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PubMed1999

Visualization and characterization of GB virus-C particles: evidence for a nucleocapsid.

GB virus type C (GBV-C) is a member of the hepacivirus genus within the Flaviviradae. Persistent GBV-C infection is common in humans, yet it remains unclear if GBV-C causes any disease. Although GBV-C infection has been associated with acute non-A to non-E post-transfusion hepatitis, it does not appear to cause chronic hepatitis. GBV-C is closely related to hepatitis C virus (HCV), but indirect evidence suggests that it does not encode a core protein at the amino terminus of the open reading frame (ORF). This has led to speculation that GBV-C does not have a nucleocapsid. We evaluated the buoyant density of GBV-C, and found very low density particles consistent with virions, and intermediate density particles consistent with nucleocapsids in GBV-C-infected people. In addition, electron microscopy demonstrated an apparent nucleocapsid within an enveloped particle. Although these biophysical data strongly suggest that GBV-C utilizes a nucleocapsid, they do not indicate the origin of the protein content of this particle. To assess this, we evaluated patient plasma for reactivity with a synthetic oligopeptide representing a conserved region near the amino terminus of the predicted ORF. Specific antibody was detected in some individuals, similar to data of Feucht et al. who identified antibody against a recombinant core protein in GBV-C-infected people. These data indicate that GBV-C particles contain nucleocapsids. At least in some patients, the region upstream of the GBV-C E1 protein coding region appears to be expressed, and this region may represent the structural protein of the nucleocapsid.

Amino Acid Sequence↗

PubMed1999

A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.

Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction.

Adenosine Triphosphate↗

PubMed1990

Localization of P protein binding sites on the Sendai virus nucleocapsid.

Previous studies have shown that the molecules of P protein associated with transcriptionally active Sendai virus nucleocapsids are arranged in discrete clusters. Our study investigates whether or not this localized distribution is due to the existence of only a few P protein binding sites on the nucleocapsid core. We used immunoelectron microscopy to examine whether additional P proteins could bind at locations between the groups of endogenous P proteins. To differentiate between endogenous and added proteins, we constructed a recombinant gene which instructs the in vitro synthesis of a chimeric protein containing the carboxyl-terminal nucleocapsid-binding region of P protein, fused to chloramphenicol acetyltransferase (CAT). Immunogold labelling, using an antibody to the CAT moiety, revealed at the electron microscope level, that the chimeric product bound to nucleocapsids at many sites located over the entire length of the nucleocapsid. This indicated that the localized distribution of P protein molecules is not due to a limited number of P protein binding sites on the nucleocapsid core.

Binding Sites↗

PubMed2005

Immunostimulatory potential of hepatitis B nucleocapsid preparations: lipopolysaccharide contamination should not be overlooked.

The nucleocapsid of hepatitis B virus (HBV) allows insertions of heterologous peptides and even complete proteins. Because of its outstanding capacity to induce B-cell, T-helper and cytotoxic T-cell responses, this structure is considered to be an important instrument for future vaccine development. Most of the evidence for the unique immunogenic qualities of nucleocapsids has been generated in mice, which are not natural hosts of HBV. Moreover, most nucleocapsid preparations used in these studies were produced in a recombinant manner in Escherichia coli. Such preparations have been shown to contain lipopolysaccharide (LPS). Not unexpectedly, it is shown here that contaminating LPS, rather than the nucleocapsid structure itself, is responsible for the activation of human antigen-presenting cells. Careful examination of the literature dealing with the immunogenicity of HBV nucleocapsids suggests that the possible presence of LPS has been largely ignored or underestimated in several studies. This raises doubts on some of the underlying mechanisms that have been proposed to explain the unique immunogenicity of the HBV nucleocapsid.

Cells, Cultured↗

PubMed1997

An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.

Amino Acid Sequence↗

PubMed1994

Amino acid requirements of the nucleocapsid protein of HIV-1 for increasing catalytic activity of a Ki-ras ribozyme in vitro.

The nucleocapsid protein NCp7 of HIV-1 is a single-stranded nucleic acid binding protein with several functions such as specific recognition, dimerization and packaging of viral RNA, tRNA annealing to viral RNA and protection against nucleases. Since some of these functions involve annealing and double-stranded RNA-melting activity we applied the nucleocapsid protein to a hammerhead ribozyme specific for the activated Ki-ras mRNA in vitro, which carries at its mutated codon 12 a GUU site. A synthetic ribozyme containing 2'-O-allyl-modified nucleotides and alternatively in vitro transcribed ribozymes were used. At a one to one molar ratio of substrate to ribozyme almost no cleavage is observed at 37 degrees C. Presence of a synthetic nucleocapsid protein significantly increases the catalytic activity of the ribozyme. Kinetic analyses by means of single and multiple turnover reactions performed at various substrate to ribozyme ratios lead to only a slight stimulation of the rate constants for single turnover reactions. The rate constants in multiple turnover reactions, however, are stimulated up to 17-fold by the presence of the nucleocapsid protein. The activating region of the nucleocapsid protein was characterized by a number of mutants. The mutants demonstrate that activation requires both basic amino acid clusters as evidenced by point mutations. Deletion mutants indicate that the second zinc finger is totally dispensable and that replacement of the first zinc finger by a glycine-glycine spacer only slightly reduces the enhancing effect of the nucleocapsid protein on the ribozyme.

Amino Acid Sequence↗

PubMed1993

Penetration of Autographa californica nuclear polyhedrosis virus nucleocapsids into IPLB Sf 21 cells induces actin cable formation.

The budded form of Autographa californica nuclear polyhedrosis virus functionally enters cells by adsorptive endocytosis. During the period of virus uptake into Spodoptera frugiperda IPLB Sf21 cells (1 to 4 hr postinfection), filamentous F-actin cables, visualized by fluorescence microscopy, were formed within the cytoplasm of infected cells. Cable formation appeared to be a direct effect of viral inoculum in that the numbers of observed cables increased with an increase in multiplicity of infection, and cable formation was not dependent on protein synthesis. Cable induction was first apparent around 30 min postinfection, although uptake of virus into endocytic vesicles began much earlier. Chloroquine, a chemical that prevents release of nucleocapsids from endosomes, inhibited cable formation. Similarly, when virus adsorbed to the exterior of cells was exposed to neutralizing antibody, release of nucleocapsids from endosomes and subsequent cable induction were prevented. Careful observation of co-labeled F-actin cables and nucleocapsids revealed a single nucleocapsid at an end of most cables. These results suggested that nucleocapsids or nucleocapsid-associated proteins of budded virus induced the polymerization or bundling of actin following their penetration into the cytoplasm from endocytic vesicles.

Actins↗

PubMed1993

Semliki Forest virus core protein fragmentation: its possible role in nucleocapsid disassembly.

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243-255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.

Aedes↗

PubMed1987

Transcriptionally defective nucleocapsids of vesicular stomatitis virus from cells treated with indomethacin.

Indomethacin blocks the biosynthesis of vesicular stomatitis virus (VSV) at the level of primary transcription, RNA replication, and protein synthesis (P. K. Mukherjee and R. W. Simpson (1985), Virology 140, 188-191). Nucleocapsids of infecting virus particles recovered from indomethacin-treated cells were analyzed for in vitro transcriptase activity. Incorporation of [3H]UTP in mixtures containing nucleocapsids from HEp-2 cells pretreated with 10(-3) M indomethacin was inhibited approximately 80% compared to control reactions containing nucleocapsids from untreated infected cells. The level of inhibition of in vitro transcriptase activity of viral nucleocapsids from drug-treated cultures varied according to the cell line used for infection. After indomethacin removal, cells regained their ability to produce enzymatically competent viral-transcribing complexes unless they were subsequently exposed to metabolic inhibitors such as actinomycin D or alpha-amanitin. Enzymatically defective nucleocapsids from indomethacin-treated cells showed enhanced in vitro transcriptase activity in the presence of modulators of prostaglandins and cyclic nucleotides. Electrophoretic analysis of product from in vitro transcriptase reactions revealed that these defective nucleocapsids are unable to synthesize VSV messenger RNA or normal size leader RNA species but only smaller transcripts of undetermined identity.

Animals↗

PubMed1990

Purified phi 6 nucleocapsids are capable of productive infection of host cells with partially disrupted outer membranes.

Purified nucleocapsids of bacteriophage phi 6, lacking the phage lipid envelope, are unable to infect intact Pseudomonas syringae host cells. A method for studying the process by which a naked virus particle, the phi 6 nucleocapsid, penetrates the host cytoplasmic membrane was developed. Host cells were rendered competent for nucleocapsid infection by treatment with repeated washings with salt and sucrose and the subsequent addition of lysozyme. This treatment disrupts the outer membrane, permitting the nucleocapsid to reach the cytoplasmic membrane and to infect the cell. The nucleocapsid infection is blocked by monoclonal antibodies raised against the nucleocapsid shell protein P8.

Absorption↗

PubMed1991

Antigenicity of hantavirus nucleocapsid proteins expressed in E. coli.

DNA clones representing the small genomic segment of Nephropathia epidemica virus strain Hällnäs B1 (NEV) and Hantaan virus strain 76-118 (HTV) encoding their nucleocapsid proteins were inserted into the E. coli vector pIN-III-ompA for secretion of proteins into the periplasmic space. The complete HTV and NEV nucleocapsid proteins and two truncated versions of the NEV nucleocapsid proteins were expressed as fusion proteins. Unexpectedly, all products accumulated as insoluble aggregates. Most of the ompA signal peptide remained uncleaved. However, nucleocapsid fusion proteins could be purified from the insoluble fraction by extraction with 8 M urea followed by separation on SDS-PAGE and electroelution. Rabbits were immunized with the eluted proteins and the resulting antibodies reacted specifically with authentic viral nucleocapsid proteins of HTV and NEV. The recombinant nucleocapsid proteins were found to react specifically with various hantavirus-immune sera, but not with human control sera, indicating their suitability as potential diagnostic antigens. This is the first report on the expression of a protein of a NEV serotype strain of hantaviruses by use of recombinant DNA techniques.

Antigens, Viral↗

PubMed1996

Stopped-flow, classical, and dynamic light scattering analysis of matrix protein binding to nucleocapsids of vesicular stomatitis virus.

During the process of assembly of enveloped viruses, binding of the nucleoprotein core of the virus (nucleocapsid) to the host membrane is mediated by the viral matrix (M) protein. Light scattering properties of vesicular stomatitis virus (VSV) nucleocapsids and nucleocapsid-M protein (NCM) complexes assembled in vivo were determined following solubilizaton of the virion envelope with detergents at varying ionic strength to vary the extent of M protein binding. Three factors were found to contribute to the light scattering properties of VSV nucleocapsids: their conformation, extent of self-association, and amount of bound M protein. All three were affected by changes in ionic strength but could be distinguished by several parameters. Conformational changes in nucleocapsids and NCM complexes occurred rapidly (millisecond time scale) upon changing salt concentration and were reflected in changes in the angular dependence of light scattering intensity (i.e., changes in radius of gyration, RG). Changes in extent of self-association occurred relatively slowly (seconds to minutes time scale) and could be distinguished by the concentration dependence of the apparent molecular mass and diffusion coefficient of the NCM complex. Changes in M protein binding occurred on an intermediate time scale (t1/2 approximately one s) and reflected changes in both molecular mass and RG. The data presented here provide criteria for assessing binding of M protein to nucleocapsids under conditions of minimal perturbation of the NCM complex assembled in vivo and at low protein concentrations so that self-association of the NCM complex was minimal and reversible.

Animals↗

PubMed1980

Isolation and partial characterization of two forms of cytoplasmic nucleocapsids from measles virus-infected cells.

Two species of measles virus nucleocapsids with distinct buoyant densities were isolated from infected AV3 cell homogenates by isopycnic CsCl gradient centrifugation. The more buoyant or 'light' form of the nucleocapsid had a density of 1.26 to 1.28 g/ml, whereas the less buoyant or 'heavy' nucleocapsid species had a density of 1.30 g/ml. Analysis of the two nucleocapsid species by SDS--polyacrylamide gel electrophoresis showed that both forms possessed two phosphorylated polypeptides with mol. wt. of 69000 and 60000. The heavy form of nucleocapsid consisted solely of these two polypeptide species, while the light form of nucleocapsid had two additional associated polypeptides (VP4, mol.wt. 52000, and 45K, mol. wt. 45000). Ultrastructural and immunofluorescent studies suggest that the two isolated capsid forms represent the two morphologically distinct capsid species observed in vivo. This paper discusses the possible relationship between the two capsid forms and assembly of the virus.

Amnion↗

PubMed1987

Biochemical evidence for the oligomeric arrangement of bovine rotavirus nucleocapsid protein and its possible significance in the immunogenicity of this protein.

The nucleocapsid protein of bovine rotavirus was shown to exist in trimeric units in both the virus particle and in infected cells, with the subunits linked by non-covalent interactions. These trimeric units complex further by disulphide bridges into larger units which may represent the hexameric structures observed by electron microscopy. Visualization of various nucleocapsid protein complexes was also achieved on polyacrylamide gels by treating virus preparations with urea at 37 degrees C or boiling in the presence and absence of 2-mercaptoethanol. Since virus particles devoid of nucleic acid were also broken down into trimeric subunits by such treatments, assembly of virus particles appears not to require an RNA-protein interaction. Four nucleocapsid-specific monoclonal antibodies with low neutralizing ability reacted with the monomeric (45,000 mol. wt., 45K), dimeric (90K), trimeric (135K) and trimeric pair (270K) subunits, indicating that a site responsible for neutralization is probably exposed after assembly of these subunits. Analysis of radiolabelled virus revealed that a high proportion (80%) of infectious particles could be immunoprecipitated by these monoclonal antibodies, suggesting that the virus particles are either partially double-shelled or have the nucleocapsid exposed on the surface. The monoclonal antibodies also cross-reacted with the nucleocapsid proteins of simian (SA11), pig (OSU), bovine (NCDV and UK) and human (Wa and ST4) rotaviruses in an immunoblot ELISA reaction. Since these six viruses belong to two different subgroups, it is likely that the antibodies did not recognize the subgroup-specific site, but a shared exposed antigenic determinant. Due to the hexameric configuration of the nucleocapsid in virus particles the neutralizing epitope may be repeatedly presented and, therefore, may contribute to the immunogenicity of this protein.

Animals↗

PubMed1974

Replication of measles virus: distinct species of short nucleocapsids in cytoplasmic extracts of infected cells.

Cytoplasmic extracts of Vero cells infected with wild-strain Edmonston measles virus were found to contain two and probably three distinct species of nucleocapsids. Species sedimenting at 200 and 110S contained RNA which sedimented at 50 and 16 to 18S, respectively. The third nucleocapsid species which sedimented at 170S was not present in all experiments and was not characterized in detail. Essentially all 200 and 170S, as well as a portion of the 110S, nucleocapsids were membrane associated and probably present in part in cell-associated virions. Five of six plaque purified strains derived from wild-type Edmonston virus produced only 200S nucleocapsids. One of these five plaque-purified strains subsequently produced both 200 and 110S nucleocapsids after being passaged by using undiluted inocula. These results suggest that measles virus may produce distinct classes of defective virus containing short nucleocapsids and subgenomic viral RNA.

Animals↗

PubMed1980

Characterization of an extremely basic protein derived from granulosis virus nucleocapsids.

Nucleocapsids were isolated from purified enveloped nucleocapsids of Plodia interpunctella granulosis virus by treatment with Nonidet P-40. When analyzed on sodium dodecyl sulfate-polyacrylamide gels, the nucleocapsids consisted of eight polypeptides. One of these, a major component with a molecular weight of 12,500 (VP12), was selectively extracted from the nucleocapsids with 0.25 M sulfuric acid. Its electrophoretic mobility on acetic acid-urea gels was intermediate to that of cellular histones and protamine. Amino acid analysis showed that 39% of the amino acid residues of VP12 were basic: 27% were arginine and 12% were histidine. The remaining residues consisted primarily of serine, valine, and isoleucine. Proteins of similar arginine content also were extracted from the granulosis virus of Pieris rapae and from the nuclear polyhedrosis viruses of Spodoptera frugiperda and Autographa californica. The basic polypeptide appeared to be virus specific because it was found in nucleocapsids and virus-infected cells but not in uninfected cells. VP12 was not present in polypeptide profiles of granulosis virus capsids, indicating that it was an internal or core protein of the nucleocapsids. Electron microscopic observations suggested that the basic protein was associated with the viral DNA in the form of a DNA-protein complex.

Journal Article↗

PubMed1984

Isolation and characterization of measles virus intracellular nucleocapsid RNA.

Protocols have been established for the preparation of large amounts of pure measles virus intracellular nucleocapsids. As a result, it has been possible to routinely achieve nucleocapsid RNA yields of approximately 200 micrograms (from approximately 5 X 10(8) infected cells). Electrophoretic analysis of this RNA under denaturing conditions revealed a single species whose mass was estimated at approximately 4.8 X 10(6) daltons. Electron microscopic assessment of nucleocapsid RNA contour lengths corroborated the electrophoretic size determination. Total nucleocapsid RNA was shown to contain both negative- and positive-stranded species distributed in a ratio of 2 to 3 genome polarity molecules for each antigenome RNA. Hybridization studies established that all of the virus-specified polyadenylated RNAs were encoded by the negative-stranded nucleocapsid RNA and, therefore, that this nucleocapsid RNA was the measles genome. Examination of the measles virus-specified, polyadenylated transcription products by HCHO-agarose gel electrophoresis revealed at least nine distinct RNA species (rather than the six predicted measles mRNAs). The significance of these observations is discussed.

Capsid↗

PubMed1991

Assembly and transcription of synthetic vesicular stomatitis virus nucleocapsids.

The functional template for transcription of vesicular stomatitis virus (VSV) RNA is a ribonucleoprotein particle (nucleocapsid) consisting of the negative-strand sense genomic RNA completely encapsidated by the viral nucleocapsid (N) protein. As an approach to create nucleocapsids in vitro, we demonstrate here the specific encapsidation by purified N protein of in vitro-synthesized RNA sequences representing the 5' end of both the negative- and positive-strand VSV genome-length RNAs. As few as 19 nucleotides from the 5'-end of positive-strand RNA allowed maximal encapsidation, although the 5' terminal 10 nucleotides would allow partial (50%) encapsidation. Sequences downstream of the binding site can be of any origin. Specific encapsidation of VSV sequences was dependent on the presence of uninfected cell cytoplasmic extracts or poly(A). The synthetic nucleocapsids have the properties of RNase resistance and a buoyant density typical of wild-type VSV nucleocapsids. We have encapsidated a synthetic virionlike RNA species which contained just the terminal sequences of the virion RNA: the N encapsidation signal from the 5' end and the leader gene from the 3' end. This assembled nucleocapsid could function in vitro as a transcription template for the VSV RNA polymerase.

Animals↗