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PubMed1978

Assembly of Semliki Forest virus nucleocapsid: detection of a precursor in infected cells.

The synthesis of Semliki Forest virus nucleocapsid in infected cells was studied by labelling the virus RNAs with 3H-uridine for different periods at various phases of infection. Short pulses (10 to 20 min) revealed the accumulation of 42S RNA in a ribonucleoprotein which sedimented at about 90S (90S RNP) and contained only small amounts of capsid protein. Only after longer pulses was the labelled 42S RNA found in the virus nucleocapsid, suggesting that the 90S RNP may be its precursor. The life time of the 90S RNP was long in the early phases of infection and short in the late phases, reflecting the increased rate of assembly of the nucleocapsid during infection. The 90S RNP was the only 42S RNA containing RNP found in cells infected with temperature sensitive mutants deficient in nucleocapsid formation or wild type infected cells treated with cycloheximide to inhibit nucleocapsid assembly.

Capsid↗

PubMed1979

Comparison of the structural properties of Sindbis and Semliki forest virus nucleocapsids.

The envelope spikes of Sindbis and Semliki Forest virus are arranged in a T = 4 icosahedral surface lattice and, by deduction, it has been suggested that the nucleocapsid proteins are similarly arranged. After treatment of the virions with a non-ionic detergent the released nucleocapsids sediment in sucrose gradients at about 160S and 150S and have densities in CsCl of 1.42 g/ml and 1.425 g/ml, respectively, for Sindbis and Semliki Forest virus. At pH 6.0 Sindbis nucleocapsids do not contract like those of Semliki Forest virus. Nucleocapsids of both viruses are sensitive to the action of ribonuclease but only those of Semliki Forest virus undergo a drastic structural rearrangement due to the treatment. EDTA treatment in hypotonic conditions results in a decrease in the S-value for both particles. Electron micrographs show that the SFV nucleocapsids are partly 'unfolded' while those of Sindbis appear slightly contracted after exposure to EDTA.

Capsid↗

PubMed1980

Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. I. The nucleocapsid protein.

Twenty-one hybridoma cultures, obtained through the fusion of mouse myeloma cells with splenocytes of BALB/c mice immunized with either rabies virus or Mokola virus, secreted monoclonal antibodies specific for the nucleocapsid of the inducer virus. They displayed different specificities for the nucleocapsids of rabies and rabies-related viruses and could be classified into eight groups which are likely to correspond to different antigenic determinants on the nucleocapsid. Four strains of fixed rabies virus (CVS, ERA, Flury-LEP and Kelev) could not be differentiated by the nucleocapsid-specific hybridoma antibody. The Flury-HEP virus (derived from Flury-LEP) as well as the rabies-related viruses Mokola, Lagos bat and Duvenhage, showed marked differences in their reactivities with hybridoma antibodies to nucleocapsid. A selected panel of three of these hybridomas may be used for a rapid differential diagnosis among all members of the Lyssavirus group.

Animals↗

PubMed1982

Intracellular nucleocapsid RNA of mumps virus.

Intracellular nucleocapsids, virtually free of host proteins and nucleic acids, were purified from cells acutely infected with several strains of mumps virus. VIral RNA obtained from the nucleocapsids was predominantly a single 50S species, with 28S RNA occurring as a secondary peak in the RNA profile. Nucleocapsids yielded approx. 9 micrograms 50S RNA per 10(9) infected cells. The 50S RNA was partially self-complementary, indicating that mumps viral nucleocapsids include full-length plus (+) sense antigenomes. Self-annealing and competition hybridization assays with 50S RNA suggest that 28S RNA includes duplexes consisting of full-length (+) and minus (-) sense 50S RNA. Properties of the viral RNA did not vary with the method of RNA isolation, time post-infection at which nucleocapsids were harvested, nor the strain of mumps virus.

Animals↗

PubMed1984

The molecular length of measles virus RNA and the structural organization of measles nucleocapsids.

Full-length measles virus RNA molecules isolated from purified virions or nucleocapsids and examined by electron microscopy were 5.12(+/- 0.12) micron in length, corresponding to a molecular weight of 5.2 (+/- 0.1) X 10(6). Purified virions examined by negative staining in the electron microscope exhibited a pleomorphic range of particle sizes varying in diameter between 300 nm and 1000 nm. Purified nucleocapsids had dimensions of 21 nm (diameter) X 1254(+/- 7) nm (length) and a central core of diameter about 5 nm. Full-length nucleocapsids were composed of 204 (+/- 3) protein discs. The pitch of the nucleocapsid helix was calculated to be 6.1 nm and the helix angle, alpha, to be 8 degrees 16'. Approximate volume calculations indicate that each enveloped virus particle contains multiple nucleocapsids.

Animals↗

PubMed1996

Nucleocapsid protein 10 activates dimerization of the RNA of Moloney murine leukaemia virus in vitro.

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.

Base Sequence↗

PubMed1975

Determination of measles virus-specific nucleocapsid antibodies by means of counterimmunoelectrophoresis.

A counterimmunoelectrophoretic method for quantitative determination of antibodies against measles virus nucleocapsid is described. Unfractionated material from measles virus infected cell cultures was used as antigen, diluted to give only a nucleocapsid-specific precipitate. Purified nucleocapsid and a rabbit hyperimmune serum against this antigen was used to establish the specificity of the test. The measles virus specific antibody titres determined by the method were comparable to those found in nucleocapsid complement fixation tests. It is concluded that the technique described offers a simple and sensitive method to determine nucleocapsid specific antibodies.

Animals↗

PubMed1978

Measles virus nucleocapsids: large-scale purification and use in radioimmunoassays.

Nucleocapsids in quantities approaching 1 mg were purified from 109 measles virus-infected cells. They contained one polypeptide species with a molecular weight of 59,000. Antiserum was raised in rabbits against purified nucleocapsids and used in a competitive radioimmunoassay. Because of their instability, purified nucleocapsids were not suitable for use in such an assay. Instead partially purified nucleocapsids from HEp-2 cells persistently infected with measles virus and labeled in vitro with [35S]methionine were used as the source of radioactive antigen. The radioimmunoassay thus developed measured less than 5 ng of nucleocapsids in infected cells.

Animals↗

PubMed1974

Proteolytic cleavage of subunits of the nucleocapsid of the paramyxovirus simian virus 5.

The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. In most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. It is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain. The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus.

Amino Acids↗

PubMed1976

Proteins of Epstein-Barr Virus. II. Electrophoretic analysis of the polypeptides of the nucleocapsid and the glucosamine- and polysaccharide-containing components of enveloped virus.

Two series of experiments were undertaken to identify the topological location of the structural polypeptides of Epstein-Barr virus. In the first series of experiments, nucleocapsids prepared by detergent treatment of enveloped virus with Nonidet P-40 and sodium deoxycholate were found to be composed of seven polypeptides, VP2, 6, 7.5, 24, 27, 31, ANd 33, which ranged in molecular weight from over 200 X 10(3) to 28 X 10(3). Nine other polypeptides, VP 4, 7, 8, 10, 15, 16, 23, 28, and 29, could be identified in preparations of Epstein-Barr virus nucleocapsids, but the relative amount of this second group of polypeptides was less in preparations of nucleocapsids than in preparations of enveloped virus. The incomplete removal of these polypeptides from enveloped virus by detergent treatment suggests that some of these polypeptides may be components of the envelope or tegument that lie in close proximity to the outer surface of the nucleocapsid In the second series of experiments periodic acid-Schiff-staining and glucosamine-containing components were identified with similar electrophoretic mobility to several of the polypeptides of enveloped virus (VP 5, 8, 9, 11, 12, 13, 14, 15, 16, 17, 28, and 29) that were completely or incompletely removed from purified virus preparations by detergent treatment. The similarity between the polypeptide composition of the nucleocapsids of Epstein-Barr virus and herpes simplex virus was in contrast to the dissimilarity between the nonnucleocapsid polypeptides of Epstein-Barr virus and herpes simplex virus.

Capsid↗

PubMed1980

Isolation of coronavirus envelope glycoproteins and interaction with the viral nucleocapsid.

The two envelope glycoproteins and the viral nucleocapsid of the coronavirus A59 were isolated by solubilization of the viral membrane with Nonidet P-40 at 4 degrees C followed by sucrose density gradient sedimentation. Isolated E2 consisted of rosettes of peplomers, whereas E1, the membrane glycoprotein, was irregular and amorphous. Under certain conditions significant interactions occurred between components of Nonidet P-40-disrupted virions. Incubation of the Nonidet P-40-disrupted virus at 37 degrees C resulted in formation of a complex between one of the viral glycoproteins, E1, and the viral nucleocapsid. This was caused by a temperature-dependent conformational change in E1, resulting in aggregation of E1 and interaction with the viral RNA in the nucleocapsid. E1 also bound rRNA. The E1-nucleocapsid complexes can be distinguished on sucrose and Renografin density gradients from native viral nucleocapsids. The separation of the membrane glycoprotein E1 from the peplomeric glycoprotein E2 permitted preparation of antisera against these isolated proteins. A model is proposed for the arrangement of the three major structural proteins in the coronavirus A59 virion in relation to the viral envelope and RNA.

Capsid↗

PubMed1981

Host cell factors involved in the production of slowly sedimenting nucleocapsids in measles virus-infected cells.

A decrease in the sedimentation rates of the measles virus nucleocapsid, and the RNA contained within, were observed during acute measles virus infection when the growth conditions of Vero cells were altered. The change in sedimentation rates of virus nucleocapsids in these experiments was apparently due to the physiological state of the cell and was independent of the history of the measles virus used for infection since: (i) the same virus stock was used to infect cells from which nucleocapsids were prepared, (ii) nucleocapsid sedimentation rates were rapid when Vero cells freshly revived from liquid nitrogen were infected, but nucleocapsid profiles showed no decrease in the amount of slowly sedimenting material using the same cells and changing the virus preparation used for infection. Frequent cell splittings and numerous medium changes were among the growth factors which appeared to correlate to slowly sedimenting particle production. Changes in the amount of self-complementarity of the measles virus RNA were also observed under these conditions.

Animals↗

PubMed1987

The nucleocapsid of the lipid-containing double-stranded RNA bacteriophage phi 6 contains a protein skeleton consisting of a single polypeptide species.

The nucleocapsid of the enveloped double-stranded RNA bacteriophage phi 6 was isolated by extraction with the nonionic detergent Triton X-114 and subjected to disruption analysis with chelating and protein-denaturing agents. The subnucleocapsid particles were separated in rate-zonal sucrose gradients, and their ultrastructure and protein composition were analyzed. The role of divalent cations in the nucleocapsid structure was studied by using a precipitation assay of the isolated nucleocapsid proteins. The phi 6 nucleocapsid had a cagelike skeleton consisting of a single polypeptide species (P1). Two other proteins (P2 and P4) were associated with the P1 cage. These three early proteins were previously known to be involved in the RNA synthesis machinery of the virus. The stability of the nucleocapsid surface lattice consisting of protein P8 was dependent on Ca2+ ions.

Bacteriophages↗

PubMed1989

The Sendai virus nucleocapsid exists in at least four different helical states.

Sendai virus nucleocapsids have been observed by electron microscopy to coexist in three different helical pitch conformations, 5.3, 6.8, and 37.5 nm. The 5.3- and 6.8-nm conformations are present both in uranyl acetate negatively stained preparations and in tantalum-tungsten metal-shadowed preparations, whereas the 37.5-nm conformation, which has not been previously reported, is present only in the shadowed preparations. The 5.3-nm pitch conformation appears to be a mixture of two discrete structural states, with a small difference in the twist of the structure between the two. We have used image reconstruction techniques on an averaged data set from eight negatively stained nucleocapsids to produce a three-dimensional reconstruction at 2.4-nm resolution of the structure in one of the 5.3-nm pitch states. There are 13.07 nucleocapsid protein (NP) subunits in each turn of the helix in this state. The helical repeat is 79.5 nm, containing 196 subunits in 15 turns of the left-handed 5.3-nm helix. The arrangement of subunits produces a 5.0-nm-diameter hollow core which forms an internal helical groove. The RNA accounts for about 3% of the mass of the nucleocapsid, and so its location is not conspicuous in the reconstruction. Because of the RNA remains associated with the NP subunits during mRNA transcription and genome replication, structural transitions in the nucleocapsid may determine the accessibility of the genome to polymerases. Alternatively, the large hollow core and internal helical groove we have reconstructed may allow access to the RNA even in the tightly coiled 5.3-nm pitch conformation.

Capsid↗

PubMed1990

Phosphorylation of NS protein by vesicular stomatitis virus nucleocapsids: lack of effect during RNA synthesis and separation of kinase from L protein.

The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.

Adenosine↗

PubMed1993

Characterization of functional Sp1 transcription factor binding sites in the hepatitis B virus nucleocapsid promoter.

The hepatitis B virus nucleocapsid minimal promoter contains sequence elements which are similar to the Sp1 transcription factor binding site consensus sequence. The interaction of these regulatory elements with Sp1 was examined by DNase I footprinting with purified Sp1 protein and DNase I footprinting and gel retardation analysis with nuclear extracts from human cell lines and was examined functionally with transient transfection assays in human hepatoma and Drosophila melanogaster Schneider line-2 cells. DNase I footprinting identified two regions of the nucleocapsid promoter, representing three recognition elements, that bound purified Sp1. Gel retardation analysis with Huh7 nuclear extracts demonstrated that each of the three recognition elements bound the same or similar transcription factor(s) as that recognized by the Sp1 consensus sequence recognition element. The function of the nucleocapsid promoter elements was examined by transient transfection assays in D. melanogaster Schneider line-2 cells by using these binding sites cloned into a minimal promoter element. Each of these regulatory regions transactivated transcription from the minimal promoter element in response to exogenously expressed Sp1. In addition, the second Sp1 site was shown to be an essential element of the nucleocapsid promoter in human hepatoma cells. This demonstrates that the hepatitis B virus nucleocapsid promoter contains three functional Sp1 binding sites which may contribute to the level of transcription from this promoter during viral infection.

Animals↗

PubMed1994

Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids.

We demonstrated recently that a fraction of the matrix (M) protein of vesicular stomatitis virus (VSV) binds tightly to cellular membranes in vivo when expressed in the absence of other VSV proteins. This membrane-associated M protein was functional in binding purified VSV nucleocapsids in vitro. Here we show that the membrane-associated M protein is largely associated with a membrane fraction having the density of plasma membranes, indicating membrane specificity in the binding. In addition, we analyzed truncated forms of M protein to identify regions responsible for membrane association and nucleocapsid binding. Truncated M protein lacking the amino-terminal basic domain still associated with cellular membranes, although not as tightly as wild-type M protein, and could not bind nucleocapsids. In contrast, deletion of the carboxy-terminal 14 amino acids did not disrupt stable membrane association or nucleocapsid interaction. These results suggest that the amino terminus of M protein either interacts directly with membranes and nucleocapsids or stabilizes a conformation that is required for M protein to mediate both of these interactions.

Amino Acid Sequence↗

PubMed2000

Nucleic acid-dependent cross-linking of the nucleocapsid protein of Sindbis virus.

The assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. Although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. Through the use of both truncated and mutant Sindbis virus nucleocapsid proteins and a variety of cross-linking reagents, a possible nucleic acid-protein assembly intermediate has been detected. The cross-linked species, a covalent dimer, has been detected only in the presence of nucleic acid and with capsid proteins capable of binding nucleic acid. Optimum nucleic acid-dependent cross-linking was seen at a protein-to-nucleic-acid ratio identical to that required for maximum binding of the capsid protein to nucleic acid. Identical results were observed when cross-linking in vitro assembled core particles of both Sindbis and Ross River viruses. Purified cross-linked dimers of truncated proteins and of mutant proteins that failed to assemble were found to incorporate into assembled core particles when present as minor components in assembly reactions, suggesting that the cross-linking traps an authentic intermediate in nucleocapsid core assembly. Endoproteinase Lys-C mapping of the position of the cross-link indicated that lysine 250 of one capsid protein was cross-linked to lysine 250 of an adjacent capsid protein. Examination of the position of the cross-link in relation to the existing model of the nucleocapsid core suggests that the cross-linked species is a cross-capsomere contact between a pentamer and hexamer at the quasi-threefold axis or is a cross-capsomere contact between hexamers at the threefold axis of the icosahedral core particle and suggests several possible assembly models involving a nucleic acid-bound dimer of capsid protein as an early step in the assembly pathway.

Capsid↗