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AbstractThe outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

Dysregulated immune cell responses have been linked to the severity of coronavirus disease 2019 (COVID-19), but the specific viral factors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were currently unknown. Herein, we reveal that the Ig-like fold ectodomain of the viral protein SARS-CoV-2 ORF7a interacts with high efficiency to CD14 + monocytes in human peripheral blood, compared to pathogenic protein SARS-CoV ORF7a. The crystal structure of SARS-CoV-2 ORF7a at 2.2 Å resolution reveals three remarkable changes on the amphipathic side of the four-stranded β-sheet, implying a potential functional interface of the viral protein. Importantly, SARS-CoV-2 ORF7a coincubation with CD14 + monocytes ex vivo triggered a decrease in HLA-DR/DP/DQ expression levels and upregulated significant production of proinflammatory cytokines, including IL-6, IL-1β, IL-8, and TNF-α. Our work demonstrates that SARS-CoV-2 ORF7a is an immunomodulating factor for immune cell binding and triggers dramatic inflammatory responses, providing promising therapeutic drug targets for pandemic COVID-19.

Coronavirus disease 2019 (COVID-19) has caused massive disruptions to society and the economy, and the transcriptional regulatory mechanisms behind the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are poorly understood. Herein, we determined the crystal structure of the SARS-CoV-2 nucleocapsid protein C-terminal domain (CTD) at a resolution of 2.0 Å, and demonstrated that the CTD has a comparable distinct electrostatic potential surface to equivalent domains of other reported CoVs, suggesting that the CTD has novel roles in viral RNA binding and transcriptional regulation. Further in vitro biochemical assays demonstrated that the viral genomic intergenic transcriptional regulatory sequences (TRSs) interact with the SARS-CoV-2 nucleocapsid protein CTD with a flanking region. The unpaired adeno dinucleotide in the TRS stem-loop structure is a major determining factor for their interactions. Taken together, these results suggested that the nucleocapsid protein CTD is responsible for the discontinuous viral transcription mechanism by recognizing the different patterns of viral TRS during transcription.

Although human antibodies elicited by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-reactive antibodies. Herein, we isolate and profile a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who has dominant antibody responses to the SARS-CoV-2 N protein rather than to the SARS-CoV-2 spike (S) protein. The complex structure of the N protein RNA binding domain with the highest binding affinity mAb (nCoV396) reveals changes in the epitopes and antigen’s allosteric regulation. Functionally, a virus-free complement hyperactivation analysis demonstrates that nCoV396 specifically compromises the N protein-induced complement hyperactivation, which is a risk factor for the morbidity and mortality of COVID-19 patients, thus laying the foundation for the identification of functional anti-N protein mAbs.