The SARS-CoV-2 nucleocapsid protein preferentially binds long and structured RNAs

The SARS-CoV-2 nucleocapsid protein (NCAP) functions in viral RNA genome packaging, virion assembly, RNA synthesis and translation, and regulation of host immune response. RNA-binding is central to these processes. Little is known how NCAP selects its binding partners in the myriad of host and viral RNAs. To address this fundamental question, we employed electrophoresis mobility shift and competition assays to compare NCAP binding to RNAs that are of SARS-CoV-2 vs. non-SARS-CoV-2, long vs. short, and structured vs. unstructured. We found that although NCAP can bind all RNAs tested, it primarily binds structured RNAs, and their association suppresses strong interaction with single-stranded RNAs. NCAP prefers long RNAs, especially those containing multiple structures separated by single-stranded linkers that presumably offer conformational flexibility. Additionally, all three major regions of NCAP bind RNA, including the low complexity domain and dimerization domain that promote formation of NCAP oligomers, amyloid fibrils and liquid-liquid phase separation. Combining these observations, we propose that NCAP-NCAP interactions that mediate higher-order structures during packaging also drive recognition of the genomic RNA and call this mechanism recognition-by-packaging. This study provides a biochemical basis for understanding the complex NCAP-RNA interactions in the viral life cycle and a broad range of similar biological processes.

Download Full-text

Structural Basis for SARS-CoV-2 Nucleocapsid Protein Recognition by Single-Domain Antibodies

Frontiers in Immunology ◽

10.3389/fimmu.2021.719037 ◽

2021 ◽

Vol 12 ◽

Author(s):

Qiaozhen Ye ◽

Shan Lu ◽

Kevin D. Corbett

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Binding Domain ◽

Single Domain ◽

Structural Basis ◽

Dimerization Domain ◽

Health Event ◽

Single Domain Antibodies ◽

Rna Binding Domain ◽

Antigen Tests

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.

Download Full-text

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.

Download Full-text

Recombinant SARS-CoV-2 Nucleocapsid Protein: Expression, Purification, and Its Biochemical Characterization and Utility in Serological Assay Development to Assess Immunological Responses to SARS-CoV-2 Infection

Pathogens ◽

10.3390/pathogens10081039 ◽

2021 ◽

Vol 10 (8) ◽

pp. 1039

Author(s):

Da Di ◽

Mythili Dileepan ◽

Shamim Ahmed ◽

Yuying Liang ◽

Hinh Ly

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Biochemical Characterization ◽

Free Form ◽

Bacterial Cells ◽

Serum Samples ◽

Mobility Shift ◽

Independent Manner ◽

Serological Assay ◽

Terminal Domain

The SARS-CoV-2 nucleocapsid protein (N) binds a single-stranded viral RNA genome to form a helical ribonucleoprotein complex that is packaged into virion particles. N is relatively conserved among coronaviruses and consists of the N-terminal domain (NTD) and C-terminal domain (CTD), which are flanked by three disorganized regions. N is highly immunogenic and has been widely used to develop a serological assay as a diagnostic tool for COVID-19 infection, although there is a concern that the natural propensity of N to associate with RNA might compromise the assay’s specificity. We expressed and purified from bacterial cells two recombinant forms of SARS-CoV-2 N, one from the soluble fraction of bacterial cell lysates that is strongly associated with bacterial RNAs and the other that is completely devoid of RNAs. We showed that both forms of N can be used to develop enzyme-linked immunosorbent assays (ELISAs) for the specific detection of human and mouse anti-N monoclonal antibodies (mAb) as well as feline SARS-CoV-2 seropositive serum samples, but that the RNA-free form of N exhibits a slightly higher level of sensitivity than the RNA-bound form to react to anti-N mouse mAb. Using the electrophoretic mobility shift assay (EMSA), we also showed that N preferentially binds ssRNA in a sequence-independent manner and that both NTD and CTD of N contribute to RNA-binding activity. Collectively, our study describes methods to express, purify, and biochemically characterize the SARS-CoV-2 N protein and to use it for the development of serological assays to detect SARS-CoV-2 infection.

Download Full-text

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.

Download Full-text

Structural basis for SARS-CoV-2 Nucleocapsid protein recognition by single-domain antibodies

10.1101/2021.06.01.446591 ◽

2021 ◽

Author(s):

Qiaozhen Ye ◽

Shan Lu ◽

Kevin D Corbett

Keyword(s):

Nucleocapsid Protein ◽

Rna Binding ◽

Binding Domain ◽

Single Domain ◽

Structural Basis ◽

Dimerization Domain ◽

Health Event ◽

Single Domain Antibodies ◽

Rna Binding Domain ◽

Antigen Tests

The COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, is the most severe public health event of the twenty-first century. While effective vaccines against SARS-CoV-2 have been developed, there remains an urgent need for diagnostics to quickly and accurately detect infections. Antigen tests, particularly those that detect the abundant SARS-CoV-2 Nucleocapsid protein, are a proven method for detecting active SARS-CoV-2 infections. Here we report high-resolution crystal structures of three llama-derived single-domain antibodies that bind the SARS-CoV-2 Nucleocapsid protein with high affinity. Each antibody recognizes a specific folded domain of the protein, with two antibodies recognizing the N-terminal RNA binding domain and one recognizing the C-terminal dimerization domain. The two antibodies that recognize the RNA binding domain affect both RNA binding affinity and RNA-mediated phase separation of the Nucleocapsid protein. All three antibodies recognize highly-conserved surfaces on the Nucleocapsid protein, suggesting that they could be used to develop affordable diagnostic tests to detect all circulating SARS-CoV-2 variants.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.74.21.9946-9952.2000 ◽

2000 ◽

Vol 74 (21) ◽

pp. 9946-9952 ◽

Cited By ~ 71

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.

Download Full-text

Potential coupling between SARS-CoV-2 replicative fitness and interactions of its nucleoprotein with human 14-3-3 proteins

10.1101/2021.12.23.474009 ◽

2021 ◽

Author(s):

Kristina V Tugaeva ◽

Andrey A. Sysoev ◽

Jake L. R. Smith ◽

Richard B Cooley ◽

Alfred A. Antson ◽

...

Keyword(s):

Fluorescence Anisotropy ◽

Nucleocapsid Protein ◽

Structural Basis ◽

Genome Packaging ◽

Rich Region ◽

Virion Assembly ◽

Replicative Fitness ◽

Multisite Phosphorylation ◽

Human Proteins

The SARS-CoV-2 nucleocapsid protein (N) is responsible for viral genome packaging and virion assembly. Being highly abundant in the host cell, N interacts with numerous human proteins and undergoes multisite phosphorylation in vivo. When phosphorylated within its Ser/Arg-rich region, a tract highly prone to mutations as exemplified in the Omicron and Delta variants, N recruits human 14-3-3 proteins, potentially hijacking their functions. Here, we show that in addition to phosphorylated Ser197, an alternative, less conserved phosphosite at Thr205 not found in SARS-CoV N binds 14-3-3 with micromolar affinity and is in fact preferred over pS197. Fluorescence anisotropy reveals a distinctive pT205/pS197 binding selectivity towards the seven human 14-3-3 isoforms. Crystal structures explain the structural basis for the discovered selectivity towards SARS-CoV-2 N phosphopeptides, and also enable prediction for how N variants interact with 14-3-3, suggesting a link between the strength of this interaction and replicative fitness of emerging coronavirus variants.

Download Full-text

The SARS-CoV-2 Nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein

10.1101/2020.07.30.228023 ◽

2020 ◽

Cited By ~ 1

Author(s):

Shan Lu ◽

Qiaozhen Ye ◽

Digvijay Singh ◽

Elizabeth Villa ◽

Don W. Cleveland ◽

...

Keyword(s):

Phase Separation ◽

Rna Binding ◽

M Protein ◽

Rna Packaging ◽

Dimerization Domain ◽

Virion Assembly ◽

N Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

C Terminus

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that a centrally located 40 amino acid intrinsically disordered domain drives phase separation of N protein when bound to RNA, with the morphology of the resulting condensates affected by inclusion in the RNA of the putative SARS-CoV-2 packaging signal. The SARS-CoV-2 M protein, normally embedded in the virion membrane with its C-terminus extending into the virion core, independently induces N protein phase separation that is dependent on the N protein's C-terminal dimerization domain and disordered region. Three-component mixtures of N+M+RNA form condensates with mutually exclusive compartments containing N+M or N+RNA, including spherical annular structures in which the M protein coats the outside of an N+RNA condensate. These findings support a model in which phase separation of the N protein with both the viral genomic RNA and the SARS-CoV-2 M protein facilitates RNA packaging and virion assembly.

Download Full-text