Abstract
Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.
Keywords: Covid-19; Nucleocapsid; Oligomerization; RNA binding; SARS coronavirus.
【저자키워드】 COVID-19, nucleocapsid, SARS Coronavirus, RNA binding, oligomerization, 【초록키워드】 coronavirus disease, SARS-CoV-2, Coronavirus disease 2019, coronavirus, pandemic, Cryo-electron microscopy, severe acute respiratory syndrome Coronavirus, Health crisis, RNA, Nucleocapsid phosphoprotein, Protein, Health, electron microscopy, Viral, nucleocapsid, SARS Coronavirus, respiratory, crystal structure, pathogenicity, oligomerization, antiviral target, virulence, genomic, moderate, thermal stability, function, binding, C-terminal domain, conformational change, viral genome, crystal structures, immunogenic determinant, acute respiratory syndrome, solution, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, complex, differential scanning fluorimetry, immunogenic, ultracentrifugation, ssRNA, SARS-CoV-2 N, full-length, tendency, single-stranded, initial, develop, caused, provide, demonstrated, SARS-CoV N, multifunctional, CTD, scattering, the SARS-CoV-2, 【제목키워드】 coronavirus, respiratory, acute respiratory syndrome, domain,