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PubMed2001

The humoral immune response to recombinant nucleocapsid antigen of canine distemper virus in dogs vaccinated with attenuated distemper virus or DNA encoding the nucleocapsid of wild-type virus.

This study compared the humoral immune response against the nucleocapsid-(N) protein of canine distemper virus (CDV) of dogs vaccinated with a multivalent vaccine against parvo-, adeno-, and parainfluenza virus and leptospira combined with either the attenuated CDV Onderstepoort strain (n = 15) or an expression plasmid containing the N-gene of CDV (n = 30). The vaccinations were applied intramuscularly three times at 2-week intervals beginning at the age of 6 weeks. None of the pre-immune sera recognized the recombinant N-protein, confirming the lack of maternal antibodies at this age. Immunization with DNA vaccine for CDV resulted in positive serum N-specific IgG response. However, their IgG (and IgA) titres were lower than those of CDV-vaccinated dogs. Likewise, DNA-vaccinated dogs did not show an IgM peak. There was no increase in N-specific serum IgE titres in either group. Serum titres to the other multivalent vaccine components were similar in both groups.

Animals↗
PubMed2003

Functional replacement of nucleocapsid flanking regions by heterologous counterparts with divergent primary sequences: effects of chimeric nucleocapsid on the retroviral replication cycle.

Nucleocapsid (NC) proteins in most retroviruses have a well-conserved Cys-His box(es) as well as more divergent flanking regions that are rich in basic residues. Mutations in the flanking regions can affect RNA packaging, virus assembly, and reverse transcription of the viral RNA. To gain a further understanding of the roles of NC flanking regions in the retroviral replication cycle, we generated and characterized chimeric gag-pol expression constructs derived from murine leukemia virus and spleen necrosis virus by replacing an NC flanking region from one virus with the counterpart from the other virus. We found that all four chimeras were able to generate virions, package viral RNA, and complete the viral replication cycle. Two chimeras had mild defects in virus assembly that correlated with a decrease in the isoelectric points of NCs, suggesting that the basic nature of NC is important in virus assembly. This finding indicates that, although the primary sequences of these flanking regions have little homology, the heterologous sequences are functional both as part of the Gag polyprotein and as processed NC protein.

Amino Acid Sequence↗
PubMed1992

Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1.

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein.

The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined. The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A). The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365. For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP. It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro. Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream. The epitope of the remaining one was in the N-terminal half of the NP.

Amino Acid Sequence↗
PubMed1994

Respiratory syncytial virus nucleocapsid protein (N) expressed in insect cells forms nucleocapsid-like structures.

The gene coding for the N protein of RSV strain Long has been cloned and sequenced. It was introduced behind the polyhedrin promoter of the shuttle vector pVL941 and baculoviruses containing the N gene were constructed by homologous recombination. Infection of Spodoptera frugiperda 9 cells resulted in the production of large amounts of a protein similar in size and antigenicity to the authentic N protein. The baculovirus expressed N protein was concentrated in the cytoplasm of the insect cells and could be extracted at low salt concentration. Nucleocapsid structures similar to those purified from RSV-infected cells could be observed by electron microscopy after negative staining of cellular extracts.

Animals↗
PubMed1995

Analysis of second-site revertants of a murine coronavirus nucleocapsid protein deletion mutant and construction of nucleocapsid protein mutants by targeted RNA recombination.

The Alb4 mutant of the coronavirus mouse hepatitis virus (MHV) is both temperature sensitive and thermolabile owing to a deletion in the gene encoding its nucleocapsid (N) protein. The deletion removes 29 amino acids that constitute a putative spacer region preceding the carboxyl-terminal domain of the protein. As a step toward understanding the structure and function of the MHV N protein, we isolated multiple independent revertants of Alb4 that totally or partially regained the ability to form large (wild-type-sized) plaques at the nonpermissive temperature. The N proteins of these revertant viruses concomitantly regained the ability to bind to RNA in vitro at a temperature that was restrictive for RNA binding by Alb4 N protein. Sequence analysis of the N genes of the revertants revealed that each contained a single second-site point mutation that compensated for the effects of the deletion. All reverting mutations were clustered within a stretch of 40 amino acids centered some 80 residues on the amino side of the Alb4 deletion, within a domain to which the RNA-binding activity of N had been previously mapped. By means of a targeted RNA recombination method that we have recently developed, two of the reverting mutations were introduced into a wild-type MHV genomic background. The resulting recombinants were stable and showed no gross phenotypic differences from the wild type. A detailed analysis of one, however, revealed that it was at a selective disadvantage with respect to the wild type.

Amino Acid Sequence↗
PubMedDate not supplied

[Detection of antibodies to the nucleocapsid protein of Hepatitis C virus by immunoenzyme analysis using synthetic peptides of nucleocapsid N-terminal part].

Solid-phase synthesis of 7 linear peptides overlapping the immunodominant N-terminal region of hepatitis C virus (HCV) core protein (aa 7-75) was carried out. Their antigen reactivity was studied by non-competitive ELISA with sera from patients with chronic HCV infection. Three B-epitopes located within aa 7-19, 20-34, and 39-75 appeared to be the most immunoreactive. Use of the set of synthetic peptides in ELISA detected anti-HCV core antibodies with 93% efficiency in comparison with ELISA and commercial anti-HCV Recomblot based on the use of commercial recombinant HCV core protein.

Enzyme-Linked Immunosorbent Assay↗
PubMed1986

Localization of P, NP, and M proteins on Sendai virus nucleocapsid using immunogold labeling.

The distribution of NP, P, and M proteins on Sendai virus nucleocapsids purified from cells and virions were studied by immunogold staining using monoclonal antibodies. NP molecules were found uniformly along the entire length of both cytosol and virion derived nucleocapsids. This observation is in accord with the earlier proposals that NP molecules maintained the structural integrity of the nucleocapsid. The distribution of P in nucleocapsids derived from the cytosol differed from the distribution in those originating from virions. In nucleocapsids derived from the cytosol, P molecules occurred in 4 to 10 discreet clusters at varying locations along the length of the nucleocapsid. In contrast, on nucleocapsids derived from virions, P molecules were uniformly distributed over the entire length of the nucleocapsid. These observations suggest that the distribution of P depends on the functional state of the nucleocapsid. The occurrence of P clusters at different locations on intracellular nucleocapsids indicates that P is a mobile molecule; this suggestion is consistent with P's role in viral RNA synthesis. The distribution of the matrix (M) protein also depended on where the nucleocapsids were derived from. Large quantities of M protein were found along the entire length of nucleocapsids derived from the cytosol, while in virion nucleocapsids, many fewer molecules of M were observed. The large amounts of M on the nucleocapsids originating from the cytosol supports the hypothesis that M protein mediates the recognition between the nucleocapsid and the envelope glycoproteins.

Animals↗
PubMed1994

Determinants of serotype specificity in transcription of vesicular stomatitis virus synthetic nucleocapsids.

Transcription of the nucleocapsid template of vesicular stomatitis virus (VSV) is serotype specific, since the viral RNA polymerase of the VSV Indiana (Ind) serotype transcribes the Ind nucleocapsid but not the nucleocapsid of the VSV New Jersey (NJ) serotype and, similarly, the NJ RNA polymerase transcribes only the NJ nucleocapsid. We have prepared synthetic nucleocapsid templates from various combinations of purified leader gene RNA and N protein of these two VSV serotypes for analysis of serotype specificity in vitro. In agreement with previous observations, in vitro transcription of synthetic homologous nucleocapsids with the 3' terminal leader RNA gene of either the (+) or (-) strand sense and the N protein of the same serotype is also serotype specific. We show with chimeric nucleocapsids, where the RNA and N protein are of different serotypes, that both components of the template are important for specificity, although the specifics depend on the serotype from which the RNA polymerase is derived. The Ind RNA polymerase will transcribe only the homologous nucleocapsids where both the RNA and N protein are of the Ind serotype, and not the chimeric nucleocapsids. In contrast, the NJ RNA polymerase is active not only on the homologous synthetic nucleocapsid, but also gives significant levels of transcription as long as one of the nucleocapsid components (N protein or RNA) is of the NJ serotype. The divergent RNA sequence of the distal portion of the leader gene (nt22 to 50/51) seems to be a major determinant for specificity, since transcription of synthetic nucleocapsids containing just the conserved proximal 1-22 nucleotides is significantly less serotype-restricted. Restriction appears to occur at the level of elongation of the product rather than initiation of RNA synthesis, since synthesis of template-length RNA, but not reiterated small initiation products, is inhibited. In addition, the serotype of the P protein subunit of the RNA polymerase also contributes to the serotype specificity of transcription, since the NJ P protein can bind equally to NJ and Ind nucleocapsids, while Ind P protein binds preferentially to Ind nucleocapsids.

Animals↗
PubMed1979

Assembly of vesicular stomatitis virus nucleocapsids in vivo: a kinetic analysis.

Pulse-chase labeling and cell fractionation were used to examine the pathways taken by the three nucleocapsid polypeptide species of vesicular stomatitis virus into nucleocapsids and then into virions. An improved method of polyacrylamide gel electrophoresis resolved nucleocapsid polypeptides N and NS from cellular actin, facilitating accurate quantitation of the viral polypeptides. Contrary to previous belief, the rate of NS synthesis was found to be a constant fraction of total virus protein synthesis throughout infection, indicating a consistent mechanism of virus protein synthesis regulation. In the kinetic studies, each polypeptide species displayed the following characteristic behavior. (i) Structural polypeptide N was the only species that entered a metabolically active soluble pool before assembly into nucleocapsids. The size of this pool increased with time after infection, causing an increasing delay in the appearance of pulse-labeled N molecules in nucleocapsids. (ii) Throughout infection, the entire complement of L molecules entered nucleocapsids immediately after their synthesis, without diversion through a soluble pool. (iii) Although 75% of newly synthesized molecules of the transcriptase-associated protein NS entered a soluble pool, they never emerged from the compartment. At all times after infection, about 25% of the NS molecules bypassed the soluble pool and entered nucleocapsids directly after their synthesis, as if in concert with L. These results indicate that VSV nucleocapsid assembly in vivo is a stepwise process, comprising an initial condensation of N with the viral RNA, followed by attachment of L and NS, analogous to the stepwise assembly of Sendai virus nucleocapsids. (D. W. Kingsbury, C.-H. Hsu, and K. G. Murti. Virology 91:86-94, 1978). About half of the intracellular nucleocapsids were recovered in a form that sedimented at anomalously low centrifugal forces, reflecting an association with large cellular organelles. This attachment was mediated mainly by electrostatic forces, since these "bound" nucleocapsids were released by elevated salt concentrations. The kinetic behavior of nucleocapsid polypeptides was the same in both fractions, providing no evidence for a division of nucleocapsid functions between cellular compartments.

Actins↗
PubMed2001

Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein.

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.

Animals↗
PubMed1983

Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids.

The association of newly synthesized vesicular stomatitis virus proteins into nucleocapsid structures was examined in a cell-free system that supports concurrent viral protein synthesis, transcription, and RNA replication. The vesicular stomatitis virus proteins synthesized by this system associated with the newly replicated RNA to form structures that banded in CsCl gradients with marker nucleocapsids. In reactions lacking nucleocapsid templates to program RNA synthesis, the newly synthesized proteins did not associate into nucleocapsid structures. The newly synthesized proteins associated with nucleocapsids were analyzed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate after separation from non-associated proteins by chromatography on Bio-Gel A15M agarose columns. The results of this analysis showed that newly synthesized L, NS, and N proteins associated into nucleocapsids in the in vitro system. In addition, a small amount of newly synthesized M protein was stably bound to the nucleocapsids. The molar ratio of the associated, newly synthesized proteins was 2:350:1,000:10 (L:NS:N:M). More than 90% of the newly synthesized NS protein that associated with nucleocapsids in vitro was of the NS2 subspecies, as assayed by DEAE-cellulose column chromatography. The stability of the association of the newly synthesized proteins with nucleocapsids in the system mimicked that of the association of viral proteins with nucleocapsids from infected cells as measured by salt sensitivity. These data indicate that nucleocapsids were assembled from newly synthesized proteins within our in vitro system and that the molar ratio of assembled proteins was similar to that observed for virion nucleocapsids.

Capsid↗
PubMed1991

Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids.

The purpose of these experiments was to study the physical structure of the nucleocapsid-M protein complex of vesicular stomatitis virus by analysis of nucleocapsid binding by wild-type and mutant M proteins and by limited proteolysis. We used the temperature-sensitive M protein mutant tsO23 and six temperature-stable revertants of tsO23 to test the effect of sequence changes on M protein binding to the nucleocapsid as a function of NaCl concentration. The results showed that M proteins from wild-type, mutant, and three of the revertant viruses had similar NaCl titration curves, while the curve for M proteins from the other three revertants differed significantly. The altered NaCl dependence of M protein was correlated with a single amino acid substitution from Phe to Leu at position 111 compared with the original temperature-sensitive mutant and was not correlated with a substitution of Gly to Glu at position 21 in tsO23 and the revertants. To determine whether protease cleavage sites in the M protein were protected by interaction with the nucleocapsid, nucleocapsid-M protein complexes were subjected to limited proteolysis with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. The initial trypsin and chymotrypsin cleavage sites, located after amino acids 19 and 20, respectively, were as accessible to proteases when M protein was bound to the nucleocapsid as when it was purified, indicating that this region of the protein does not interact directly with the nucleocapsid. Furthermore, trypsin or chymotrypsin treatment released the M protein fragments from the nucleocapsid, presumably due to conformational changes following proteolysis. V8 protease cleaved the M protein at position 34 or 50, producing two distinct fragments. The M protein fragment produced by V8 protease cleavage at position 34 remained associated with the nucleocapsid, while the fragment produced by cleavage at position 50 was released from the nucleocapsid. These results suggest that the amino-terminal region of the M protein around amino acid 20 does not interact directly with the nucleocapsid and that conformational changes resulting from single-amino-acid substitutions at other sites in the M protein are important for this interaction.

Amino Acid Sequence↗
PubMed1999

Assembly of nucleocapsids with cytosolic and membrane-derived matrix proteins of vesicular stomatitis virus.

During budding of vesicular stomatitis virus (VSV), the viral matrix (M) protein binds the viral nucleocapsid to the host plasma membrane and condenses the nucleocapsid into the tightly coiled nucleocapsid-M protein (NCM) complex observed in virions. In infected cells, the viral M protein exists mostly as a soluble molecule in the cytoplasm, and a small amount is bound to the plasma membrane. Despite the high concentrations of M protein and intracellular nucleocapsids in the cytoplasm, they are not associated with each other except at the sites of budding. The experiments presented here address the question of why M protein and nucleocapsids associate with each other only at the plasma membrane but not in the cytoplasm of infected cells. An assay for exchange of soluble M protein into NCM complexes in vitro was used to show that both cytosolic and membrane-derived M proteins bound to virion NCM complexes with affinities similar to that observed for virion M protein, indicating that both cytosolic and membrane-derived M proteins are competent for virus assembly. However, neither cytosolic nor membrane-derived M protein bound to intracellular nucleocapsids with the same high affinity observed for virion NCM complexes. Cytosolic M protein was able to bind intracellular nucleocapsids, but with an affinity approximately eightfold less than that observed in virion NCM complexes. Membrane-derived M protein exhibited little or no binding activity for intracellular nucleocapsids. These data indicate that intracellular nucleocapsids, and not intracellular M proteins, need to undergo an assembly-initiating event in order to assemble into an NCM complex. Since neither membrane-derived nor cytosolic M protein could initiate high-affinity binding to intracellular nucleocapsids, the results suggest that another viral or host factor is required for assembly of the NCM complex observed in virions.

Animals↗
PubMed1984

Immunoassays for measles virus nucleocapsid antigen: effect of antigen-antibody complexes.

An inhibition radioimmunoassay and a two-site immunoradiometric (four-layer) assay for the quantification of measles virus nucleocapsid antigen were established and their sensitivities compared. Both assays exhibited threshold sensitivities of from 1 to 10 ng nucleocapsid antigen per ml. Concentrations of reagents used in the assays were shown to be an important factor determining the sensitivities of the assays. Purified mumps or parainfluenza 2 virus nucleocapsids did not compete with measles virus nucleocapsids in the inhibition assay but some degree of cross-reactivity with canine distemper virus nucleocapsid was observed. Pretreatment of virus-infected cells with detergent had a significant effect on the amount of antigen detectable by the assays. SDS greatly decreased the reactivity of measles virus nucleocapsid but Triton X-100 and, to a larger degree, sodium deoxycholate released antigen from the cells in quantities greater than that detected when no detergent pretreatment was employed. Complexing of antibodies to measles virus nucleocapsids in vitro decreased dramatically the threshold sensitivity of the nucleocapsid assays. The antigen-antibody complexes could be disrupted and the components separated but the conditions employed destroyed 90% of the antigenic reactivity of the nucleocapsid and also had a deleterious effect on the antibody. Nucleocapsid antigen was readily detected in brains from hamsters with acute measles encephalitis. The assays, however, were not sensitive enough for routine detection of antigen in brains from hamsters with chronic measles encephalitis.

Animals↗
PubMed1994

Selective interference with P protein binding to paramyxovirus nucleocapsids.

We previously observed that some anti-nucleoprotein monoclonal antibodies were able to displace P protein from Sendai virus (SV) nucleocapsid cores. The current work extends that observation by showing that such antibody-mediated P displacement is not unique to Sendai virus nucleocapsids, but can also occur with nucleocapsids of a related human respiratory pathogen. Anti-NP antibody prevents binding of SV P protein to nucleocapsids from human parainfluenza virus type 1 (PIV1) or from SV. Antibody also prevents binding of PIV1 P protein to PIV1 nucleocapsids, but not to SV nucleocapsids. We have also examined the stoichiometry of antibody interference with P binding, to determine how large a nucleocapsid region can be protected from P binding by a single antibody molecule. We found that approximately 40 antibody molecules per nucleocapsid complex can block attachment of most P protein. This indicates that a single antibody molecule can prevent P binding to a region representing about 65 nucleoprotein monomers on the nucleocapsid core.

Antibodies, Viral↗