scholarly journals A SARS-CoV-2 antibody curbs viral nucleocapsid protein-induced complement hyperactivation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sisi Kang ◽  
Mei Yang ◽  
Suhua He ◽  
Yueming Wang ◽  
Xiaoxue Chen ◽  
...  
Keyword(s):  
Risk Factor ◽  
Monoclonal Antibodies ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Complex Structure ◽  
Antibody Responses ◽  
N Protein ◽  
Human Antibodies ◽  
Quick Recovery ◽  
Viral Nucleocapsid

AbstractAlthough 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.

scholarly journals A COVID-19 antibody curbs SARS-CoV-2 nucleocapsid protein-induced complement hyperactivation

2020 ◽  
Author(s):  
Sisi Kang ◽  
Mei Yang ◽  
Suhua He ◽  
Yueming Wang ◽  
Xiaoxue Chen ◽  
...  
Keyword(s):  
Risk Factor ◽  
Monoclonal Antibodies ◽  
Activation Analysis ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Complex Structure ◽  
Antibody Responses ◽  
N Protein ◽  
Human Antibodies ◽  
Quick Recovery

Abstract 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.

scholarly journals A COVID-19 antibody curbs SARS-CoV-2 nucleocapsid protein-induced complement hyper-activation

2020 ◽  
Author(s):  
Sisi Kang ◽  
Mei Yang ◽  
Suhua He ◽  
Yueming Wang ◽  
Xiaoxue Chen ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Distress Syndrome ◽  
Rna Binding ◽  
Complex Structure ◽  
Antibody Responses ◽  
Spike Protein ◽  
Structural Determination ◽  
N Protein ◽  
Human Antibodies ◽  
Quick Recovery

AbstractAlthough 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.

scholarly journals Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

2004 ◽  
Vol 78 (15) ◽  
pp. 8281-8288 ◽  
Author(s):  
M. A. Mir ◽  
A. T. Panganiban
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Viruses ◽  
Sin Nombre Virus ◽  
Viral Capsid ◽  
Genomic Rna ◽  
Rna Molecules ◽  
N Protein ◽  
High Affinity ◽  
Negative Sense

ABSTRACT Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3′ and 5′ ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

scholarly journals The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

2021 ◽  
Vol 17 (9) ◽  
pp. e1009931
Author(s):  
Jorge Vera-Otarola ◽  
Estefania Castillo-Vargas ◽  
Jenniffer Angulo ◽  
Francisco M. Barriga ◽  
Eduard Batlle ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Initiation Factor ◽  
Cellular Proteins ◽  
Dependent Manner ◽  
The Andes ◽  
Viral Nucleocapsid

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.

scholarly journals Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures

2021 ◽  
Author(s):  
Christine Roden ◽  
Yifan Dai ◽  
Ian Seim ◽  
Myungwoon Lee ◽  
Rachel Sealfon ◽  
...  
Keyword(s):  
Phase Separation ◽  
Rna Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genome Packaging ◽  
Rna Motifs ◽  
Double Stranded Rna ◽  
N Protein ◽  
Binding Domains

Betacoronavirus SARS-CoV-2 infections caused the global Covid-19 pandemic. The nucleocapsid protein (N-protein) is required for multiple steps in the betacoronavirus replication cycle. SARS-CoV-2-N-protein is known to undergo liquid-liquid phase separation (LLPS) with specific RNAs at particular temperatures to form condensates. We show that N-protein recognizes at least two separate and distinct RNA motifs, both of which require double-stranded RNA (dsRNA) for LLPS. These motifs are separately recognized by N-protein's two RNA binding domains (RBDs). Addition of dsRNA accelerates and modifies N-protein LLPS in vitro and in cells and controls the temperature condensates form. The abundance of dsRNA tunes N-protein-mediated translational repression and may confer a switch from translation to genome packaging. Thus, N-protein's two RBDs interact with separate dsRNA motifs, and these interactions impart distinct droplet properties that can support multiple viral functions. These experiments demonstrate a paradigm of how RNA structure can control the properties of biomolecular condensates.

scholarly journals Saudi Arabian SARS-CoV-2 genomes implicate a mutant Nucleocapsid protein in modulating host interactions and increased viral load in COVID-19 patients

2021 ◽  
Author(s):  
Tobias Mourier ◽  
Muhammad Shuaib ◽  
Sharif Hala ◽  
Sara Mfarrej ◽  
Fadwa Alofi ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Viral Load ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Genomic Sequence ◽  
Mass Spectrometry Analysis ◽  
Interferon Response ◽  
Sequence Information ◽  
Effective Population ◽  
N Protein

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. The availability of patient hospital records is crucial for linking the genomic sequence information to virus function during the course of infections. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. From the assembled sequences, we estimate the SARS-CoV-2 effective population size and infection rate and outline the epidemiological dynamics of import and transmission events during this period in Saudi Arabia. We show that two consecutive mutations (R203K/G204R) in the SARS-CoV-2 nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein by mass-spectrometry analysis. Furthermore, analysis of the host cell transcriptome suggests that the mutant N protein results in dysregulated interferon response genes. We provide crucial information in linking the R203K/G204R mutations in the N protein as a major modulator of host-virus interactions and increased viral load and underline the potential of the nucleocapsid protein as a drug target during infection.

scholarly journals IgG Antibodies Develop to Spike but Not to the Nucleocapsid Viral Protein in Many Asymptomatic and Light COVID-19 Cases

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1945
Author(s):  
Maria Tutukina ◽  
Anna Kaznadzey ◽  
Maria Kireeva ◽  
Ilya Mazo
Keyword(s):  
Disease Severity ◽  
Nucleocapsid Protein ◽  
Receptor Binding Domain ◽  
Igg Antibodies ◽  
N Protein ◽  
Main Target ◽  
Viral Nucleocapsid ◽  
Different Levels ◽  
Antibody Ratio ◽  
Severe Lung

Since SARS-CoV-2 appeared in late 2019, many studies on the immune response to COVID-19 have been conducted, but the asymptomatic or light symptom cases were somewhat understudied as respective individuals often did not seek medical help. Here, we analyze the production of the IgG antibodies to viral nucleocapsid (N) protein and receptor-binding domain (RBD) of the spike protein and assess the serum neutralization capabilities in a cohort of patients with different levels of disease severity. In half of light or asymptomatic cases the antibodies to the nucleocapsid protein, which serve as the main target in many modern test systems, were not detected. They were detected in all cases of moderate or severe symptoms, and severe lung lesions correlated with respective higher signals. Antibodies to RBD were present in the absolute majority of samples, with levels being sometimes higher in light symptom cases. We thus suggest that the anti-RBD/anti-N antibody ratio may serve as an indicator of the disease severity. Anti-RBD IgG remained detectable after a year or more since the infection, even with a slight tendency to raise over time, and the respective signal correlated with the serum capacity to inhibit the RBD interaction with the ACE-2 receptor.

scholarly journals Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

2020 ◽  
Author(s):  
Corinne A. Lutomski ◽  
Tarick J. El-Baba ◽  
Jani R. Bolla ◽  
Carol V. Robinson
Keyword(s):  
Antibody Response ◽  
Nucleocapsid Protein ◽  
Electron Transfer Dissociation ◽  
Rna Binding ◽  
Vaccine Development ◽  
Cyclophilin A ◽  
Proteolytic Processing ◽  
Full Length ◽  
Oligomeric State ◽  
N Protein

AbstractThe SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

scholarly journals 1H, 13C, and 15N backbone chemical shift assignments of the C-terminal dimerization domain of SARS-CoV-2 nucleocapsid protein

2020 ◽  
Author(s):  
Sophie M. Korn ◽  
Roderick Lambertz ◽  
Boris Fürtig ◽  
Martin Hengesbach ◽  
Frank Löhr ◽  
...  
Keyword(s):  
Chemical Shift ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Drug Binding ◽  
Chemical Shift Assignments ◽  
Binding Studies ◽  
Dimerization Domain ◽  
N Protein ◽  
Intrinsically Disordered ◽  
Backbone Chemical Shift

AbstractThe current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.

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