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PubMed2006

Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization.

Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.

Amino Acid Sequence↗

PubMed1997

Identification of a specific interaction between the coronavirus mouse hepatitis virus A59 nucleocapsid protein and packaging signal.

The coronavirus mouse hepatitis virus (MHV) is an enveloped positive stranded RNA virus. In infected cells MHV produces a 3' coterminal nested set of subgenomic messenger RNAs. Only the genomic RNA, however, is encapsidated by the nucleocapsid protein and incorporated in infectious MHV virions. It is believed that an RNA packaging signal (Ps), present only in the genomic RNA, is responsible for this selectivity. Earlier studies mapped this signal to a 69-nt stem-loop structure positioned in the 3' end of ORF1b. The selective encapsidation mechanism probably initiates by specific interaction of the packaging signal with the nucleocapsid protein. In this study we demonstrate the in vitro interaction of the MHV-A59 nucleocapsid protein with the packaging signal of MHV using gel retardation and UV cross-linking assays. This interaction was observed not only with the nucleocapsid protein from infected cells but also with that from purified virions and from cells expressing a recombinant nucleocapsid protein. The specificity of the interaction was demonstrated by competition experiments with nonlabeled Ps containing RNAs, tRNA, and total cytoplasmic RNA. The results indicated that no virus specific modification of the N-protein or the presence of other viral proteins are required for this in vitro intervention. The assays described in this report provide us with a powerful tool for studying encapsidation (initiation) in more detail.

Animals↗

PubMed1998

The role of nucleocapsid of HIV-1 in virus assembly.

The role of the nucleocapsid protein of HIV-1 Gag in virus assembly was investigated using Gag truncation mutants, a nucleocapsid deletion mutant, and point mutations in the nucleocapsid region of Gag, in transfected COS cells, and in stable T-cell lines. Consistent with previous investigations, a truncation containing only the matrix and capsid regions of Gag was unable to assemble efficiently into particles; also, the pelletable material released was lighter than the density of wild-type HIV-1. A deletion mutant lacking p7 nucleocapsid but containing the C-terminal p6 protein was also inefficient in particle release and released lighter particles, while a truncation containing only the first zinc finger of p7 could assemble more efficiently into virions. These results clearly show that p7 is indispensable for virus assembly and release. Some point mutations in the N-terminal basic domain and in the basic linker region between the two zinc fingers, which had been previously shown to have reduced RNA binding in vitro [Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. (1996). J. Virol. 70: 771-777], were shown to reduce virus assembly dramatically when expressed in full-length viral clones. A fusion protein consisting of matrix and capsid fused to a heterologous viral protein known to have nonspecific RNA binding activity [Ribas, J. C., Fujimura, T., and Wickner, R. B. (1994) J. Biol. Chem. 269: 28420-28428] released pelletable material slightly more efficiently than matrix and capsid alone, and these particles had density higher than matrix and capsid alone. These results demonstrate the essential role of HIV-1 nucleocapsid in the virus assembly process and show that the positively charged N terminus of p7 is critical for this role.

Amino Acid Sequence↗

PubMed1990

Localization of nucleocapsid associated polypeptides in measles virus-infected cells by immunogold labelling after resin embedding.

The nucleo-, phospho- and matrix protein of measles virus were localized at high resolution within infected cells by use of post-embedding immunogold labelling techniques. In general, labelling with monospecific antibodies as well as with a polyvalent rabbit anti-measles hyperimmune antiserum revealed measles virus polypeptides to be distributed non-randomly within infected cells with the label largely confined to specific sites, namely inclusions of nucleocapsids and assembled virus structures at the plasma membrane. Immunogold double labelling indicated that the phosphoprotein strictly co-localized with the nucleoprotein in cytoplasmic inclusions of nucleocapsids and in budding virions, whereas intranuclear inclusions of nucleocapsids were devoid of phosphoprotein labelling. Antibodies to the matrix protein clearly labelled assembled virus structures at the plasma membrane but exhibited no significant cytoplasmic or intranuclear reaction. The data indicate that the composition of nucleocapsids varies with the cellular compartment with which they are associated, supporting the view of a rapid assembly of paramyxovirus nucleocapsid polypeptides, and emphasize the proposed selective role of the matrix protein in virus assembly and budding at the plasma membrane.

Capsid↗

PubMed1995

Human immunodeficiency virus nucleocapsid protein stimulates strand transfer from internal regions of heteropolymeric RNA templates.

We have examined the influence of HIV nucleocapsid protein (NCp) on strand transfer from internal regions of a heteropolymeric RNA template. The system consisted of a DNA-primed 225 nucleotide RNA donor template, on which reverse transcriptase initiated primer extension, and a 189 nucleotide RNA acceptor template, to which extended primers could transfer. The last 133 nucleotides on the 3' end of the acceptor were homologous to an internal region on the donor, limiting homologous strand transfer to this region. Primers extended to the end of the donor were 212 nucleotides while those transferred to, and extended on the acceptor were 259 nucleotides in length. The rate of strand transfer and the level of transfer products increased several-fold when nucleocapsid was included in the reactions. The increase was due, at least in part, to the transfer to, and extension on the acceptor, of incompletely elongated primer-extension products that were "chased" into transfer products in the presence of nucleocapsid. Nucleocapsid did not directly influence reverse transcriptase elongation as the enzyme processivity (number of nucleotides incorporated before the enzyme dissociates from the primer-template) was approximately the same in the presence and absence of nucleocapsid.

Capsid↗

PubMed1984

Structural and functional analysis of Sendai virus nucleocapsid protein NP with monoclonal antibodies.

Monoclonal antibodies specific for Sendai virus nucleocapsid protein NP were used to map the antigenic structure of NP and to investigate the role of NP in transcription. Using nine anti-NP antibodies in competitive-binding (CB) assays, it was found that the NP molecule contained at least two topographically distinct antigenic sites. By Western blot analysis, one of the NP epitopes belonging to antigenic site I was localized to a Mr 34,000 (34K) trypsin digest fragment, and another to a Mr 48,000 (48K) fragment which remained associated with the nucleocapsid. The other antibodies which define antigenic site I did not react with either fragment; however, the results of CB would indicate that their epitopes were in a region on the tertiary structure of the NP molecule that is closely proximal to these fragments. The 48K and 34K fragments on the published NP amino acid sequence have been tentatively identified. Since the 34K and 48K fragments bind antibody, it appears that nucleocapsid-bound NP may be folded into a configuration which places at least some of these sequences on the surface of the nucleocapsid structure. Six antibodies representing both antigenic sites were purified for functional studies. All the antibodies inhibited nucleocapsid transcription in vitro to the same extent (greater than 90%); however, they differed in the amount of antibody required to produce the same effect. Within site I, antibodies producing maximum inhibition were divided into three groups: three antibodies inhibited at relatively low concentrations (0.17 microgram), one antibody inhibited at an intermediate range (0.43 microgram), and another required a 10-fold higher concentration (1.73 microgram) to produce the same effect. The antibody which detected the 48K trypsin digest fragment was the one which fell into the intermediate range for transcription inhibition, while the antibody that detected the 34K fragment was in the low range. Thus, antigenic site I, defined by CB and trypsin digestion studies, can be defined further into three subsites which appear to differ in their involvement in the transcription process. Antigenic site II was defined by a single antibody which also inhibited transcription by greater than 90%.

Antibodies, Monoclonal↗

PubMed1986

The intracellular half-lives of nonreplicating nucleocapsids of DI particles of wild type and mutant strains of vesicular stomatitis virus.

We have used defective interfering (DI) particles purified free of all infectious virus to determine the intracellular biological stability of nonreplicating nucleocapsids of vesicular stomatitis virus (VSV). Following infection of BHK-21 cells or tc-7 cells with a low multiplicity of pure DI particles, we superinfected them at varying times afterward with a high multiplicity of infectious helper virus to allow replication of those DI particle nucleocapsids retaining biological activity. Careful quantitation of DI particles in the yields from each time point showed that the biological half-life of intracellular VSV Indiana wild type DI particle nucleocapsids was 6 hr in BHK-21 cells and 5.3 hr in tc-7 cells (not significantly different). However, a DI particle from a temperature-sensitive mutant (tsG31) of VSV exhibited a biological nucleocapsid half-life of 12.5 hr and a DI particle isolated following 5 years of persistent infection had a half-life of 18 hr. These findings have significance for the stability of DI particle activity in vivo during acute infections where virus and DI particles are not always present together in the same cells due to cycling interactions. The increased half-life of DI nucleocapsids after years of persistent infection contrasts with the decreased stability and debilitation generally observed in infectious virus from persistent infection. Finally, transcapsidation studies showed that the intracellular half-life differences between DI particles are due mainly to their RNA genomes rather than to the N protein which encapsidates them.

Animals↗

PubMed1986

Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein.

The sequence of 1690 nucleotides from the 5' end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5' sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5' end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

Amino Acid Sequence↗

PubMed1989

Intra- and extracellular distribution and immunochemical characterization of hepatitis B virus nucleocapsid proteins produced by a human hepatoma cell line transfected with cloned viral DNA.

Hepatitis B virus (HBV)-related antigens produced by the human hepatoma cell line (HB611 cell), which had been transfected with a cloned HBV DNA and established as a stable producer of HBV (T. Tsurimoto, A. Fujiyama, and K. Matsubara, 1987, Proc. Natl. Acad. Sci. USA 84, 444-448), were investigated immunochemically and morphologically. All HBV-related antigens, HBV surface (HBsAg), e (HBeAg), and core (HBcAg), were semiquantitatively examined by the respective reversed passive hemagglutination assay (RPHA). RPHAs for HBcAg and for HBeAg were characterized as reacting only to the core particles and to the free form of nucleocapsid proteins, respectively. The amounts of HBsAg and nucleocapsid protein in culture medium were roughly related to the number of viable cells. The amount of core particles was, instead, proportional to the number of dead cells. Relative amounts of HBsAg, core particles, and nucleocapsid proteins in culture medium, cell surface, and cell lysate were determined and it was found that HBsAg and nucleocapsid proteins were effectively secreted into culture medium but core particles were not. Molecular species of nucleocapsid proteins were identified to be p17 and p18 (HBeAg polypeptides) in the culture medium and HBeAg polypeptides and p21.5 (HBcAg polypeptide) in the cytosol fraction. The p21.5 was preferentially found in the nuclear fraction.

Carcinoma, Hepatocellular↗

PubMed1991

Two noncontiguous regions of Sendai virus P protein combine to form a single nucleocapsid binding domain.

Binding of Sendai virus P protein to viral nucleocapsids requires amino acids in two separate regions of P protein. Both required regions are near the carboxyl terminus, and they are separated by a region which is expendable for binding (K. W. Ryan and A. Portner, 1990, Virology 174, 515-521). To examine the topography of these regions in the folded P protein molecule we mapped the epitopes present in several undenatured P proteins with overlaping deletions near their carboxyl termini. The epitopes recognized by two monoclonal antibodies were each composed of both protein regions necessary for binding, indicating that these two regions are each required at some point during the folding of P protein. To determine if these protein regions interact directly in forming the nucleocapsid binding domain, we constructed a deleted P gene which encodes a protein comprising only these two regions with all other P protein sequences deleted. This protein was able to bind to nucleocapsids, demonstrating that these two regions alone are sufficient to form the nucleocapsid-binding domain. In addition, this protein formed the folded epitopes comprising the two nucleocapsid-binding regions, indicating that the two regions interact directly with each other to form a single folded structure. The involvement of this binding domain in viral mRNA synthesis was examined by testing the ability of each monoclonal antibody to inhibit the in vitro transcription activity of full-size P protein. Several antibodies to epitopes near the binding domain were found to be potent inhibitors of viral transcription, showing that these regions contribute to P protein's role in mRNA synthesis.

Animals↗

PubMed1987

Topological organization of Sindbis virus capsid protein in isolated nucleocapsids.

Sindbis virus nucleocapsids were isolated from mature virions by a two-step purification method. Detergent-treated virions were sedimented in sucrose gradients and the nucleocapsid peaks chromatographed on RNase-free Sephadex G-200. The purified nucleocapsids displayed several morphologies when examined in the electron microscope. These morphologies, and the results of double-angle shadowing, suggest that the core of this enveloped virus has the shape of a regular icosahedron with a triangulation number of 4. Peptide mapping of capsid protein obtained from nucleocapsids that had been radioiodinated by a variety of means, indicated that of the four tyrosine residues in the protein, only Tyr180 was exposed at the surface of the icosahedral structure. The other three residues were not exposed on the outer surface of the nucleocapsid shell, nor on the surface of capsid protein itself, implying that they were buried within the folded protein.

Capsid↗

PubMed1989

The mumps virus nucleocapsid mRNA sequence and homology among the Paramyxoviridae proteins.

The nucleotide sequence of mumps virus nucleocapsid protein (NP) mRNA has been determined from two overlapping cDNA clones and confirmed by partial sequencing of the mRNA and the genome. The mRNA contains 1844 nucleotides excluding poly(A) and encodes a protein of 553 amino acids with a calculated molecular weight of 61,792. A comparison of the mumps virus nucleocapsid protein sequence with that of other paramyxoviruses revealed a moderate degree of homology (33.1%) with the Newcastle disease virus (NDV) only. The nucleocapsid proteins of all paramyxoviruses studied to date, excluding that of the genus pneumovirus, have a conserved sequence of six amino acids (Ser-Tyr-Ala-Met-Gly-Val) except that of NDV which has a mismatch of two amino acids (Ser-Phe-Ala-Met-Gly-Met) in that sequence. In addition, there is another conserved region of seven amino acids (Phe-Ala-Pro-Gly-X-Tyr-Pro) in the nucleocapsid proteins of mumps virus, Sendai virus and parainfluenza virus type 3. The nucleocapsid proteins of measles virus and canine distemper virus (CDV) also have this conserved region but with three conservative amino acid changes.

Amino Acid Sequence↗

PubMed1992

Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein.

Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.

Animals↗

PubMed1983

Identification of envelope and nucleocapsid proteins of infectious bovine rhinotracheitis virus by SDS-polyacrylamide gel electrophoresis.

Infectious bovine rhinotracheitis (IBR) virus was purified by rate zonal and isopycnic centrifugation in potassium tartrate gradients. Viral nucleocapsids were isolated from purified virions by treatment with the nonionic detergent Triton X-100 followed by high speed centrifugation. This treatment was shown to produce a suspension of 74% completely de-enveloped nucleocapsids, 24% incompletely de-enveloped nucleocapsids, and 2% whole virions. The viral nucleocapsids contained DNA and banded at a density of 1.25 g/cm3. Analysis of the viral polypeptides by gradient SDS-polyacrylamide gel electrophoresis revealed that 33 virion proteins, ranging in molecular weight from 13,000 to 275,000 dalton, were present in the complete virus particle. Detergent treatment of the virus quantitatively removed two of the major proteins (vp8, 90,000 dalton, and vp13, 73,000 dalton) and partially removed eleven other proteins. Fifteen viral polypeptides appeared to remain firmly associated with the viral nucleocapsids.

Animals↗

PubMed1995

Measles virus nucleocapsid protein can function in Sendai virus defective interfering particle genome synthesis in vitro.

The Sendai virus P and L proteins, the viral RNA polymerase, and the nucleocapsid protein, NP, synthesized in a transient mammalian expression system support the replication of Sendai virus defective interfering particle (DI) genome RNA in vitro. We have shown that the measles virus nucleocapsid protein, N, can substitute for the Sendai NP protein in genome synthesis. The chimeric product nucleocapsids, which contained Sendai RNA encapsidated with measles N protein, were atypical since they were sensitive to micrococcal nuclease digestion, unlike wild-type Sendai or measles nucleocapsids. The utilization of measles N protein required the endogenous Sendai virus RNA polymerase, since DI nucleocapsids free of polymerase were not replicated. Although both Sendai virus NP and P proteins and measles N and P proteins formed complexes when they were coexpressed, sedimentation analysis showed that measles N protein self-assembled and did not form a complex when expressed with the Sendai P protein. Furthermore, when the Sendai P-L polymerase complex was provided separately, measles N protein alone synthesized DI genome RNA in the absence of Sendai P protein. These data suggest that the self-assembled form of measles N protein functions in Sendai DI genome synthesis.

Capsid↗

PubMed2003

Synthesis of mumps virus nucleocapsid protein in yeast Pichia pastoris.

The expression of mumps virus nucleocapsid protein in yeast Pichia pastoris was investigated. Viral nucleocapsid proteins usually elicit a strong long-term humoral immune response in patients and experimental animals. Therefore, the detection of antibodies specific to mumps virus nucleoprotein can play an important role in immunoassays for mumps diagnosis. For producing a high-level of recombinant mumps virus nucleoprotein the expression system of yeast P. pastoris was employed. The recombinant nucleocapsid protein was purified by cesium chloride ultracentrifugation of yeast lysates. Electron microscopy of the purified recombinant nucleocapsid protein revealed a herring-bone structure similar to the one discovered in mammalian cells infected with mumps virus. The yield of purified nucleocapsid-like particles from P. pastoris constituted 2.1 mg per 1 g of wet biomass and was considerably higher in comparison to the other expression systems.

Animals↗

PubMed1997

Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein.

Nucleocapsid p7 (NCp7) proteins of human immunodeficiency virus type 1 (HIV-1) contain two zinc binding domains of the sequence Cys-(X)2-Cys-(X)4-His-(X)4-Cys (CCHC). The spacing pattern and metal-chelating residues (3 Cys, 1 His) of these nucleocapside CCHC zinc fingers are highly conserved among retroviruses. These CCHC domains are required during both the early and late phases of retroviral replication, making them attractive targets for antiviral agents. toward that end, we have identified a number of antiviral chemotypes that electrophilically attack the sulfur atoms of the zinc-coordinating cysteine residues of the domains. Such nucleocapside inhibitors were directly virucidal by preventing the initiation of reverse transcription and blocked formation of infectious virus from cells through modification of CCHC domains within Gag precursors. Herein we report that azodicarbonamide (ADA) represents a new compound that inhibits HIV-1 and a broad range of retroviruses by targeting the the nucleocapsid CCHC domains. Vandevelde et al. also recently disclosed that ADA inhibits HIV-1 infection via an unidentified mechanism and that ADA was introduced into Phase I/II clinical trials in Europe for advanced AIDS. These studies distinguish ADA as the first known nucleocapsid inhibitor to progress to human trials and provide a lead compound for drug optimization.

Anti-HIV Agents↗

PubMed1983

Coronavirus JHM: nucleotide sequence of the mRNA that encodes nucleocapsid protein.

A DNA copy of the mRNA that encodes the nucleocapsid protein of Mouse Hepatitis Virus JHM has been cloned into pAT153. The DNA copy specifically inhibited the synthesis in vitro of the nucleocapsid protein. The cDNA was subcloned into M13 vectors and the entire sequence, 1767 bases including a 15 base terminal poly (A) tract, has been determined by chain-terminator sequencing. The sequence contained an open-reading frame that could encode a basic protein of mol.wt. 49700. From the predicted sequence it was apparent that the nucleocapsid protein has 5 basic regions, two of which are located near the middle of the sequence, a serine-rich region was also located, a feature which may be of functional importance as the nucleocapsid protein is phosphorylated at serine residues. The carboxy terminus of the nucleocapsid protein was found to be acidic. The 5' non-coding sequence contained a triple repeat of the pentamer AATCT, a structural feature which may play a significant role during the production of subgenomic viral mRNAs.

Amino Acid Sequence↗