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 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 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 Essential Amino Acids of the Hantaan Virus N Protein in Its Interaction with RNA

2005 ◽  
Vol 79 (15) ◽  
pp. 10032-10039 ◽  
Author(s):  
William Severson ◽  
Xiaolin Xu ◽  
Michaela Kuhn ◽  
Nina Senutovitch ◽  
Mercy Thokala ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Essential Amino Acids ◽  
Full Length ◽  
Hantaan Virus ◽  
Deletion Mapping ◽  
Mobility Shift ◽  
N Protein ◽  
Lysine Residues

ABSTRACT The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

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 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 Using nucleocapsid proteins to investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses

2020 ◽  
Author(s):  
Noah Avery Schuster
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Close Association ◽  
Phylogenetic Analyses ◽  
N Gene ◽  
Nucleocapsid Proteins ◽  
Nucleocapsid Gene ◽  
N Protein ◽  
Global Pandemic ◽  
The Relationship

An initial outbreak of coronavirus disease 2019 (COVID-19) in China has resulted in a massive global pandemic causing well over 16,500,000 cases and 650,000 deaths worldwide. The virus responsible, SARS-CoV-2, has been found to possess a very close association with Bat-CoV RaTG13 and Pangolin-CoV MP789. The nucleocapsid protein can serve as a decent model for determining phylogenetic, evolutionary, and structural relationships between coronaviruses. Therefore, this study uses the nucleocapsid gene and protein to further investigate the relationship between SARS-CoV-2 and closely related bat and pangolin coronaviruses. Sequence and phylogenetic analyses have revealed the nucleocapsid gene and protein in SARS-CoV-2 are both closely related to those found in Bat-CoV RaTG13 and Pangolin-CoV MP789. Evidence of recombination was detected within the N gene, along with the presence of a double amino acid insertion found in the N-terminal region. Homology modeling for the N-Terminal Domain revealed similar structures but distinct electrostatic surfaces and topological variations in the β-hairpin that likely reflect specific adaptive functions. In respect to SARS-CoV-2, two amino acids (S37 and A267) were found to exist only in its N protein, along with an extended β-hairpin that bends towards the nucleotide binding site. Collectively, this study strengthens the relationship among SARS-CoV-2, Bat-CoV RaTG13, and Pangolin-CoV MP789, providing additional insights into the structure and adaptive nature of the nucleocapsid protein found in these coronaviruses. Furthermore, these data will enhance our understanding of the complete history behind SARS-CoV-2 and help assist in antiviral 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.

scholarly journals Interactome Analysis of the Nucleocapsid Protein of SARS-CoV-2 Virus

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1155
Author(s):  
Xiaoqin Zheng ◽  
Zeyu Sun ◽  
Liang Yu ◽  
Danrong Shi ◽  
Miaojin Zhu ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Affinity Purification ◽  
Human Cells ◽  
Genome Replication ◽  
N Protein ◽  
Global Pandemic ◽  
Interaction Partners ◽  
Viral Genome Replication

SARS-CoV-2 infection has caused a global pandemic that has severely damaged both public health and the economy. The nucleocapsid protein of SARS-CoV-2 is multifunctional and plays an important role in ribonucleocapsid formation and viral genome replication. In order to elucidate its functions, interaction partners of the SARS-CoV-2 N protein in human cells were identified via affinity purification and mass spectrometry. We identified 160 cellular proteins as interaction partners of the SARS-CoV-2 N protein in HEK293T and/or Calu-3 cells. Functional analysis revealed strong enrichment for ribosome biogenesis and RNA-associated processes, including ribonucleoprotein complex biogenesis, ribosomal large and small subunits biogenesis, RNA binding, catalysis, translation and transcription. Proteins related to virus defence responses, including MOV10, EIF2AK2, TRIM25, G3BP1, ZC3HAV1 and ZCCHC3 were also identified in the N protein interactome. This study comprehensively profiled the viral–host interactome of the SARS-CoV-2 N protein in human cells, and the findings provide the basis for further studies on the pathogenesis and antiviral strategies for this emerging infection.

scholarly journals SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans

2020 ◽  
Author(s):  
Nitin T. Supekar ◽  
Asif Shajahan ◽  
Anne S. Gleinich ◽  
Daniel Rouhani ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Point Of Care ◽  
Variable Mass ◽  
Phosphorylation Site ◽  
Post Translational Modifications ◽  
N Protein ◽  
Point Of Care Diagnostics ◽  
Glycosylation Sites

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

scholarly journals Adjuvant Potential of CD24 on Immunogenicity and Lethal Challenge Protection of a DNA vector Expressing Nucleocapsid Protein of Crimean Congo Hemorrhagic Fever Virus

2018 ◽  
Author(s):  
Touraj Aligholipour Farzani ◽  
Alireza Hanifehnezhad ◽  
Katalin Foldes ◽  
Koray Ergunay ◽  
Erkan Yilmaz ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Hemorrhagic Fever ◽  
Fever Virus ◽  
Foreign Gene ◽  
Lethal Challenge ◽  
Crimean Congo Hemorrhagic Fever ◽  
N Protein ◽  
Hemorrhagic Fever Virus ◽  
Dna Vector

Crimean Congo hemorrhagic fever virus (CCHFV) is the causative agent of a globally-spread tick-borne zoonotic infection with an eminent risk of fatal human disease. Imminent public health threat posed by disseminated virus activity and lack of an approved therapeutic make CCHFV an urgent target for vaccine development. We described the construction of a DNA vector expressing nucleocapsid protein (N) of CCHFV (pV-N13) and investigated its potential to stimulate cytokine and total/specific antibody responses in BALB/c and challenge experiment in IFNAR-/- mice. Due to lack of sufficient antibody stimulation towards N protein, we have selected CD24 protein as a potential adjuvant which has proliferative effect on B and T cells. Overall, our N expressing construct when administered solely or in combination with pCD24 vector elicited significant cellular and humoral responses in BALB/c, despite variations in particular cytokines and total antibodies. However, the stimulated antibodies produced due to expression of N protein have shown no neutralizing ability in VNA. Furthermore, challenge experiments were revealed protection potential of N expressing construct in IFNAR -/- mice model. In conclusion, we have shown that CD24 has prominent effect as a genetic adjuvant when co-delivers with a synergic foreign gene expressing vector. Besides, targeting of S segment of CCHFV can be considered as a practical way in developing vaccine against this virus due to its ability to induce immune response which leads to protection in challenge assays in IFN-gamma defective mice models.

Sign in / Sign up

Export Citation Format

Share Document