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 SARS-CoV-2 Nucleocapsid Protein Targets RIG-I-Like Receptor Pathways to Inhibit the Induction of Interferon Response

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 530
Author(s):  
Soo Jin Oh ◽  
Ok Sarah Shin
Keyword(s):  
Nucleocapsid Protein ◽  
Nuclear Translocation ◽  
Nonstructural Protein ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Antiviral Immune Response ◽  
N Protein ◽  
Nonstructural Protein 1 ◽  
Polycytidylic Acid

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) that has resulted in the current pandemic. The lack of highly efficacious antiviral drugs that can manage this ongoing global emergency gives urgency to establishing a comprehensive understanding of the molecular pathogenesis of SARS-CoV-2. We characterized the role of the nucleocapsid protein (N) of SARS-CoV-2 in modulating antiviral immunity. Overexpression of SARS-CoV-2 N resulted in the attenuation of retinoic acid inducible gene-I (RIG-I)-like receptor-mediated interferon (IFN) production and IFN-induced gene expression. Similar to the SARS-CoV-1 N protein, SARS-CoV-2 N suppressed the interaction between tripartate motif protein 25 (TRIM25) and RIG-I. Furthermore, SARS-CoV-2 N inhibited polyinosinic: polycytidylic acid [poly(I:C)]-mediated IFN signaling at the level of Tank-binding kinase 1 (TBK1) and interfered with the association between TBK1 and interferon regulatory factor 3 (IRF3), subsequently preventing the nuclear translocation of IRF3. We further found that both type I and III IFN production induced by either the influenza virus lacking the nonstructural protein 1 or the Zika virus were suppressed by the SARS-CoV-2 N protein. Our findings provide insights into the molecular function of the SARS-CoV-2 N protein with respect to counteracting the host antiviral immune response.

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 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 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.

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 RNA Binding Properties of Bunyamwera Virus Nucleocapsid Protein and Selective Binding to an Element in the 5′ Terminus of the Negative-Sense S Segment

2000 ◽  
Vol 74 (21) ◽  
pp. 9946-9952 ◽  
Author(s):  
Jane C. Osborne ◽  
Richard M. Elliott
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Metal Chelate ◽  
Rnase A ◽  
Mobility Shift ◽  
N Protein ◽  
Encapsidation Signal ◽  
Negative Sense ◽  
Bunyamwera Virus

ABSTRACT The genome of Bunyamwera virus (BUN) (familyBunyaviridae, genus Bunyavirus) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocapsid protein (N). Previous studies have suggested that the encapsidation signal may reside within the 5′ terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and purified by metal chelate chromatography. An RNA probe containing the 5′-terminal 32 and 3′-terminal 33 bases of the BUN S (small) genome segment was used to investigate binding by the N protein in vitro using gel mobility shift and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a degree of cooperativity in N protein binding. RNA-N complexes were resistant to digestion with up to 1 μg of RNase A per ml. Competition assays with a variety of viral and nonviral RNAs identified a region within the 5′ terminus of the BUN S segment for which N had a high preference for binding. This site may constitute the signal for initiation of encapsidation by N.

scholarly journals Colocalization and Interaction of the Porcine Arterivirus Nucleocapsid Protein with the Small Nucleolar RNA-Associated Protein Fibrillarin

2003 ◽  
Vol 77 (22) ◽  
pp. 12173-12183 ◽  
Author(s):  
Dongwan Yoo ◽  
Sarah K. Wootton ◽  
Gang Li ◽  
Cheng Song ◽  
Raymond R. Rowland
Keyword(s):  
Amino Acids ◽  
Host Cell ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Specific Interaction ◽  
Cell Protein ◽  
Small Nucleolar Rna ◽  
N Protein ◽  
Cell Functions ◽  
Nucleolar Rna

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione S-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.

scholarly journals RNA Binding Domain of Jamestown Canyon Virus S Segment RNAs

2007 ◽  
Vol 81 (24) ◽  
pp. 13754-13760 ◽  
Author(s):  
Monica M. Ogg ◽  
Jean L. Patterson
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Binding Domain ◽  
Binding Potential ◽  
Deletion Mutants ◽  
Wild Type ◽  
Binding Assays ◽  
N Protein ◽  
Jamestown Canyon Virus ◽  
California Serogroup

ABSTRACT Jamestown Canyon virus (JCV) is a member of the Bunyaviridae family, Orthobunyavirus genus, California serogroup. Replication and, ultimately, assembly and packaging rely on the process of encapsidation. Therefore, the ability of viral RNAs (vRNAs) (genomic and antigenomic) to interact with the nucleocapsid protein (N protein) and the location of this binding domain on the RNAs are of interest. The questions to be addressed are the following. Where is the binding domain located on both the vRNA and cRNA strands, is this RNA bound when double or single stranded, and does this identified region have the ability to transform the binding potential of nonviral RNA? Full-length viral and complementary S segment RNA, as well as 3′ deletion mutants of both vRNA and cRNA, nonviral RNA, and hybrid viral/nonviral RNA, were analyzed for their ability to interact with bacterially expressed JCV N protein. RNA-nucleocapsid interactions were examined by UV cross-linking, filter binding assays, and the generation of hybrid RNA to help define the area responsible for RNA-protein binding. The assays identified the region responsible for binding to the nucleocapsid as being contained within the 5′ half of both the genomic and antigenomic RNAs. This region, if placed within nonviral RNA, is capable of altering the binding potential of nonviral RNA to levels seen with wild-type vRNAs.

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