Biochemical characterization of SARS-CoV-2 nucleocapsid protein

Abstract The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients' sera, proving the importance of this antigen in host immunity and diagnostics.

[1]  Matthew R. McKay,et al.  Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies , 2020, bioRxiv.

[2]  Wei-Lun Chang,et al.  Modular organization of SARS coronavirus nucleocapsid protein , 2005, Journal of biomedical science.

[3]  Torsten Schwede,et al.  SWISS-MODEL: homology modelling of protein structures and complexes , 2018, Nucleic Acids Res..

[4]  Chi-Ju Chen,et al.  Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates , 2006, Vaccine.

[5]  I. Yu,et al.  Crystal Structure of the Severe Acute Respiratory Syndrome (SARS) Coronavirus Nucleocapsid Protein Dimerization Domain Reveals Evolutionary Linkage between Corona- and Arteriviridae* , 2006, Journal of Biological Chemistry.

[6]  Ping Li,et al.  Diagnosis of Severe Acute Respiratory Syndrome (SARS) by Detection of SARS Coronavirus Nucleocapsid Antibodies in an Antigen-Capturing Enzyme-Linked Immunosorbent Assay , 2003, Journal of Clinical Microbiology.

[7]  Qi Jin,et al.  Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19) , 2020, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[8]  V. Deubel,et al.  Characterization and application of monoclonal antibodies against N protein of SARS-coronavirus , 2005, Biochemical and Biophysical Research Communications.

[9]  P. Minor,et al.  WHO Expert Committee on Biological Standardization. , 2004, World Health Organization technical report series.

[10]  Caiying Zhang,et al.  Structure determination of CAMP factor of Mobiluncus curtisii and insights into structural dynamics. , 2019, International journal of biological macromolecules.

[11]  Chun-Yuan Chen,et al.  Structure of the SARS Coronavirus Nucleocapsid Protein RNA-binding Dimerization Domain Suggests a Mechanism for Helical Packaging of Viral RNA , 2007, Journal of Molecular Biology.

[12]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[13]  Maxim V. Petoukhov,et al.  New developments in the ATSAS program package for small-angle scattering data analysis , 2012, Journal of applied crystallography.

[14]  A. Rohou,et al.  Structures of autoinhibited and polymerized forms of CARD9 reveal mechanisms of CARD9 and CARD11 activation , 2019, Nature Communications.

[15]  A. Samad,et al.  X-ray crystal structure of putative transcription regulator lmo2088 from Listeria monocytogenes. , 2019, Biochemical and biophysical research communications.

[16]  Chin-Kun Hu,et al.  Multiple Nucleic Acid Binding Sites and Intrinsic Disorder of Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein: Implications for Ribonucleocapsid Protein Packaging , 2008, Journal of Virology.

[17]  Kai Zhao,et al.  A pneumonia outbreak associated with a new coronavirus of probable bat origin , 2020, Nature.

[18]  S. G. Anderson.  W.H.O. Expert Committee on Biological Standardization: Twenty-sixth Report: World Health Organization Technical Report Series , 1976 .

[19]  H. Feldmann,et al.  Characterization of protein–protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus , 2004, Virus Research.

[20]  Tai-Huang Huang,et al.  Transient Oligomerization of the SARS-CoV N Protein – Implication for Virus Ribonucleoprotein Packaging , 2013, PloS one.

[21]  A. Gunasekera,et al.  Structure of the N-terminal RNA-binding domain of the SARS CoV nucleocapsid protein. , 2004, Biochemistry.

[22]  N. Bastien,et al.  Analysis of multimerization of the SARS coronavirus nucleocapsid protein , 2004, Biochemical and Biophysical Research Communications.

[23]  E. Holmes,et al.  Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding , 2020, The Lancet.

[24]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[25]  Yajuan Li,et al.  Crystal structure and activation mechanism of DR3 death domain , 2019, The FEBS journal.

[26]  Y. Leo,et al.  Profiles of Antibody Responses against Severe Acute Respiratory Syndrome Coronavirus Recombinant Proteins and Their Potential Use as Diagnostic Markers , 2004, Clinical Diagnostic Laboratory Immunology.

[27]  J. Koenderink.  Q… , 2014, Les noms officiels des communes de Wallonie, de Bruxelles-Capitale et de la communaute germanophone.

[28]  Xin Jia,et al.  Structure–function analyses reveal the molecular architecture and neutralization mechanism of a bacterial HEPN–MNT toxin–antitoxin system , 2018, The Journal of Biological Chemistry.

[29]  Raymond C. Stevens,et al.  Ribonucleocapsid Formation of Severe Acute Respiratory Syndrome Coronavirus through Molecular Action of the N-Terminal Domain of N Protein , 2007, Journal of Virology.

[30]  Kaixian Chen,et al.  The nucleocapsid protein of SARS coronavirus has a high binding affinity to the human cellular heterogeneous nuclear ribonucleoprotein A1 , 2005, FEBS Letters.

[31]  P. Lim,et al.  Antibody Response of Patients with Severe Acute Respiratory Syndrome (SARS) Targets the Viral Nucleocapsid , 2004, The Journal of infectious diseases.

[32]  O. Tsang,et al.  Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study , 2020, The Lancet Infectious Diseases.

[33]  Zhigang Tian,et al.  Functional exhaustion of antiviral lymphocytes in COVID-19 patients , 2020, Cellular & Molecular Immunology.

[34]  Krishna Shankara Narayanan,et al.  Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal , 2003, Journal of Virology.

[35]  Syed Faraz Ahmed,et al.  Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies , 2020, Viruses.

[36]  C. A. Koetzner,et al.  Identification of In Vivo-Interacting Domains of the Murine Coronavirus Nucleocapsid Protein , 2009, Journal of Virology.