The nucleocapsid protein of SARS-associated coronavirus inhibits B23 phosphorylation.
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ABSTRACT:
SUBMITTER: Zeng Y
PROVIDER: S-EPMC7092856 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A novel coronavirus has been associated with a worldwide outbreak of atypical pneumonia referred to as Severe Acute Respiratory Syndrome (SARS-CoV). SARS-CoV nucleocapsid (N) protein has been cloned sequenced and expressed in Escherichia coli strain. Purified N protein was used to measure the SARS-CoV specific IgG antibodies from 16 SARS-CoV infected patients' sera and from 131 control subjects using ELISA assay. Specific antibody responses to the purified recombinant N protein after 10, 20, and 30 days of disease onset were observed in 13 of 16 (81.3%), 16 of 16 (100%) and 16 of 16 (100%) SARS patients sera, respectively. Comparison of detection results with a commercially available diagnostic kit coated with a mixture of SARS-CoV viral proteins showed 9 of 16 (56.3%), 13 of 16 (81.3%), and 15 of 16 (93.7%) positive responses, respectively. None of 131 control sera gave positive reaction in either assay. This data suggests that the N protein of SARS-CoV is immunodominant and this ELISA based test assay for detecting the SARS-CoV N antigen may hold a significant value for SARS diagnosis.
| S-EPMC7130062 | biostudies-literature
Project description:The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses."
| S-EPMC7113676 | biostudies-literature
Project description:The coronavirus nucleocapsid (N) is a multifunctional phosphoprotein that encapsidates the genomic RNA into a helical nucleocapsid within the mature virion. The protein also plays roles in viral RNA transcription and/or replication and possibly viral mRNA translation. Phosphorylation is one of the most common post-translation modifications that plays important regulatory roles in modulating protein functions. It has been speculated for sometime that phosphorylation could play an important role in regulation of coronavirus N protein functions. As a first step toward positioning to address this we have identified the amino acids that are phosphorylated on the mouse hepatitis coronavirus (MHV) A59 N protein. High performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was used to identify phosphorylated sites on the N protein from both infected cells and purified extracellular virions. A total of six phosphorylated sites (S162, S170, T177, S389, S424 and T428) were identified on the protein from infected cells. The same six sites were also phosphorylated on the extracellular mature virion N protein. This is the first identification of phosphorylated sites for a group II coronavirus N protein.
| S-EPMC2001268 | biostudies-literature
Project description:Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N-N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N-N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein-protein interaction) in N-N interaction compared to that observed in either M-M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.
| S-EPMC7111152 | biostudies-literature
Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for SARS infection. Nucleocapsid protein (NP) of SARS-CoV (SARS_NP) functions in enveloping the entire genomic RNA and interacts with viron structural proteins, thus playing important roles in the process of virus particle assembly and release. Protein-protein interaction analysis using bioinformatics tools indicated that SARS_NP may bind to human cyclophilin A (hCypA), and surface plasmon resonance (SPR) technology revealed this binding with the equilibrium dissociation constant ranging from 6 to 160nM. The probable binding sites of these two proteins were detected by modeling the three-dimensional structure of the SARS_NP-hCypA complex, from which the important interaction residue pairs between the proteins were deduced. Mutagenesis experiments were carried out for validating the binding model, whose correctness was assessed by the observed effects on the binding affinities between the proteins. The reliability of the binding sites derived by the molecular modeling was confirmed by the fact that the computationally predicted values of the relative free energies of the binding for SARS_NP (or hCypA) mutants to the wild-type hCypA (or SARS_NP) are in good agreement with the data determined by SPR. Such presently observed SARS_NP-hCypA interaction model might provide a new hint for facilitating the understanding of another possible SARS-CoV infection pathway against human cell.
| S-EPMC7092810 | biostudies-literature
Project description:The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5alpha and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8(+) CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS.
| S-EPMC7111813 | biostudies-literature
Project description:Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, and, as of yet, none of the currently available broad-spectrum drugs or vaccines can effectively control these diseases. Host antiviral proteins play an important role in inhibiting viral proliferation. One of the isoforms of cytoplasmic poly(A)-binding protein (PABP), PABPC4, is an RNA-processing protein, which plays an important role in promoting gene expression by enhancing translation and mRNA stability. However, its function in viruses remains poorly understood. Here, we report that the host protein, PABPC4, could be regulated by transcription factor SP1 and broadly inhibits the replication of CoVs, covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. PABPC4 recruited the E3 ubiquitin ligase MARCH8/MARCHF8 to the N protein for ubiquitination. Ubiquitinated N protein was recognized by the cargo receptor NDP52/CALCOCO2, which delivered it to the autolysosomes for degradation, resulting in impaired viral proliferation. In addition to regulating gene expression, these data demonstrate a novel antiviral function of PABPC4, which broadly suppresses CoVs by degrading the N protein via the selective autophagy pathway. This study will shed light on the development of broad anticoronaviral therapies. IMPORTANCE Emerging coronaviruses (CoVs) can cause severe diseases in humans and animals, but none of the currently available drugs or vaccines can effectively control these diseases. During viral infection, the host will activate the interferon (IFN) signaling pathways and host restriction factors in maintaining the innate antiviral responses and suppressing viral replication. This study demonstrated that the host protein, PABPC4, interacts with the nucleocapsid (N) proteins from eight CoVs covering four genera (Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus) of the Coronaviridae family. PABPC4 could be regulated by SP1 and broadly inhibits the replication of CoVs by targeting the nucleocapsid (N) protein through the autophagosomes for degradation. This study significantly increases our understanding of the novel host restriction factor PABPC4 against CoV replication and will help develop novel antiviral strategies.
| S-EPMC8510267 | biostudies-literature
Project description:For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another.
| S-EPMC289081 | biostudies-other
Project description:In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia and subsequently proven to be the causative agent of the disease now referred to as severe acute respiratory syndrome (SARS). The complete genome of the SARS coronavirus (SARS-CoV) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) shares little homology with other members of the coronavirus family. To determine if the N protein is involved in the regulation of cellular signal transduction, an ELISA-based assay on transcription factors was used. We found that the amount of transcription factors binding to promoter sequences of c-Fos, ATF2, CREB-1, and FosB was increased by the expression of SARS-CoV N. Since these factors are related to AP-1 signal transduction pathway, we investigated whether the AP-1 pathway was activated by SARS-CoV N protein using the PathDetect system. The results demonstrated that the expression of N protein, not the membrane protein (M), activated AP-1 pathway. We also found that SARS-CoV N protein does not activate NF-kappaB pathway, demonstrating that activation of important cellular pathways by SAS-CoV N protein is selective. Thus our data for the first time indicate that SARS-CoV has encoded a strategy to regulate cellular signaling process.
| S-EPMC7111052 | biostudies-literature
Project description:Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate. Specific anti-N scFvs were isolated by panning against a recombinant nucleocapsid protein and reactivity was confirmed with phage-ELISA. Sequence analysis indicated that two of the isolated anti-N scFv clones were identical and displayed a high homology with an scFv specific for interleukin 11 (IL-11), an anti-inflammatory cytokine derived from bone marrow stroma cells. In a neutralization assay, IL-11-induced STAT 3 phosphorylation in rat intestinal epithelial IEC-18 cells was completely suppressed by the anti-N scFv clone L9N01.
| S-EPMC7092895 | biostudies-literature