Biomarker Discovery for Betacoronaviruses through NMR-based Epitope Mapping

The SARS-CoV-2 nucleocapsid protein (N) stands out because of its conservation in betacoronaviruses, high immunogenicity, and abundant expression during infection. The N protein consists mainly of intrinsically disordered regions, contributing to its riboprotein activity. Understanding the molecular basis of recognition involving human antibodies and the N protein is critical for vaccine development, diagnostics, and antiviral therapies. We employed nuclear magnetic resonance spectroscopy to investigate the interaction between the RNA-binding domain of the N protein and purified reactive antibodies from the plasma of convalescent COVID-19 patients in the Belo Horizonte metropolitan region, Minas Gerais, Brazil. Epitope mapping was performed using a heteronuclear NMR [1H,15N]-HSQC experiment to assess changes in the chemical environment through chemical shift perturbation (CSP) and to characterize the molecular dynamics of the backbone in the presence of the antibodies on a time scale of microseconds to milliseconds using transverse relaxation (R2). Validation experiments were conducted using the MERS-CoV N protein, to assess the binding with reactive SARS-CoV-2 N protein antibodies. The identified conformational epitopes were screened and mapped on the N protein structure. Afterward, the epitope sequences were evaluated, using immunoinformatics approaches and its druggability features. Our findings successfully mapped conformational epitopes in the SARS-CoV-2 nucleocapsid structure, particularly those related to the genomic RNA interaction site. These epitopes were mainly located in exposed ordered secondary structure regions, comprising the β-strand antiparallel core, representing a conserved motif among betacoronaviruses. However, the cross-reactivity between reactive SARS-CoV-2 N antibodies and MERS N protein proved less specific, despite a few common epitope sites being mapped. Notably, the amino acid sequence in β1 of the SARS-CoV-2 N protein emerged as a promising and conserved T and B cell epitope site, and its structure was calculated using experimental data of homonuclear 2D NMR [1H-1H] TOCSY and NOESY spectra combined with in silico methods. Our work successfully correlated conformational epitopes, regarding their structure and molecular dynamics involved in antibody recognition for epitope mapping in viral proteins, providing insights into promising biomarkers for betacoronaviruses.