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The outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus continually led to worldwide human infections and deaths. Currently, there is no specific viral protein-targeted therapeutics. 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 remains unclear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although the overall structure is similar as 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., Graphical abstract The crystal structure of the N-terminal RNA-binding domain of SARS-CoV-2 nucleocapsid protein was determined by X-ray. Compared with other previously reported coronavirus nucleocapsid protein N-terminal domains, the authors have identified a unique potential RNA-binding pocket that can guide the design of novel antiviral drugs targeting SARS-CoV-2.Image 1

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 Immunoglobulin-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., Graphical abstract, Immunology; Virology

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 isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAbs) from a quick recovery coronavirus disease-19 (COVID-19) convalescent patient who had 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 mAb with the highest binding affinity (nCoV396) revealed changes in the epitopes and antigen’s allosteric regulation. Functionally, a virus-free complement hyper-activation analysis demonstrated that nCoV396 specifically compromises the N protein-induced complement hyper-activation, 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.

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.

Although human antibodies elicited by severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid (N) protein are profoundly boosted upon infection, little is known about the function of N-directed antibodies. Herein, we isolated and profiled a panel of 32 N protein-specific monoclonal antibodies (mAb) from a quick recovery coronavirus disease-19 (COVID-19) convalescent, who had dominant antibody responses to SARS-CoV-2 N protein rather than to Spike protein. The complex structure of N protein RNA binding domain with the highest binding affinity mAb nCoV396 reveals the epitopes and antigen’s allosteric changes. Functionally, a virus-free complement hyper-activation analysis demonstrates that nCoV396 specifically compromises N protein-induced complement hyper-activation, a risk factor for morbidity and mortality in COVID-19, thus paving the way for functional anti-N mAbs identification.One Sentence SummaryB cell profiling, structural determination, and protease activity assays identify a functional antibody to N protein.

Centromere protein W (CENP-W), identified as a centromeric component, plays an important role in the cell life cycle. However, how CENPW expression affects biological processes in liver cancer cells remains unknown. In this article, we found that CENPW was overexpressed in liver cancer tissues. Low CENPW expression was correlated with a better prognosis in hepatocellular carcinoma (HCC) patients, compared to high CENPW expression. The results of qRT-PCR and western blot assay showed that CENPW was effectively knocked down in HCC cells using siRNA transfection. Cell proliferation, migration, and invasion were inhibited. Cell apoptosis rates were increased. The cells were arrested in the G2/M phase of the cell cycle. Subsequently, 127 differentially expressed genes (DEGs) were identified based on RNA-seq data. GO and KEGG enrichment and PPI network analysis were performed. The novel DEGs were found and mainly enriched in nucleosome assembly and the complement system. In summary, our study indicated that overexpression of CENPW implied unfavorable prognosis and CENPW might be the potential predictive biomarker in liver cancer. Downregulation of CENPW might inhibit the HCC developmentby regulating the expression of the molecules in nucleosomes and the complement system., Graphical abstract

Dysregulated immune cell responses have been linked to the severity of Coronavirus Disease 2019 (COVID-19). However, the specific viral factor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributing to the immune-dysregulation is currently unclear. Herein, we identified the ectodomain of Ig-like fold viral proteinSARS-CoV-2 Orf7a interacted with CD14+ monocytes at the highest efficiency in human peripheral blood mononuclear cells, but not for the relative highly pathogenic protein SARS-CoV Orf7a. The 2.2 A resolutioncrystal structure of SARS-CoV-2 Orf7a reveals three remarkable changes in the amphipathic side of the four-strand β-sheet, implying the potential functional interface of the viral protein. Structure-based superimposition of SARS-CoV-2 Orf7a with SARS-CoV Orf7a - LFA1 working model suggests that SARS-CoV-2 Orf7a utilizes different binding patterns to recognize the specific immune cells. Importantly, SARS-CoV-2 Orf7a co-incubation with CD14+ monocytes ex vivo triggers a decrease of HLA-DR/DP/DQ and significant pro-inflammatory cytokines expressions, including IL-6, IL-1β, IL-8, and TNF-α. Our work demonstrates that SARS-CoV-2 Orf7a is an immunomodulating factor for immune cells binding and trigging aberrant inflammatory responses, providing promising therapeutic drug targets in the pandemic disease COVID-19. Funding: This work is supported by the National Key RD the Special Fund for Scientific and Technological Innovation Strategy of Guangdong Province of China (2018B030306029), COVID-19 Emerging Prevention Products, Research Special Fund of Zhuhai City (ZH22036302200016PWC) granted to S.C.; the Fundamental Research Funds for the Central Universities (19ykzd36) granted to J.C; and the Science and Technology Program of Guangzhou (202002030069) granted to J.C. Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: The human peripheral blood samples for the experiments were collected through The Health Management Center, The Fifth Affiliated Hospital, Sun Yat-sen University. This study was approved by The Medical Ethics Committee of The Fifth Affiliated Hospital, Sun Yat-sen University (2020-K195-1).

The worldwide epidemic of novel coronavirus disease (COVID-19) has led to a strong demand for highly efficient immunobinding to achieve rapid and accurate on-site detection of SARS-CoV-2 antibodies. However, hour-scale time-consumption is usually required to ensure the adequacy of immunobinding on expensive large instruments in hospitals, and the common false negative or positive results often occur in rapid on-site immunoassay (e.g. immunochromatography). We solved this dilemma by presenting a reciprocating-flowing immunobinding (RF-immunobinding) strategy. RF-immunobinding enabled the antibodies in fluid contacting with the corresponding immobilized antigens on substrate repeatedly during continuous reciprocating-flowing, to achieve adequate immunobinding within 60 s. This strategy was further developed into an immunoassay method for the serological detection of 13 suspected COVID-19 patients. We obtained a 100% true negative and true positive rate and a limit of quantification (LOQ) of 4.14 pg/mL. Our strategy also can be a potential support for other areas related to immunorecognition, such as proteomics, immunopharmacology and immunohistochemistry., Highlights • Our strategy that can reach saturation within 60 s may be the fastest immunobinding. • Two-step reciprocating-flowing enzyme linked immunosorbent assay needs 5 min only. • This ultra-fast immunobinding will speed up many on-site COVID-19 immunoassays. • Easy fabrication and pressure control allow portable test in less-developed regions.