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Results for “Nucleocapsid”

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PubMed2001

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

Amino Acid Sequence↗

PubMed1994

Nuclear export of recombinant baculovirus nucleocapsids through small pore or nuclear-pore-like structure in Sf9 cells.

Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complete nuclear pore complexes proper.

Animals↗

PubMed1993

HLA B44-restricted cytotoxic T lymphocytes recognizing an epitope on hepatitis C virus nucleocapsid protein.

Cytotoxic T lymphocytes have been reported to be involved in the immune clearance of virus-infected cells and in the pathogenesis of viral infection. We studied the cytotoxic T lymphocyte response to the putative nucleocapsid protein of hepatitis C virus in patients with chronic hepatitis C. Cytotoxic T lymphocytes specific for hepatitis C virus nucleocapsid protein were generated from peripheral blood lymphocytes by means of repeated stimulation with a synthetic hepatitis C virus nucleocapsid protein peptide. The cytotoxic T lymphocytes were CD8 positive and recognized an epitope in hepatitis C virus nucleocapsid protein residues 81 to 100 in association with a human leukocyte antigen class I molecule, B44. The peptide-induced cytotoxic T lymphocytes recognized target cells synthesizing hepatitis C virus nucleocapsid protein endogenously, though less efficiently than peptide-pulsed target cells. The human leukocyte antigen B44-restricted cytotoxic T lymphocyte response was observed in three of five patients with chronic hepatitis C and a human leukocyte antigen B44 molecule but in neither of two hepatitis C virus-negative healthy individuals with human leukocyte antigen B44 molecules. The results demonstrate the presence of hepatitis C virus-specific cytotoxic T lymphocytes in the peripheral blood of patients with chronic hepatitis C and provide a strategy to study the role of cytotoxic T lymphocytes in the viral clearance and the pathogenesis of hepatitis C virus infection.

Antigens, Viral↗

PubMed2004

Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice.

Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particles. Here, we demonstrate that the insertion of 45 or 120 amino acid-long segments from the N-terminus of Puumala hantavirus nucleocapsid protein into sites 1 (amino acids 80-89) and 4 (amino acids 288-295) of VP1 allowed the highly efficient formation of virus-like particles. In contrast, expression level and assembly capacity of fusions to sites 2 (amino acids 222-225) and 3 (amino acids 243-247) were drastically reduced. Immunization of BALB/c mice with chimeric virus-like particles induced a high-titered antibody response against the hantavirus nucleocapsid protein, even in the absence of any adjuvant. The strongest response was observed in mice immunized with virus-like particles harboring 120 amino acids of hantavirus nucleocapsid protein. According to the immunoglobulin subclass distribution of nucleocapsid protein-specific antibodies a mixed Th1/Th2 response was detected. The VP1 carrier itself also induced a mixed Th1/Th2 response, which was found to be reduced in mice immunized with virus-like particles harboring 120 amino acid-long inserts. In conclusion, hamster polyomavirus VP1 represents a promising carrier moiety for future vaccine development.

Animals↗

PubMed2004

Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins.

We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C. Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

Binding Sites↗

PubMed2005

Evaluation of a recombinant nucleocapsid protein-based assay for anti-SARS-CoV IgG detection.

A high throughput accurate assay for anti-SARS-CoV IgG detection is needed for large-scale epidemiological studies. The evaluation of a commercial recombinant nucleocapsid protein-based microtitre plate enzyme immunoassay, ELISARS is described. The results on 150 sera from SARS patients and 450 sera from non-SARS controls showed that this assay had a high level of sensitivity (96.2% for late serum samples) and specificity (97.8%). The performance and setup of this assay fulfills the requirement as a screening test for large-scale studies. A vast majority of SARS patients developed antibodies against the nucleocapsid protein. In some patients (10/45), a high level of anti-nucleocapsid antibody appeared very early in the course of the illness. In contrast, a minority (4 of 105 patients) never developed these antibodies. The implication of differences in antibody response to the nucleocapsid protein deserves further investigation.

Adolescent↗

PubMed1990

Association of the Sendai virus C protein with nucleocapsids.

The subcellular localization of the nonstructural protein C of Sendai virus was investigated by means of indirect immunofluorescence microscopy of Sendai virus-infected cells, using an antiserum specific for C protein. In infected cells, C protein was detected exclusively in the cytoplasm as granular fluorescence, which coincided very well with the distribution of nucleocapsid protein NP and phosphoprotein P, which were also detected with specific antisera. This suggested that these proteins are present together in inclusions, probably forming nucleocapsids. In contrast, when the NP and C proteins were individually expressed in COS cells by transfection with expression plasmids containing cDNA for these proteins, their distribution patterns in the cytoplasm were found to be quite different from each other. Protein-blot analyses of purified virions revealed the presence of a significant amount of the C protein in virions, which indicated that C protein is integrated into virions. Under conditions in which most of the envelope-associated proteins, such as HN, F, and M, were removed from the virions by a detergent, the C protein remained tightly associated with the nucleocapsids--about 40 molecules per nucleocapsid.

Animals↗

PubMed2005

Nucleocapsid protein VP15 is the basic DNA binding protein of white spot syndrome virus of shrimp.

White spot syndrome virus (WSSV) is type species of the genus Whispovirus of the new family Nimaviridae. Despite the elucidation of its genomic sequence, very little is known about the virus as only 6% of its ORFs show homology to known genes. One of the structural virion proteins, VP15, is part of the nucleocapsid of the virus and shows homology to some putative baculovirus DNA binding proteins. These DNA-binding or histone-like proteins are thought to be involved in the condensation and packaging of the genome in the nucleocapsid. Using bacterially expressed VP15 fusion proteins in ELISA and Far-Western experiments showed that VP15 interacts with itself, forming homomultimers, but not with the other major structural proteins of the WSSV virion. Antibodies against phosphorylated proteins revealed that VP15 originating from different sources was not phosphorylated. WSSV VP15 binds non-specifically to double-stranded DNA, but has a clear preference to supercoiled DNA suggesting that VP15 is involved in the packaging of the WSSV genome in the nucleocapsid. This research shed further light on the composition of WSSV virions and the function of one of its nucleocapsid proteins.

Animals↗

PubMed1995

Nucleoprotein phosphorylated on both serine and threonine is preferentially assembled into the nucleocapsids of measles virus.

The nucleoprotein (N) in the nucleocapsids of measles virus (MV) has different conformation and antigenicity than the free N-protein in MV-infected cells. These two forms of N-protein have identical methionine-containing tryptic peptides. The free N-protein contains 4 phosphorylated tryptic peptides. However, the nucleocapsid-associated N-protein has an additional phosphorylated peptide not found in the free N-protein. The free N-protein is phosphorylated only on serine residues, whereas the nucleocapsid-associated N-protein is phosphorylated on both serine and threonine residues. The MV N-protein expressed from a cloned gene in primate cells is also phosphorylated on both serine and threonine residues. These results suggest that cellular kinases phosphorylate the MV N-protein, and N-protein with phosphorylated serine and threonine is preferentially assembled into the viral nucleocapsids.

Cells, Cultured↗

PubMed2003

The nucleocapsid of the hepatitis B virus: a remarkable immunogenic structure.

The hepatitis B virus nucleocapsid or core antigen is extremely immunogenic during infection and after immunization. This review summarizes several features of the nucleocapsid which explain this exceptionally high immunogenicity: a unique three-dimensional folding, the presence of a region that interacts with immunoglobulins outside the classical antibody-binding site, the presence of many CD4+ T cell epitopes, and the presence of encapsidated nucleic acids. Because of these features, nucleocapsids efficiently interact and activate antigen presenting cells, especially nai;ve B cells. This leads to the generation of a dominant Th1 immunity phenotype and the secretion of high levels of IgM and IgG anti-nucleocapsid antibodies.

Animals↗

PubMed2006

The coiled-coil domain structure of the Sin Nombre virus nucleocapsid protein.

Hantaviruses can cause hemorrhagic fever with a renal syndrome and hantavirus pulmonary syndrome when transmitted to humans. The nucleocapsid protein of hantaviruses encapsidates viral genomic RNA and associates with transcription and replication complexes. Both the amino and carboxy termini of the nucleocapsid protein had been predicted to form trimers prior to the formation of the ribonucleoprotein. Crystal structures of amino-terminal fragments of the nucleocapsid protein showed the formation of intramolecular antiparallel coiled coils, but not intermolecular trimers. Thus, the amino-terminal part of the nucleocapsid protein is probably insufficient to initiate the trimerization of the full-length molecule.

Amino Acid Sequence↗

PubMed2006

Structural analysis of viral nucleocapsids by subtraction of partial projections.

The nucleocapsid of flavivirus particles does not have a recognizable capsid structure when using icosahedral averaging for cryo-electron microscopy structure determinations. The apparent absence of a definitive capsid structure could be due to a lack of synchronization of the symmetry elements of the external glycoprotein layer with those of the core or because the nucleocapsid does not have the same structure within each particle. A technique has been developed to determine the structure of the capsid, and possibly also of the genome, for icosahedral viruses, such as flaviviruses, using cryo-electron microscopy. The method is applicable not only to the analyses of viral cores, but also to the missing structure of multi-component complexes due to symmetry mismatches. The density contributed by external glycoprotein and membrane layers, derived from previously determined three-dimensional icosahedrally averaged reconstructions, was subtracted from the raw images of the virus particles. The resultant difference images were then used for a three-dimensional reconstruction. After appropriate test data sets were constructed and tested, the procedure was applied to examine the nucleocapsids of flaviviruses, which showed that there is no distinct protein density surrounding the genome. Furthermore, there was no evidence of any icosahedral symmetry within the nucleocapsid core.

Flavivirus↗

PubMed2006

The expression of membrane protein augments the specific responses induced by SARS-CoV nucleocapsid DNA immunization.

Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20mug of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG2a subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen, The CD4+ and CD8+ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-gamma, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection.

Animals↗

PubMed2001

Molecular characterisation of the nucleocapsid protein gene, glycoprotein gene and gene junctions of rhabdovirus 903/87, a novel fish pathogenic rhabdovirus.

The sequences of the nucleocapsid and glycoprotein genes and the gene junctions of the fish pathogenic rhabdovirus 903/87 were determined from cDNA and PCR clones. The mRNA of the nucleocapsid is most likely 1492 nucleotides long and encodes a protein of 426 amino acids, whereas the mRNA of the glycoprotein is likely to be 1682 nucleotides long and the protein 517 amino acids. When the nucleocapsid and glycoprotein genes of virus 903/87 were compared at amino acid level with other rhabdoviruses they showed the highest homology with the Vesiculovirus genus. By sequencing the junctions between the N, P, M, G and L genes it was determined that transcription start and stop codons were conserved between virus 903/87 and the vesiculoviruses. Virus 903/87 has no open reading frame coding for a non-virion gene between the glycoprotein and the polymerase gene. Phylogenetic studies based on rhabdovirus nucleocapsid and glycoprotein genes suggested that virus 903/87 is related to viruses in the Vesiculovirus genus.

Amino Acid Sequence↗

PubMed2005

Endosome-to-cytosol transport of viral nucleocapsids.

During viral infection, fusion of the viral envelope with endosomal membranes and nucleocapsid release were thought to be concomitant events. We show here that for the vesicular stomatitis virus they occur sequentially, at two successive steps of the endocytic pathway. Fusion already occurs in transport intermediates between early and late endosomes, presumably releasing the nucleocapsid within the lumen of intra-endosomal vesicles, where it remains hidden. Transport to late endosomes is then required for the nucleocapsid to be delivered to the cytoplasm. This last step, which initiates infection, depends on the late endosomal lipid lysobisphosphatidic acid (LBPA) and its putative effector Alix/AIP1, and is regulated by phosphatidylinositol-3-phosphate (PtdIns3P) signalling via the PtdIns3P-binding protein Snx16. We conclude that the nucleocapsid is exported into the cytoplasm after the back-fusion of internal vesicles with the limiting membrane of late endosomes, and that this process is controlled by the phospholipids LBPA and PtdIns3P and their effectors.

Animals↗

PubMed2002

Antivirally active MxA protein sequesters La Crosse virus nucleocapsid protein into perinuclear complexes.

Bunyaviruses replicate in the cytoplasm of infected cells. New viral particles are formed by budding of nucleocapsids into the Golgi apparatus. We have previously shown that the IFN-induced human MxA protein inhibits bunyavirus replication by an unknown mechanism. Here we demonstrate that MxA binds to the nucleocapsid protein of La Crosse virus (LACV) and colocalizes with the viral protein in cytoplasmic complexes. Electron microscopy revealed that these complexes accumulated in the perinuclear area and consisted of highly ordered fibrillary structures. A similar MxA-mediated redistribution of viral nucleocapsid proteins was detected with other bunyaviruses, such as Bunyamwera virus and Rift Valley fever virus. MxA(E645R), a carboxy-terminal mutant of MxA without antiviral activity against LACV, did not lead to complex formation. Wild-type MxA, but not MxA(E645R), was able to bind to LACV nucleocapsid protein in coimmunoprecipitation assays, demonstrating the importance of the carboxy-terminal effector domain of MxA. These results illustrate an efficient mechanism of IFN action whereby an essential virus component is trapped in cytoplasmic inclusions and becomes unavailable for the generation of new virus particles.

Animals↗

PubMed2004

Detection of Puumala hantavirus antibody with ELISA using a recombinant truncated nucleocapsid protein expressed in Escherichia coli.

A truncated recombinant nucleocapsid protein (rNp118) consisting of the first 118 amino-terminal amino acids (AA) of the Puumala hantavirus (PUUV) nucleocapsid protein expressed in Escherichia coli, was evaluated for its antigenicity and reliability as serodiagnostic antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of PUUV antibodies in human sera. The PUUV nucleocapsid protein has been shown to contain several B-cell epitopes, mapped within the first 118 amino-terminal AA. This finding makes the rNp118 an interesting recombinant protein to use as serodiagnostic antigen. The sensitivity of this new PUUV-rNp118 ELISA, was compared with those of a commercially available PUUV ELISA assay and an home-made ELISA based on a recombinant whole nucleocapsid protein of PUUV. Eighty-six human serum samples clinically suspected for PUUV-induced nephropathia epidemica, and previously screened with the reference assays, were tested. The sensitivity of the new assay was compared with that of the reference assays and an excellent correlation between the assays was found. Sera found to be negative by other methods were also negative in our assay. The ELISA based on rNp118 represents an alternative and valid test for detection of antibodies to PUUV in human sera.

Antibodies, Viral↗

PubMed2006

Visualization of intracellular transport of vesicular stomatitis virus nucleocapsids in living cells.

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the viral RNA polymerase. In previous studies, we demonstrated that insertion of 19 amino acids in the hinge region of the protein had no significant effect on P protein function. In the present study, we inserted full-length enhanced green fluorescent protein (eGFP) in frame into the hinge region of P and show that the fusion protein (PeGFP) is functional in viral genome transcription and replication, albeit with reduced activity. A recombinant vesicular stomatitis virus encoding PeGFP in place of the P protein (VSV-PeGFP), which possessed reduced growth kinetics compared to the wild-type VSV, was recovered. Using the recombinant VSV-PeGFP, we show that the viral replication proteins and the de novo-synthesized RNA colocalize to sites throughout the cytoplasm, indicating that replication and transcription are not confined to any particular region of the cytoplasm. Real-time imaging of the cells infected with the eGFP-tagged virus revealed that, following synthesis, the nucleocapsids are transported toward the cell periphery via a microtubule (MT)-mediated process, and the nucleocapsids were seen to be closely associated with mitochondria. Treatment of cells with nocodazole or Colcemid, drugs known to inhibit MT polymerization, resulted in accumulation of the nucleocapsids around the nucleus and also led to inhibition of infectious-virus production. These findings are compatible with a model in which the progeny viral nucleocapsids are transported toward the cell periphery by MT and the transport may be facilitated by mitochondria.

Animals↗