Nucleocapsid protein (NPs) of negative-sense single-stranded RNA (-ssRNA) viruses function in different stages of viral replication, transcription, and maturation. Structural investigations show that -ssRNA viruses that encode NPs preliminarily serve as structural building blocks that encapsidate and protect the viral genomic RNA and mediate the interaction between genomic RNA and RNA-dependent RNA polymerase. However, recent structural results have revealed other biological functions of -ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.
Animals ; Capsid ; metabolism ; Humans ; Lassa virus ; chemistry ; physiology ; Nucleocapsid Proteins ; chemistry ; metabolism ; Orthobunyavirus ; chemistry ; physiology ; RNA Viruses ; chemistry ; physiology

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.
Ebolavirus ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Nucleocapsid ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Virus Assembly

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.
Antibodies, Monoclonal/*immunology ; Antigens, Viral/chemistry/*immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Epitopes/immunology ; Nucleocapsid Proteins/chemistry/*immunology ; Rinderpest virus/*immunology

OBJECTIVETo clarify the genetic characteristics of N protein coding region of HRSV isolates from Beijing and GenBank downloaded sequences.

METHODSReverse transciption polymerase chain reaction (RT-PCR) was performed to amplify the N protein gene of 2 A and 2 B subgroups HRSV isolates from Beijing in the year 2004. The RT-PCR products were sequenced for N protein coding region. The sequences of N protein coding region of 4 Beijing isolates and those downloaded from GenBank were compared and analyzed.

RESULTSThe differences in number of nucleotide and deduced amino acid between 2 A Beijing 2004 isolates and prototype strain Long were 36-40 (3.1%-3.4%) and 4 (1.0%). The differences in number of nucleotide and deduced amino acid between 2 B Beijing 2004 isolates and prototype strain CH18537 were 17 (1.4%) and 1 (0.3%). The differences in number of nucleotide and deduced amino acid were 3-172 (0.25%-14.63%) and 0-18 (0-4.6%) respectively between 4 Beijing 2004 isolates and GenBank sequences.

CONCLUSIONN gene is the highly conservative gene in the HRSV genome. The variation between A and B subgroups were widely distributed in the entire gene of N protein, while the variation within the A or B subgroups HRSV was considerably lower.

Humans ; Molecular Sequence Data ; Nucleocapsid Proteins ; chemistry ; genetics ; Phylogeny ; Respiratory Syncytial Virus Infections ; virology ; Respiratory Syncytial Virus, Human ; chemistry ; classification ; genetics ; isolation & purification ; Sequence Homology, Amino Acid

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 Å resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identified through mutational analysis and identified to have a significant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The findings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.
Binding Sites ; Crystallography, X-Ray ; Mutation ; Nucleocapsid Proteins ; chemistry ; genetics ; metabolism ; Phlebovirus ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Quaternary ; RNA, Viral ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

OBJECTIVETo obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.

METHODSBALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.

RESULTSAfter cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.

CONCLUSIONThe anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Female ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; isolation & purification ; SARS Virus ; chemistry ; immunology

OBJECTIVETo develop and evaluate a multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever.

METHODSThe nucleocapsid proteins (NP) of HTN, SEO, Puu MBV, Lassa, RFV and HPS viruses expressed in prokaryotic cells and purified were coupled to 7 different xMAP fluorescent microbeads. The assay was evaluated and optimized when screened against a panel of reference sera collected from HFRS patients, and compared to commonly used MacELISA Kits.

RESULTSFor detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commonly used MacELISA kit, but it could detect different antigen specific antibodies in one reaction simultaneously.

CONCLUSIONA robust, rapid and multiplex assay based on NPs could be developed via Luminex xMAP platform for laboratory diagnosis of viral hemorrhagic fever and seroepidemiological investigation.

Antibodies, Viral ; blood ; Fluorescence ; Hemorrhagic Fevers, Viral ; blood ; immunology ; virology ; Humans ; Immunoassay ; methods ; Immunoglobulin M ; blood ; Microspheres ; Nucleocapsid Proteins ; chemistry ; immunology ; Viruses ; immunology

Objective: To establish a rapid and specific quantitative real-time PCR (qPCR) method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA (SgN) in patients with COVID-19 or environmental samples. Methods: The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data (GISAID) database. The reaction conditions were optimized by using different annealing temperature, different primers and probe concentrations and the standard curve was established. Further, the specificity, sensitivity and repeatability were also assessed. The established SgN and genomic RNA (gRNA) qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients. In the specimens with both positive gRNA and positive SgN, 25 specimens were inoculated on cells. Results: The primers and probes of SgN had good specificity for SARS-CoV-2. The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×10² copies/ml, with a coefficient of variation less than 1%. The positive rate of gRNA in 372 samples was 97.04% (361/372). The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84% (7/19) and 49.42% (169/342), respectively. The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain. Among the 25 SgN positive samples, 12 samples within 5 days of sampling time were all isolated with virus; 13 samples sampled for more than 12 days had no cytopathic effect. Conclusion: A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established. The sensitivity, specificity and repeatability of this method are good.
Humans ; SARS-CoV-2/genetics* ; COVID-19/diagnosis* ; Subgenomic RNA ; Real-Time Polymerase Chain Reaction/methods* ; RNA, Viral/genetics* ; Sensitivity and Specificity ; Nucleocapsid/chemistry* ; COVID-19 Testing

Coronaviruses are the causative agent of respiratory and enteric diseases in animals and humans. One example is SARS, which caused a worldwide health threat in 2003. In coronaviruses, the structural protein N (nucleocapsid protein) associates with the viral RNA to form the filamentous nucleocapsid and plays a crucial role in genome replication and transcription. The structure of N-terminal domain of MHV N protein also implicated its specific affinity with transcriptional regulatory sequence (TRS) RNA. Here we report the crystal structures of the two proteolytically resistant N- (NTD) and C-terminal (CTD) domains of the N protein from murine hepatitis virus (MHV). The structure of NTD in two different crystal forms was solved to 1.5 Å. The higher resolution provides more detailed structural information than previous reports, showing that the NTD structure from MHV shares a similar overall and topology structure with that of SARS-CoV and IBV, but varies in its potential surface, which indicates a possible difference in RNA-binding module. The structure of CTD was solved to 2.0-Å resolution and revealed a tightly intertwined dimer. This is consistent with analytical ultracentrifugation experiments, suggesting a dimeric assembly of the N protein. The similarity between the structures of these two domains from SARS-CoV, IBV and MHV corroborates a conserved mechanism of nucleocapsid formation for coronaviruses.
Amino Acid Sequence ; Binding Sites ; Crystallography, X-Ray ; Molecular Sequence Data ; Murine hepatitis virus ; chemistry ; metabolism ; Nucleocapsid Proteins ; chemistry ; metabolism ; Phosphoproteins ; chemistry ; metabolism ; Protein Binding ; Protein Folding ; Protein Multimerization ; Protein Structure, Secondary ; Protein Structure, Tertiary ; RNA ; metabolism ; Sequence Alignment

The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this protein. A detailed screening of peptide with ELISA demonstrated that the amino sequence from Codons 371 to 407 was the epitope site at the C-terminus of the N protein. Understanding of the epitope sites could be very significant for developing an effective diagnostic approach to SARS.
Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; chemistry ; immunology ; Humans ; Nucleocapsid Proteins ; chemistry ; immunology ; Peptide Fragments ; chemical synthesis ; Plasmids ; Recombinant Proteins ; immunology ; isolation & purification ; metabolism ; SARS Virus ; genetics ; immunology ; metabolism