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.

scholarly journals Coronavirus Nucleocapsid Protein Facilitates Template Switching and Is Required for Efficient Transcription

2009 ◽  
Vol 84 (4) ◽  
pp. 2169-2175 ◽  
Author(s):  
Sonia Zúñiga ◽  
Jazmina L. G. Cruz ◽  
Isabel Sola ◽  
Pedro A. Mateos-Gómez ◽  
Lorena Palacio ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Chaperone Activity ◽  
Protein Domain ◽  
Transmissible Gastroenteritis Virus ◽  
Deletion Mutants ◽  
Template Switching ◽  
Rna Chaperone ◽  
N Protein

ABSTRACT Purified nucleocapsid protein (N protein) from transmissible gastroenteritis virus (TGEV) enhanced hammerhead ribozyme self-cleavage and favored nucleic acid annealing, properties that define RNA chaperones, as previously reported. Several TGEV N-protein deletion mutants were expressed in Escherichia coli and purified, and their RNA binding ability and RNA chaperone activity were evaluated. The smallest N-protein domain analyzed with RNA chaperone activity, facilitating DNA and RNA annealing, contained the central unstructured region (amino acids 117 to 268). Interestingly, N protein and its deletion mutants with RNA chaperone activity enhanced template switching in a retrovirus-derived heterologous system, reinforcing the concept that TGEV N protein is an RNA chaperone that could be involved in template switching. This result is in agreement with the observation that in vivo, N protein is not necessary for TGEV replication, but it is required for efficient transcription.

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

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.

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.

Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein

1991 ◽  
Vol 11 (6) ◽  
pp. 3075-3087
Author(s):  
L Minvielle-Sebastia ◽  
B Winsor ◽  
N Bonneaud ◽  
F Lacroute
Keyword(s):  
Molecular Weight ◽  
Sequence Analysis ◽  
Decay Rate ◽  
Rna Binding ◽  
Binding Domain ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Rna Binding Domain ◽  
Temperature Sensitive Phenotype

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other.

scholarly journals Functional Replacement of a Domain in the Rubella Virus P150 Replicase Protein by the Virus Capsid Protein

2009 ◽  
Vol 83 (8) ◽  
pp. 3549-3555 ◽  
Author(s):  
Wen-Pin Tzeng ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Rna Binding ◽  
Signal Sequence ◽  
Deletion Mutants ◽  
Replicase Gene ◽  
Wild Type ◽  
C Protein ◽  
Virus Capsid Protein ◽  
Functional Replacement ◽  
Arginine Rich Motif

ABSTRACT The rubella virus (RUBV) capsid (C) protein rescues mutants with a lethal deletion between two in-frame NotI sites in the P150 replicase gene, a deletion encompassing nucleotides 1685 to 2192 of the RUBV genome and amino acids (aa) 548 to 717 of P150 (which has a total length of 1,301 aa). The complete domain rescuable by the C protein was mapped to aa 497 to 803 of P150. Introduction of aa 1 to 277 of the C protein (lacking the C-terminal E2 signal sequence) between the NotI sites in the P150 gene in a replicon construct yielded a viable construct that synthesized viral RNA with wild-type kinetics, indicating that C and this region of P150 share a common function. Further genetic analysis revealed that an arginine-rich motif between aa 60 and 68 of the C protein was necessary for the rescue of ΔNotI deletion mutants and substituted for an arginine-rich motif between aa 731 and 735 of the P150 protein when the C protein was introduced into P150. Possible common functions shared by these arginine-rich motifs include RNA binding and interaction with cell proteins.

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 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.

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