Li, Q., Cao, Z. & Rahman, P. 2020. Genetic variability of human angiotensin‐converting enzyme 2 (hACE2) among various ethnic populations. Molecular Genetics & Genomic Medicine, 8(e1344): 1-6.
47. 47. Li, F. 2016. Structure, function and evolution of coronavirus spike proteins. Annual Review of Virology, 3(1): 237-261.
48. 48. Li, F., Li, W., Farzan, M. & Harrison, S.C. 2005. Structural biology: Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science, 309(5742): 1864-1868.
49. 49. Li, W., Wong, S.-K., Li, F., Kuhn, J.H., Huang, I.C., Choe, H. & Farzan, M. 2006. Animal Origins of the Severe Acute Respiratory Syndrome Coronavirus: Insight from ACE2-S-protein interactions. Journal of Virology, 80(9): 4211-4219.
50. 50. Li, W., Zhang, C., Sui, J., Kuhn, J.H., Moore, M.J., Luo, S., Wong, S.K., Huang, I.C., Xu, K., Vasilieva, N., Murakami, A., He, Y., Marasco, W.A., Guan, Y., Choe, H. & Farzan, M. 2005. Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO Journal, 24(8): 1634-1643.
51. 51. Lovell, S.C., Davis, I.W., Arendall, W.B., De Bakker, P.I.W., Word, J.M., Prisant, M.G., Richardson, J.S. & Richardson, D.C. 2003. Structure validation by Cα geometry: φ,ψ and Cβ deviation. Proteins: Structure, Function and Genetics, 50(3): 437-450.
52. 52. Lu, Y., Neo, T.L., Liu, D.X. & Tam, J.P. 2008. Importance of SARS-CoV spike protein Trp-rich region in viral infectivity. Biochemical and Biophysical Research Communications, 371(3): 356-360.
53. 53. Lumangtad, L.A. & Bell, T.W. 2020. The signal peptide as a new target for drug design. Bioorganic and Medicinal Chemistry Letters, 30(10): 127115.
54. 54. Mahajan, M., Chatterjee, D., Bhuvaneswari, K., Pillay, S. & Bhattacharjya, S. 2018. NMR structure and localization of a large fragment of the SARS-CoV fusion protein: Implications in viral cell fusion. Biochimica et Biophysica Acta - Biomembranes, 1860(2): 407-415.
55. 55. Mahmudpour, M., Roozbeh, J., Keshavarz, M., Farrokhi, S. & Nabipour, I. 2020. COVID-19 cytokine storm: The anger of inflammation. Cytokine, 133(155151): 1-10.
56. 56. Marquez, A., Wysocki, J., Pandit, J. & Batlle, D. 2020. An update on ACE2 amplification and its therapeutic potential. Acta Physiologica, e13513: 1-14.
57. 57. Mason, J.M. & Arndt, K.M. 2004. Coiled coil domains: Stability, specificity, and biological implications. ChemBioChem, 5(2): 170-176.
58. 58. Mount, D.W. 2008. Using BLOSUM in sequence alignments. Cold Spring Harbor Protocols, 3(6): 1. https://doi.org/10.1101/pdb.top39
59. 59. NCBI. 2019. NCBI Virus. Www.Ncbi.Nlm.Nih.Gov/Labs/Virus. https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/
60. 60. Ou, J., Zhou, Z., Dai, R., Zhang, J., Lan, W., Zhao, S., Wu, J., Seto, D., Cui, L., Zhang, G. & Zhang, Q. 2020. Emergence of RBD mutations from circulating SARS-CoV-2 strains with enhanced structural stability and higher human ACE2 receptor affinity of the spike protein. BioRxiv, 2020.03.15.991844. https://doi.org/10.1101/2020.03.15.991844
61. 61. Ou, X., Liu, Y., Lei, X., Li, P., Mi, D., Ren, L., Guo, L., Guo, R., Chen, T., Hu, J., Xiang, Z., Mu, Z., Chen, X., Chen, J., Hu, K., Jin, Q., Wang, J. & Qian, Z. 2020. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature Communications, 11(1620): 1-12.
62. 62. Ou, X., Zheng, W., Shan, Y., Mu, Z., Dominguez, S.R., Holmes, K.V. & Qian, Z. 2016. Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins. Journal of Virology, 90(12): 5586-5600.
63. 63. Peisajovich, S.G. & Shai, Y. 2003. Viral fusion proteins: Multiple regions contribute to membrane fusion. Biochimica et Biophysica Acta - Biomembranes, 1614(1): 122-129.
64. 64. Perin, P.M., Haid, S., Brown, R.J.P., Doerrbecker, J., Schulze, K., Zeilinger, C., Schaewen, M., Heller, B., Vercauteren, K., Luxenburger, E., Baktash, Y.M., Vondran, F.W.R., Speerstra, S., Awadh, A., Mukhtarov, F., Schang, L.M., Kirschning, A., Müller, R., Guzman, C.A., Kaderali, L., Randall, G., Meuleman, P., Ploss, A. & Pietschmann, T. 2016. Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1. Hepatology, 63(1): 49-62.
65. 65. Perlman, S. 2020. Another decade, another coronavirus. In New England Journal of Medicine, 382(8): 760-762.
66. 66. Phan, T. 2020. Novel coronavirus: From discovery to clinical diagnostics. Infection, Genetics and Evolution, 79(104211): 1-2.
67. 67. Remmert, M., Biegert, A., Hauser, A. & Söding, J. 2012. HHblits: Lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nature Methods, 9(2): 173-175.
68. 68. Santos, R.A.S., Oudit, G.Y., Verano-Braga, T., Canta, G., Steckelings, U.M. & Bader, M. 2019. The renin-angiotensin system: going beyond the classical paradigms. American Journal of Physiology-Heart and Circulatory Physiology, 316(5): 958-970.
69. 69. Shang, J., Wan, Y., Luo, C., Ye, G., Geng, Q., Auerbach, A. & Li, F. 2020a. Cell entry mechanisms of SARS-CoV-2. PNAS, 117(21): 11727-11734.
70. 70. Shang, J., Wan, Y., Liu, C., Yount, B., Gully, K., Yang, Y., Auerbach, A., Peng, G., Baric, R. & Li, F. 2020b. Structure of mouse coronavirus spike protein complexed with receptor reveals mechanism for viral entry. PLoS Pathogens, 16(3): e1008392.
71. 71. Shang, J., Ye, G., Shi, K., Wan, Y., Luo, C., Aihara, H., Geng, Q., Auerbach, A. & Li, F. 2020c. Structural basis of receptor recognition by SARS-CoV-2. Nature, 581, 221-224.
72. 72. Srivastava, A., Bandopadhyay, A., Das, D., Pandey, R.K., Singh, V., Khanam, N., Srivastava, N., Singh, P.P., Dubey, P.K., Pathak, A., Gupta, P., Rai, N., Sultana, G.N.N. & Chaubey, G. 2020. Genetic association of ACE2 rs2285666 polymorphism with Covid-19 spatial distribution in India. Frontiers in Genetics, 11(564741): 1-6.
73. 73. Torresi, J. 2008. Hepatitis B antiviral resistance and vaccine escape: Two sides of the same coin. Antiviral Therapy, 13(3): 337-340.
74. 74. Vermeire, K., Bell, T.W., Van Puyenbroeck, V., Giraut, A., Noppen, S., Liekens, S., Schols, D., Hartmann, E., Kalies, K.U. & Marsh, M. 2014. Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation. PLoS Biology, 12(12): e1002011.
75. 75. Walls, A.C., Park, Y.J., Tortorici, M.A., Wall, A., McGuire, A.T. & Veesler, D. 2020. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell, 181(2): 281-292.
76. 76. Walls, A.C., Tortorici, M.A., Snijder, J., Xiong, X., Bosch, B.J., Rey, F.A. & Veesler, D. 2017. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proceedings of the National Academy of Sciences of the United States of America, 114(42): 11157-11162.
77. 77. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., De Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R. & Schwede, T. 2018. SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Research, 46(W1): W296-W303.
78. 78. Wilkins, M.R., Gasteiger, E., Bairoch, A., Sanchez, J.C., Williams, K.L., Appel, R.D. & Hochstrasser, D.F. 1999. Protein identification and analysis tools in the ExPASy server. Methods in Molecular Biology, 112: 531-552.
79. 79. Worldometer. 2020. Coronavirus Cases. In Worldometer (pp. 1-22).
80. 80. Wrapp, D., Wang, N., Corbett, K.S., Goldsmith, J.A., Hsieh, C.L., Abiona, O., Graham, B.S. & McLellan, J.S. 2020. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483): 1260-1263.
81. 81. Wu, F., Zhao, S., Yu, B., Chen, Y.-M., Wang, W., Hu, Y., Song, Z.-G., Tao, Z.-W., Tian, J.-H., Pei, Y.-Y., Yuan, M.-L., Zhang, Y.-L., Dai, F.-H., Liu, Y., Wang, Q.-M., Zheng, J.-J., Xu, L., Holmes, E.C. & Zhang, Y.Z. 2020. Complete genome characterisation of a novel coronavirus associated with severe human respiratory disease in Wuhan, China. BioRxiv, 2020.01.24.919183. https://doi.org/10.1101/2020.01.24.919183
82. 82. Yang, L., Cheng, Y., Zhao, X., Wei, H., Tan, M., Li, X., Zhu, W., Huang, W., Chen, W., Liu, J., Li, Z., Shu, Y. & Wang, D. 2019. Mutations associated with egg adaptation of influenza A(H1N1)pdm09 virus in laboratory based surveillance in China, 2009-2016. Biosafety and Health, 1(1): 41-45.
83. 83. Zhang, M., Zeng, C.Q.-Y., Dong, Y., Ball, J.M., Saif, L.J., Morris, A.P. & Estes, M.K. 1998. Mutations in Rotavirus Nonstructural Glycoprotein NSP4 Are Associated with Altered Virus Virulence. Journal of Virology, 72(5): 3666-3672.
84. 84. Zhou, P., Yang, X.-L., Wang, X.G., Hu, B., Zhang, L., Zhang, W., Si, H.R., Zhu, Y., Li, B., Huang, C.L., Chen, H.D., Chen, J., Luo, Y., Guo, H., Jiang, R.-Di, Liu, M.Q., Chen, Y., Shen, X.R., Wang, X., Zheng, X.S., Zhao, K., Chen, Q.J., Deng, F., Liu, L.L., Yan, B., Zhan, F., Wang, Y., Xiao, G.F. & Shi, Z.L. 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 579(7798): 270-273.
85. 85. Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., Niu, P., Zhan, F., Ma, X., Wang, D., Xu, W., Wu, G., Gao, G.F. & Tan, W. (2020). A novel coronavirus from patients with pneumonia in China, 2019. New England Journal of Medicine, 382(8): 727-733.