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

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.

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Trimeric Hantavirus Nucleocapsid Protein Binds Specifically to the Viral RNA Panhandle

Journal of Virology ◽

10.1128/jvi.78.15.8281-8288.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8281-8288 ◽

Cited By ~ 53

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.

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

10.1101/2021.06.14.448452 ◽

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.

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Identification of four unconventional kinetoplastid kinetochore proteins KKT22–25 in Trypanosoma brucei

Open Biology ◽

10.1098/rsob.190236 ◽

2019 ◽

Vol 9 (12) ◽

pp. 190236 ◽

Cited By ~ 6

Author(s):

Olga O. Nerusheva ◽

Patryk Ludzia ◽

Bungo Akiyoshi

Keyword(s):

Trypanosoma Brucei ◽

Chromosome Segregation ◽

Rna Binding ◽

Affinity Purification ◽

Potential Interaction ◽

Interacting Proteins ◽

Interaction Partners ◽

Spindle Microtubules ◽

Acetyltransferase Domain ◽

Mitosis And Meiosis

The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1–20). In addition, 12 proteins (KKT-interacting proteins 1–12, KKIP1–12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22–25, in Trypanosoma brucei . KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N -acetyltransferase domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

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Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10894-10904.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10894-10904 ◽

Cited By ~ 32

Author(s):

Robert V. Intine ◽

Miroslav Dundr ◽

Alex Vassilev ◽

Elena Schwartz ◽

Yingmin Zhao ◽

...

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Affinity Purification ◽

Close Association ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Rna Recognition Motif ◽

Translational Machinery ◽

Terminal Domain

ABSTRACT La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D. J. Kenan and J. D. Keene, Nat. Struct. Mol. Biol. 11 :303-305, 2004). While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known. In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366. We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation. Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin. Moreover, La lacking the SBM does not localize to nucleoli. Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay. The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

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

10.21203/rs.3.rs-106760/v1 ◽

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.

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

10.1101/2021.05.06.21256706 ◽

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.

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

10.1101/2020.10.06.328112 ◽

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.

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

10.1101/2020.06.25.172312 ◽

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.

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

Biomolecular NMR Assignments ◽

10.1007/s12104-020-09995-y ◽

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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Antibody Response against the SARS-CoV-2 Nucleocapsid Protein and Its Subdomains—Identification of Pre-Immunization Status by Human Coronaviruses with Multipanel Nucleocapsid Fragment Immunoblotting

COVID ◽

10.3390/covid1010009 ◽

2021 ◽

Vol 1 (1) ◽

pp. 105-114

Author(s):

Sahra Pajenda ◽

Sebastian Kapps ◽

Thomas Reiter ◽

Raimundo Freire ◽

Veronique A. J. Smits ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Posterior Part ◽

Rapid Identification ◽

Diagnostic Methods ◽

Serum Samples ◽

N Protein ◽

Humoral Immune ◽

Humoral Immune Responses ◽

Global Pandemic ◽

Human Coronaviruses

A novel beta coronavirus that emerged in late December 2019 triggered a global pandemic. Diagnostic methods for rapid identification of infected individuals were established in new biotechnological approaches. Vaccine production and application to individuals and measurement of SARS-CoV-2 antibodies also began. Serum samples from 240 health care workers were collected at three-month intervals over nine months. Indirect SARS-CoV-2 nucleocapsid IgG ELISA tests were used to identify humoral immune responses. All seropositive individuals and those with borderline ELISA values were tested with a specifically generated multipanel nucleocapsid fragment immunoblot. Of the 240 individuals, 24 showed seroconversion in ELISA after experiencing COVID-19. All of them showed a positive reaction against the full-length nucleocapsid protein in the immunoblot. The highest reactivity was seen either against fragment N(100–300) or in a minority against the posterior part N(200–419). In general, the staining pattern of COVID-19 patients showed four phenotypes. In contrast, three individuals classified as borderline by ELISA reacted exclusively with fragments N(1–220) and N(100–300) containing the octamer amino acid sequence FYYLGTGP, which is identical in human coronaviruses sharing this sequence with SARS-CoV-2. These represent a unique and thus fifth phenotype. This work suggests the existence of distinct phenotypic patterns of IgG production towards N-protein subdomains.

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