Serial Expression of the Truncated Fragments of the Nucleocapsid Protein of CCHFV and Identification of the Epitope Region

The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nucleocapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of NP was detected with a polyclonal serum(rabbit)and 2 monoclonal(mAbs)(14B7 and 43E5)against CCHFV by Western-blot analyses. The results showed that the three expressed constructs,which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum.The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.

Indirect Enzyme-Linked Immunosorbent Assay Based on the Nucleocapsid Protein of SARS-like Coronaviruses

The nucleocapsid protein(N) is a major structural protein of coronaviruses. The N protein of bat SARS-like coronavirus(SL-CoV) has a high similarity with that of SARS-CoV. In this study,the SL-CoV N protein was expressed in Escherichia coli,purified and used as antigen. An Indirect Enzyme-Linked Immunosorbent Assay(indirect ELISA) was developed for detection of SARS-or SL-CoV infections in bat populations. The detection of 573 bat sera with this indirect ELISA demonstrated that SL-CoVs consistently circulate in Rhinilophus species,further supporting the proposal that bats are natural reservoirs of SL-CoVs. This method uses 1-2 μl of serum sample and can be used for preliminary screening of infections by SARS-or SL-CoV with a small amount of serum sample.

Expression of Hantaan virus 26 kD fragment of nucleocapsid protein in insect cells and prelimimary study on its immunogenicity

Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.

Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus

The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

Characterization of Influenza H5N1 Nucleocapsid Protein for Potential Vaccine Design

Avian influenza, subtype H5N1, causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. Nucleocapsid protein (NP) is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymphocyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physico-chemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Study on the Cytotoxic T Lymphocytes Clone Specific for the Nucleocapsid Protein of Hantaan Virus from Peripheral Blood in Patients with Hemorrhagic Fever with Renal Syndrome

In order to elucidate the molecular and immunological mechanisms as well as the pathogenesis of hemorrhagic fever with renal syndrome (HFRS), the CD8 + cytotoxic T lymphocytes (CTL) clone was established directly from peripheral blood mononuclear cells (PBMC) of patients with HFRS. The activities of CTL were detected as usual with EBV-transformed lymphoblastoid cell line (BLCL) as target cells. The results showed that the CTL clone could recognized and killed the target cells with specificity of nucleocapsid protein of Hantaan virus (HTNVNP) with the cytotoxicity percentages of 50.2%, 25.4% and 39.0% respectively. These results demonstrated that the antigenic epitopes of HTNVNP mainly located on the C-terminal of the viral nucleocapsid protein.

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid protein is a potential antiviral drug target,serving multiple critical functions during the viral life cycle.However,the structural information of SARS-CoV-2 nucleocapsid protein remains unclear.Herein,we have determined the 2.7 A crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein.Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain,the surface electrostatic potential characteristics between them are distinct.Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside theβ-sheet core.Complemented by in vitro binding studies,our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain,guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

A versatile building block:the structures and functions of negative-sense single-stranded RNA virus nucleocapsid proteins

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 bio-logical functions of-ssRNA viruses that extend our understanding of the versatile roles of virally encoded NPs.

The nucleocapsid protein of rice stripe virus in cell nuclei of vector insect regulates viral replication

Rice stripe virus(RSV)transmitted by the small brown planthopper causes severe rice yield losses in Asian countries.Although viral nuclear entry promotes viral replication in host cells,whether this phenomenon occurs in vector cells remains unknown.Therefore,in this study,we systematically evaluated the presence and roles of RSV in the nuclei of vector insect cells.We observed that the nucleocapsid protein(NP)and viral genomic RNAs were partially transported into vector cell nuclei by utilizing the importin a nuclear transport system.When blocking NP nuclear localization,cytoplasmic RSV accumulation significantly increased.In the vector cell nuclei,NP bound the transcription factor YY1 and affected its positive regulation to FAIM.Subsequently,decreased FAIM expression triggered an antiviral caspase-dependent apoptotic reaction.Our results reveal that viral nuclear entry induces completely different immune effects in vector and host cells,providing new insights into the balance between viral load and the immunity pressure in vector insects.

Structures of the N- and C-terminal domains of MHV-A59 nucleocapsid protein corroborate a conserved RNA-protein binding mechanism in coronavirus

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 Nterminal 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 1.5Å.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-Åresolution 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.

Mass Spectrometry Analysis of SARS-CoV-2 Nucleocapsid Protein Reveals Camouflaging Glycans and Unique Post-Translational Modifications

The devastating coronavirus disease 2019(COVID-19)pandemic has prompted worldwide efforts to study structural biological traits of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)and its viral components.Compared to the Spike protein,which is the primary target for currently available vaccines or antibodies,knowledge about other virion structural components is incomplete.Using high-resolution mass spectrometry,we report a comprehensive post-translational modification(PTM)analysis of nucleocapsid phosphoprotein(NCP),the most abundant structural component of the SARS-CoV-2 virion.In addition to phosphoryl groups,we show that the SARS-CoV-2 NCP is decorated with a variety of PTMs,including N-glycans and ubiquitin.Based on newly identified PTMs,refined protein structural models of SARS-CoV-2 NCP were proposed and potential immune recognition epitopes of NCP were aligned with PTMs.These data can facilitate the design of novel vaccines or therapeutics targeting NCP,as valuable alternatives to the current vaccination and treatment paradigm that is under threat of the ever-mutating SARS-CoV-2 Spike protein.

Antigenicity of severe fever with thrombocytopenia syndrome virus nucleocapsid protein and its potential application in the virus serodiagnosis

Dear Editor,Severe fever with thrombocytopenia syndrome virus(SFTSV)is a newly identified viral pathogen of the genus Phlebovirus in the family Bunyaviridae(Sun et al.,2012).SFTSV was first identified from patient serum samples in China(Li et al.,2013;Ning et al.,2015).SFTSV can cause a severe hemorrhagic fever-like disease with a reported case fatality rate ranging from 2.5%

The Epitope Study on the SARS-CoV Nucleocapsid Protein

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.

Comparative Immunization in BALB/c Mice with Recombinant Replication-Defective Adenovirus Vector and DNA Plasmid Expressing a SARS-CoV Nucleocapsid Protein Gene

In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus (SARS-CoV)-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ (IFN-γ) secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific (IFN-γ) secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirus boosting could be used as a potential SARS-CoV vaccine.

The C-Terminal Portion of the Nucleocapsid Protein Demonstrates SARS-CoV Antigenicity

In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC)gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter.Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls.The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Genetic and antigenic characterization of recombinant nucleocapsid proteins derived from canine coronavirus and canine respiratory coronavirus in China

To characterize the antigenicity of nucleocapsid proteins(NP) derived from canine coronavirus(CCo V) and canine respiratory coronavirus(CRCo V) in China, the N genes of CCo V(CCo V-BJ70) and CRCo V(CRCo V-BJ202) were cloned from swabs obtained from diseased pet dogs in Beijing and then sequenced. The recombinant NPs(r NPs) were expressed in Escherichia coli and purified by nickel-affinity column and size exclusion chromatography. Sequencing data indicated that the N genes of CCo V-BJ70 and CRCo V-BJ202 belonging to two distinctly different groups were relatively conserved within each subgroup. Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) results showed that r NPs of CCo V and CRCo V were expressed efficiently and isolated with a final purity of over 95%. Western blot analysis revealed the r NP from CRCo V could cross-react with mice antisera against human coronavirus(HCo V-229 E, NL63, OC43, HKU1), while r NP of CCo V had cross-reactivity with only anti-sera against viruses belonging to the same group(HCo V-229 E and NL63). In summary, CCo V and CRCo V r NPs were successfully expressed in E. coli and showed antigenic cross-reactivity with antisera raised against human coronaviruses. These findings indicate that further serologic studies on coronavirus infections at the animal-human interface are needed.

Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 15-mer phage-displayed library

Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we cloned,expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then,the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly,all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding pep-tides,designated separately as SNA1,SNA2,SNA4,SNA5,SNA9 and SNG11,were selected for se-quencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4,SNA2,SNA9 and SNA1. In addition,the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also,the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program,and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur sub-units (UCRI or UQCR),otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely,in the 2Fe-2S redox centre of UCRI,there were 6 residues GGW(Y)F(Y)CP compatible to the residues (po-sition 2→7,GGWFCP7) of the NH2-terminal of the 15-mer peptide,which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here,the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.

稳定表达猪德尔塔冠状病毒N蛋白的Vero细胞系的建立及鉴定

为获得稳定表达猪德尔塔冠状病毒(PDCoV)核衣壳(N)蛋白的非洲绿猴肾(Vero)细胞系,本研究将PDCoV-N基因克隆入慢病毒载体中,获得重组质粒pLVX-PDCoV-N,利用慢病毒包装系统转染293T细胞,包装成表达N蛋白的慢病毒颗粒,慢病毒感染Vero细胞,嘌呤霉素加压筛选目的细胞。RT-PCR扩增N基因和测序表明细胞系基因组中存在N蛋白编码序列,Western blot和IFA试验表明N蛋白可在细胞系中稳定表达。应用制备的细胞系对临床PDCoV阳性血清样品进行检测,与ELISA检测结果符合率达到100%。本研究成功建立了稳定表达PDCoV N蛋白的Vero细胞系,为PDCoV N蛋白生物学特性研究和PDCoV的临床检测、流行病学调查奠定了基础。

牡蛎疱疹病毒结构蛋白真核表达系统构建及多聚化特性

牡蛎疱疹病毒(OsHV-1)在全球范围内导致牡蛎、扇贝与蚶类的大规模死亡,成为双壳贝类养殖产业的重要威胁。为了解OsHV-1的结构与致病机制。本研究利用人胚胎肾细胞(HEK293t),构建OsHV-1主要核衣壳蛋白(ORF104和ORF33)的真核表达系统,并对ORF104和ORF33潜在相互作用进行分析。实验首先通过特异性PCR扩增技术得到orf 104和orf 33的基因序列,根据其编码蛋白的理化性质、跨膜区与三维结构等生物信息学分析结果,选择pCDNA3.1(+)构建两种基因的重组表达质粒。重组质粒经大肠杆菌扩增、提取后,利用转染试剂Lipo8000™将pCDNA3.1(+)-orf 104与pCDNA3.1(+)-orf 33分别单独或共转染至HEK293t。然后,将转染后的细胞培养18 h后裂解收集蛋白。最后利用蛋白免疫印迹(Western blot,WB)与负染电镜检测两种目的蛋白的表达情况。结果显示,实验成功构建了OsHV-1衣壳蛋白ORF104和ORF33的重组表达质粒载体,通过真核细胞表达得到大小约为135与35 ku的目的蛋白。研究表明,表达质粒可在真核表达系统中实现蛋白单独转染与共转染,共转染蛋白间可能存在相互作用的趋势并形成多聚体,其中,ORF33自身即可形成分子质量不同的多聚体。本研究首次利用真核表达系统开展OsHV-1关键结构蛋白的表达,为进一步开展该病毒结构蛋白功能与互作,以及病毒入侵机制研究奠定基础。

基于重组核衣壳蛋白的猪德尔塔冠状病毒间接ELISA检测方法的建立

目的:建立一种关于猪德尔塔冠状病毒(Porcine Deltacoronavirus, PDCoV)抗体的检测方法。方法:对猪德尔塔冠状病毒的核衣壳蛋白(N)进行原核表达,通过Western Blot、SDS-PAGE等方法,对重组蛋白进行鉴定;在获得正确表达的重组蛋白后,以此为抗原,对ELISA反应条件进行优化,并对其特异性、敏感性、重复性进行测试,最终建立ELISA检测方法,并对临床样品进行检测。结果:优化后的抗原包被浓度为2μg/孔,37℃孵育1 h;1%BSA 37℃封闭1 h;一抗稀释最佳浓度为1∶1 600,孵育时间为1 h;酶标二抗最佳孵育时间为60 min。重组蛋白能与猪德尔塔冠状病毒血清发生特异性反应,所建立的检测方法具有优良的敏感性、特异性、重复性。结论:本研究建立了一种针对猪德尔塔冠状病毒的间接ELISA检测方法,为猪德尔塔冠状病毒的抗体水平检测及疾病的防控提供了一种有效的方法。