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At least 19 recordsLinked to original sources

PubMed1998

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486-497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 A long and thin molecule that is spaced at around 34 A along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.

Animals↗

PubMed1993

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.

Animals↗

PubMed1993

Structural studies of the enveloped dsRNA bacteriophage phi 6 of Pseudomonas syringae by Raman spectroscopy. II. Nucleocapsid structure and thermostability of the virion, nucleocapsid and polymerase complex.

Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage phi 6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy. The spectra show that proteins of the phi 6 virion are collectively deficient in beta-sheet secondary structure. In particular, the major protein (P8) of the outer spherical shell of the phi 6 nucleocapsid exhibits a secondary structure dominated largely by alpha-helix and irregular conformations. The absence of appreciable beta-structure in the P8 subunit suggests a tertiary conformation lacking the beta-barrel motif common to subunits of most other spherical viral capsids. In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly alpha-helical. The results thus indicate a largely alpha-helical secondary structure for the major subunit (P1) of the phi 6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell. Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged phi 6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core. Also, we find that proteins of the phi 6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993). The present results suggest that both the architectural principles and modes of protein-RNA interaction in the phi 6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses. Both Raman and circular dichroism spectra indicate that the dsRNA genome of phi 6 is an A-form structure. The Raman marker bands signify the presence only of C3'-endo/anti nucleoside conformers. The Raman signature of dsRNA, revealed in the spectrum of the phi 6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

Bacteriophage phi 6↗

PubMed2006

X-ray structures of the N- and C-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation.

Coronaviruses cause a variety of respiratory and enteric diseases in animals and humans including severe acute respiratory syndrome. In these enveloped viruses, the filamentous nucleocapsid is formed by the association of nucleocapsid (N) protein with single-stranded viral RNA. The N protein is a highly immunogenic phosphoprotein also implicated in viral genome replication and in modulating cell signaling pathways. We describe the structure of the two proteolytically resistant domains of the N protein from infectious bronchitis virus (IBV), a prototype coronavirus. These domains are located at its N- and C-terminal ends (NTD and CTD, respectively). The NTD of the IBV Gray strain at 1.3-A resolution exhibits a U-shaped structure, with two arms rich in basic residues, providing a module for specific interaction with RNA. The CTD forms a tightly intertwined dimer with an intermolecular four-stranded central beta-sheet platform flanked by alpha helices, indicating that the basic building block for coronavirus nucleocapsid formation is a dimeric assembly of N protein. The variety of quaternary arrangements of the NTD and CTD revealed by the analysis of the different crystal forms delineates possible interfaces that could be used for the formation of a flexible filamentous ribonucleocapsid. The striking similarity between the dimeric structure of CTD and the nucleocapsid-forming domain of a distantly related arterivirus indicates a conserved mechanism of nucleocapsid formation for these two viral families.

Animals↗

PubMed2001

Identification of a cytotoxic T-cell epitope on the recombinant nucleocapsid proteins of Rinderpest and Peste des petits ruminants viruses presented as assembled nucleocapsids.

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.

Amino Acid Motifs↗

PubMed1994

The carboxy-terminal domain of Sendai virus nucleocapsid protein is involved in complex formation between phosphoprotein and nucleocapsid-like particles.

The replicative unit of Sendai virus, the nucleocapsid, is composed of the viral genomic RNA and the viral proteins NP, P, and L. P and L proteins form a polymerase complex and are associated with the nucleocapsid core (NP/RNA complex). The NP protein has recently been shown to be composed of two domains. While the amino-terminal domain I encapsidates RNA and gives rise to the characteristic helical nucleocapsid structure, the precise function of the carboxy-terminal domain II remained unclear. It was however supposed to be involved in binding the polymerase complex. To test this hypothesis, we established a cosedimentation assay which detects complex formation between P protein and nucleocapsid-like (NC-like) particles. Four mutant NC-like particles all carrying amino acid deletions in domain II of the NP protein were tested. Complex formation was abolished after deletion of amino acids 426-497 or 456-524, while deletions of amino acids 400-415 or 414-439 had no influence on the interaction. The results indicate that domain II of NP protein is involved in binding P protein to nucleocapsids. The function and position of a putative P protein binding site in domain II are discussed.

Animals↗

PubMed1990

Antigenic cross-reaction between a varicella-zoster virus nucleocapsid protein encoded by gene 40 and a herpes simplex virus nucleocapsid protein.

Human sera from varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) seropositive individuals contain antibody to a 155-kilodalton (155 kDa) viral protein. In this study, we show that monoclonal antibodies (mAb10.1 and mAb1A1.4) prepared against VZV and HSV-1 proteins, respectively, reacted with nuclear antigens and recognized a 155 kDa protein in the infected cells. Immunoprecipitation of whole virions and viral nucleocapsids with these mAbs showed that the 155 kDa protein is located in VZV and HSV-1 nucleocapsids. In addition, immunofluorescence and cross-reaction experiments revealed the antigenic cross-reactivity between the VZV and HSV-1 155 kDa nucleocapsid proteins. To map the coding region of the VZV 155 kDa protein, a truncated DNA fragment from the predicted open reading frame 40 was cloned into an in vitro transcription vector (pGEM). The RNA transcribed from the inserted DNA was translated in vitro and immunoprecipitated with mAb10.1. The reactivity of the in vitro translation products with mAb10.1 indicated that the 155 kDa nucleocapsid protein is encoded by VZV gene 40. These findings demonstrated that the VZV 155 kDa nucleocapsid protein encoded by gene 40 induces humoral response which cross-reacts with both VZV and HSV.

Antibodies, Monoclonal↗

PubMed1989

Form-determining functions in Sindbis virus nucleocapsids: nucleosomelike organization of the nucleocapsid.

Purified intact Sindbis virus nucleocapsids were treated at different pH values or with various concentrations of divalent cations, cation chelators, salt, or formamide. The resulting structures were examined by velocity sedimentation, electron microscopy, and protein-protein cross-linking. Changes in each of the test conditions led to alterations in the sedimentation profile of treated nucleocapsids. Appropriate concentrations of formamide or divalent cations generated beaded strandlike structures similar in morphology to those generated from adenovirus cores and nucleosomes. The capsid protein and RNA remained associated with each other at NaCl concentrations less than or equal to 1 M or after treatment of the structures with alkaline pH up to and including pH 10.7. Protein and RNA were dissociated by salt concentrations of greater than 1 M, suggesting that the arginine-rich, amino-terminal portion of the capsid protein is responsible for binding the RNA. Protein-protein cross-linking also indicated that the capsid proteins remained associated in small aggregates under some of the conditions that caused dissociation of the nucleocapsid and suggested the presence of more than one type of protein-protein interaction in the nucleocapsids. Collectively, these data suggest that, like histones and adenovirus core proteins, the Sindbis virus capsid protein serves to package segments of the genome into nucleoprotein beads which are capable of interacting with each other to form the nucleocapsid structure.

Capsid↗

PubMed1993

Measles virus nucleocapsid protein expressed in insect cells assembles into nucleocapsid-like structures.

The gene encoding the major nucleocapsid, N, protein of measles virus has been inserted into a baculovirus vector under the control of the polyhedrin promoter. Insect cells infected with this recombinant baculovirus synthesize high levels of measles N protein, up to 40% of total soluble cell protein. The recombinant protein is recognized by sera from convalescent patients, vaccinees and patients with subacute sclerosing panencephalitis and thus could form the basis of a simple diagnostic assay. Nucleocapsid-like structures, similar to those found in mammalian cells infected with measles virus, can be observed in both the nucleus and cytoplasm of the infected insect cells. These have many structural features in common with nucleocapsids found in measles virus-infected cells, but are longer (up to 2 microns) and have a lower buoyant density. Measles N protein thus appears to be capable of assembling into nucleocapsid-like structures in the absence of measles virion RNA or other viral proteins.

Animals↗

PubMed2006

Transmission electron microscopy reveals an optimal HIV-1 nucleocapsid aggregation with single-stranded nucleic acids and the mature HIV-1 nucleocapsid protein.

HIV-1 nucleocapsid protein (NCp7) condenses the viral RNA within the mature capsid. In a capsid-free system, NCp7 promotes an efficient mechanism of aggregation with both RNA and DNA. Here, we show an analysis of these macromolecular complexes by dark-field imaging using transmission electron microscopy. Thousands of mature NCp7 proteins co-aggregate with hundreds of single-stranded circular DNA molecules (ssDNA) within minutes, as observed with poly(rA). These co-aggregates are highly stable but dynamic structures, as they dissociate under harsh conditions, and after addition of potent ssDNA or NCp7 competitive ligands. The N-terminal domain and zinc fingers of NCp7 are both required for efficient association. Addition of magnesium slightly increases the avidity of NCp7 for ssDNA, while it strongly inhibits co-aggregation with relaxed circular double-stranded DNA (dsDNA). This DNA selectivity is restricted to mature NCp7, compared to its precursors NCp15 and NCp9. Moreover, for NCp15, the linkage of NCp7 with the Gag C-terminal p6-peptide provokes a deficiency in ssDNA aggregation, but results in DNA spreading similar to prototypical SSB proteins. Finally, this co-aggregation is discussed in a dynamic architectural context with regard to the mature HIV-1 nucleocapsid. On the basis of the present data, we propose that condensation of encapsidated RNA requires the C-terminal processing of NCp. Subsequently, disassembly of the nucleocapsid should be favoured once dsDNA is produced by HIV-1 reverse transcriptase.

Capsid Proteins↗

PubMed2003

Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein.

Bacterially expressed nucleocapsid (N) protein, from respiratory syncytial virus (RSV), was used to investigate RNA binding in a modified North-Western blotting protocol. The recombinant protein demonstrated no sequence specificity in binding RNA representing either the antigenomic leader sequence or the nonspecific sequence derived from a plasmid vector. When recombinant N was purified on CsCl gradients, two types of structure, both with densities indicating that they contained RNA, could be visualised by negative-stain electron microscopy. Structures similar to nucleocapsids (NC) from RSV-infected cells were observed, as were ring structures. A small fragment of the N (amino acids 1-92) was all that was required for the production of NC-like structures. Another mutant with an internal deletion could form rings but not NC-like structures. This suggests that this domain (amino acids 121-160) may be important for maintaining helical stability. Further analysis has also identified a potential site in the amino-terminus that may be involved in an interaction with the phosphoprotein. A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly.

Animals↗

PubMed1997

Assembly of recombinant Newcastle disease virus nucleocapsid protein into nucleocapsid-like structures is inhibited by the phosphoprotein.

A recombinant baculovirus expressing the nucleocapsid gene (NP) of Newcastle disease virus (NDV), a member of the genus Rubulavirus, has been generated and shown to express the native protein to high levels in insect cells. In contrast to the NP protein of the rubulavirus human parainfluenza virus 2, the NDV protein has been demonstrated by electron microscopy and caesium chloride gradient analysis to be capable of self-assembly in vivo to form nucleocapsid-like structures in the absence of other NDV proteins. These structures, which contained RNA that was resistant to micrococcal nuclease digestion, were also observed when the protein was expressed in E. coli, a phenomenon which was not inhibited by the presence of a 40 amino acid fusion region at the amino terminus of the protein. Further, the formation of these structures was inhibited by the co-expression of the phosphoprotein (P). Therefore, we conclude that the P protein acts as a chaperone, preventing uncontrolled encapsidation of non-viral RNA by NP protein.

Animals↗

PubMed1999

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

Amino Acid Sequence↗

PubMed2006

Nucleocapsid amino acids 211 to 254, in particular, tetrad glutamines, are essential for the interaction between the nucleocapsid and membrane proteins of SARS-associated coronavirus.

GST pull-down assays were used to characterize the SARS-CoV membrane (M) and nucleocapsid (N) interaction, and it was found that the amino acids 211-254 of N protein were essential for this interaction. When tetrad glutamines (Q) were replaced with glutamic acids (E) at positions of 240-243 of the N protein, the interaction was disrupted.

Amino Acid Substitution↗

PubMed1993

Immunization with recombinant woodchuck hepatitis virus nucleocapsid antigen or hepatitis B virus nucleocapsid antigen protects woodchucks from woodchuck hepatitis virus infection.

Woodchucks were immunized with recombinant woodchuck hepatitis virus (WHV) nucleocapsid antigen (WHcAg) or hepatitis B virus (HBV) nucleocapsid antigen (HBcAg) and challenged with 10(6) WHV ID50. Six out of six woodchucks immunized with WHcAg and four out of six immunized with HBcAg were protected from WHV infection. Woodchucks immunized with WHcAg or HBcAg developed high serum antibody titres against WHcAg or HBcAg. Antibodies against WHc and HBc displayed little cross-reactivity (< 1%). This confirms and extends previous reports of protection against homologous challenge after immunization with HBcAg/WHcAg which are both internal viral antigens. As the dominant B-cell epitope(s) on particulate WHcAg and HBcAg appear not to be conserved it also demonstrates that antibodies against HBcAg/WHcAg are not important for this protection. Woodchucks immunized with WHcAg/HBcAg reacted with a fast serum antibody response against viral envelope proteins upon challenge with WHV, indicative of functional intrastructural/intermolecular T-cell help as one potential mechanism of protection after immunization with an internal viral antigen.

Animals↗

PubMed1985

cDNA cloning of the nucleocapsid and nucleocapsid-associated protein genes of mumps virus.

cDNA clones of the mumps virus N and P messenger RNAs were isolated from an infected cell cDNA library. The N and P clones selected the two predominant polyadenylated RNAs found in mumps virus-infected cells with mol. wt. of 0.69 X 10(6) and 0.51 X 10(6), respectively. In addition, clones of the P gene hybridized to and selected mRNAs of higher mol. wt. probably representing polycistronic transcripts of the mumps genome. Hybrid-select translation experiments confirmed the specificity of the clones as representing the nucleocapsid (N) and nucleocapsid-associated protein (P) genes.

Capsid↗

PubMed1991

Sequence of the major nucleocapsid protein gene of pneumonia virus of mice: sequence comparisons suggest structural homology between nucleocapsid proteins of pneumoviruses, paramyxoviruses, rhabdoviruses and filoviruses.

The complete nucleotide sequence of gene 3 of pneumonia virus of mice has been determined, and the 5' end of the mRNA mapped using a modification of the polymerase chain reaction technique. The gene contains a single open reading frame, beginning with a 5'-proximal AUG initiation codon, encoding a polypeptide with a predicted Mr of 43141. Expression of the gene 3 protein in Escherichia coli and in vitro showed that it reacted with virus-specific antiserum and comigrated with the major nucleocapsid (N) polypeptide. The predicted amino acid sequence has extensive identity with that of the N protein of human respiratory syncytial virus. Comparisons with the amino acid sequences of N proteins of other paramyxoviruses, vesicular stomatitis virus and Ebola virus suggest that these proteins may have retained much of the same structure. These regions of conserved structure would most likely have the common functions of RNA binding and protein/protein interactions in the virus nucleocapsid.

Amino Acid Sequence↗

PubMed1999

Strict conservation of the retroviral nucleocapsid protein zinc finger is strongly influenced by its role in viral infection processes: characterization of HIV-1 particles containing mutant nucleocapsid zinc-coordinating sequences.

The retroviral nucleocapsid (NC) protein contains highly conserved amino acid sequences (-Cys-X2-Cys-X4-His-X4-Cys-) designated retroviral (CCHC) Zn2+ fingers. The NC protein of murine leukemia viruses contains one NC Zn2+ finger and mutants that were competent in metal binding (CCCC and CCHH) packaged wild-type levels of full-length viral RNA but were not infectious. These studies were extended to human immunodeficiency virus type 1 (HIV-1), a virus with two NC Zn2+ fingers. Viruses with combinations of CCHC, CCCC, and CCHH Zn2+ fingers in each position of HIV-1 NC were characterized. Mutant particles contained the normal complement of processed viral proteins. Four mutants packaged roughly wild-type levels of genomic RNA, whereas the remaining mutants packaged reduced levels. Virions with mutated C-terminal position NC fingers were replication competent. One interesting mutant, containing a CCCC Zn2+ finger in the N-terminal position of NC, packaged wild-type levels of viral RNA and showed approximately 5% wild-type levels of infectivity when examined in CD4-expressing HeLa cells containing an HIV-1 LTR/beta-galactosidase construct. However, this particular mutant was replication defective in H9 cells; all other mutants were replication defective over the 8-week course of the assay. Two long terminal repeat viral DNA species could be detected in the CCCC mutant but not in any of the other replication-defective mutants. These studies show that the N-terminal Zn2+ finger position is more sensitive to alterations than the C-terminal position with respect to replication. Additionally, the retroviral (CCHC) NC Zn2+ finger is required for early infection processes. The evolutionary pressure to maintain CCHC NC Zn2+ fingers depends mainly on its function in infection processes, in addition to its function in genome packaging.

Amino Acid Sequence↗