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1.
Noroviruses are an important cause of epidemic acute gastroenteritis and the viruses recognize human histo-blood group antigens (HBGAs) as receptors. The protruding (P) domain of noroviral capsid, the receptor-binding domain, forms subviral particles in vitro that retain the receptor-binding function. In this study we characterized the structure and HBGA-binding function of the P particle. Structure reconstruction using cryo-EM showed that the P particles are comprised of 12 P dimers that are organized in octahedral symmetry. The dimeric packing of the proteins in the P particles is similar to that in the norovirus capsid, in which the P2 subdomain with the receptor-binding interface is located at the outermost surface of the P particle. The P particles are immunogenic and reveal similar antigenic and HBGA-binding profiles with their parental virus-like particle, further confirming the shared surface structures between the two types of particles. The P particles are easily produced in E. coli and yeast and are stable, which are potentially useful for a broad application including vaccine development against noroviruses. 相似文献
2.
As a commentary on a recently published paper in Immunology, this article summaries the principle of norovirus P particle as a promising vaccine against noroviruses. It emphasizes the importance of P particle formation in the immune enhancement of the vaccine and methods for production/verification of high quality P particles which may be easily neglected by researchers. 相似文献
3.
Koho T Huhti L Blazevic V Nurminen K Butcher SJ Laurinmäki P Kalkkinen N Rönnholm G Vesikari T Hytönen VP Kulomaa MS 《Journal of virological methods》2012,179(1):1-7
Noroviruses are an important cause of epidemic acute gastroenteritis in humans. In this study the production and characterization of GII.4 norovirus virus-like particles (VLPs) in insect cells is reported. Furthermore, the expression of corresponding norovirus polyhistidine-tagged P domain protein in Escherichia coli is described. The protruding P domain of the norovirus capsid is known to contain determinants for antibody and receptor binding. Therefore, P domain proteins were studied as an alternative diagnostic tool for evaluating norovirus infection. Analyses by dynamic light scattering and cryo-electron microscopy revealed the presence of intact VLPs with an average diameter of about 40 nm. Immunostaining and ELISA assays using norovirus-specific human sera revealed that VLPs and the P domain are recognized by norovirus-specific antibodies and by their putative receptor. The VLPs and P domain protein are potentially useful in the development of diagnostic and vaccination tools for noroviruses. 相似文献
4.
目的 了解SZ9711株P粒子与唾液组织血型抗原受体(HBGAs)的结合模式.方法 从诺如病毒SZ9711株基因组中克隆P区基因片段并构建pGEX-4T-1原核表达质粒,在原核细胞中表达目的 重组蛋白并纯化,经溶血酶酶切后释放目的 蛋白.用EIA方法测定SZ9711株和VA387株P粒子与唾液HBGAs的结合情况.结果 SDS-PAGE电泳分析确定重组融合蛋白的表达,经纯化和凝血酶切后获得约38×10^3的目的 蛋白P蛋白.根据EIA分析表明,SZ9711株P粒子与先前报道的VA387株P粒子与唾液HBGAs模式相同,与A、B和O^secretor有亲和力,但与O^non-secretor亲和力非常低.同时,SZ9711与A抗原的亲和力较VA387与A抗原的亲和力低.结论 本研究利用我国分离到的SZ9711株制备的P粒子进行唾液HBGAs受体结合分析,表明与同源性较高的先前报道的VA387 P粒子结合模式相似,为今后研究诺如病毒与宿主受体之间的关系奠定实验基础. 相似文献
5.
Self-assembly of the recombinant capsid protein of a bovine norovirus (BoNV) into virus-like particles and evaluation of cross-reactivity of BoNV with human noroviruses
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None of the enteric caliciviruses except Po/Sapo/GIII/Cowden/80/US replicates in cell culture, which complicates efforts to develop control strategies or to study viral replication. To develop serological assays for bovine noroviruses (BoNVs) and to determine the cross-reactivity of BoNV with human noroviruses, we generated two recombinant baculoviruses, rCV186-OH and rJNCV, to express the capsid genes of Bo/CV186-OH/00/US (Norovirus genogroup III [GIII], genotype 2 [GIII/2]). rCV186-OH expressed the expected 57-kDa capsid protein, but rJNCV expressed a truncated capsid protein of 35 kDa. Sequence analysis of rJNCV identified a single nucleotide deletion in the P domain of the capsid gene, which introduced a stop codon at amino acid 323. The recombinant capsid protein produced by rCV186-OH but not that produced by rJNCV self-assembled into virus-like particles (VLPs) similar to native BoNV. An antibody-capture enzyme-linked immunosorbent assay (ELISA) and antigen-capture ELISA (Ag-ELISA) detected serum antibody and antigen, respectively, from calves infected with Bo/CV186-OH/00/US but not antibodies or antigens to other enteric viruses. In other tests of the GIII/2 BoNV Ag-ELISA, no cross-reactivity was observed with VLPs from one GI and four GII human noroviruses and porcine sapovirus Cowden strain. Because, like human noroviruses, BoNVs do not grow in cell culture, the BoNV VLPs will be useful in the serological assays described for the detection of BoNV antibody and antigen. Consistent with the phylogenetic analysis of the capsid genes of bovine and human noroviruses (M. G. Han, J. R. Smiley, C. Thomas, and L. J. Saif, J. Clin. Microbiol. 42:5214-5224, 2004), the results suggest that GIII/2 BoNV does not share significant antigenic relationships with the five characterized human noroviruses tested. 相似文献
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7.
Comparative study of infectious and formaldehyde-inactivated tick-borne encephalitis virus particles
I. V. Krasilnikov L. B. Elbert M. K. Khanina S. G. Sobolev 《Archives of virology》1984,79(3-4):241-253
Summary The structure and properties of infectious and formaldehyde-treated particles of tick-borne encephalitis virus, concentrated and purified by chromatography on macroporous glass, were studied.In addition to complete virions, such preparations contain some incomplete forms that differ in density, morphology and protein composition (incomplete forms do not contain nucleocapsid protein).The physico-chemical analysis of complete virions showed that formaldehyde treatment causes a) the formation of glycoprotein dimers and b) a portion of nucleocapsid protein to become tightly cross-linked with viral RNA.Formaldehyde treatment of incomplete forms resulted only in the formation of a small amount of glycoprotein dimers.Incomplete forms and glycoprotein extracted from inactivated preparations had protective and antigenic activity.With 4 Figures 相似文献
8.
The capsid protein of bacteriophage P2, encoded by the N gene, can assemble into icosahedraf capsids of two possible sizes, with diameters of 60 and 45 nm, respectively. Only the larger capsid is used by P2 itself, but the smaller one is exploited by the satellite phage P4. We have analyzed the assembly products of gpN expressed in vivo from a plasmid, i.e., in the absence of any other phage proteins, and find that gpN alone forms closed shells of both sizes, although with poor efficiency. Coexpressing gpN with gpO, the putative P2 scaffolding protein, increases the efficiency of large particle formation. In contrast, introducing the sid gene by P4 infection stimulates the assembly of small particles. Our results suggest that gpO and gpSid act competitively with respect to capsid size determination. Furthermore, we demonstrate that gpN alone undergoes the normal proteolytic maturation steps, implying that gpN processing is either autocatalytic or mediated by a host enzyme. 相似文献
9.
Summary. Cowpea mosaic virus (CPMV) spreads from cell-to-cell as virus particles through tubular structures in modified plasmodesmata which are composed
of viral movement protein (MP). Mutational analysis of the MP has revealed that the N-terminal and central regions of the
MP are involved in tubule formation and that the C-terminal domain probably has a role in the interactions with virus particles.
By constructing C-terminal deletion mutants and comoviral hybrid MPs, it was possible to delineate the C-terminal border of
the tubule-forming domain to a small region between amino acids 292 and 298. Experiments with tripartite viruses in protoplasts
indicated that the C-terminus of the MP is involved in the incorporation of virus particles in the tubule and that for efficient
incorporation of virus particles all MP molecules incorporated in a tubule need to contain a functional C-terminus. A mutant
virus coding for a MP in which the last 10 C-terminal amino acids were replaced by the green fluorescent protein (GFP) was
able to form tubules in protoplasts. These tubules did not contain virus particles, probably because the GFP interferes with
the incorporation of virions into the tubule. These results suggest a model for the structure of the tubule in which the C-terminus
of the MP is located inside the tubular structure, where it is able to interact with virus particles.
Present address: Netherlands Biotech Industry Association, P.O. Box 443, 2260 AK Leidschendam, The Netherlands.
Present address: Department of Tumor Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Received June 6, 2002; accepted September 2, 2002 相似文献
10.
Snezana Maljevic Christian Lerche Guiscard Seebohm Alexi K. Alekov reas E. Busch Holger Lerche 《The Journal of physiology》2003,548(2):353-360
Coexpression of KCNQ2 and KCNQ3 channels results in a 10-fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C-terminus as a potential interaction domain by construction of chimeras with interchanged C-termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C-terminus (Q1ctQ2) showed an 8-fold increase in current amplitude, and Q1ctQ3 a 3-fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C-terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C-terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C-terminus for a dominant-negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A-domain, plus either 40 residues downstream of the A-domain (B-domain) or the proximal C-terminus between S6 and the A-domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10-fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage-dependent activation. The proximal part of the KCNQ3 C-terminus was necessary to produce these effects. Our results indicate that specific parts of the C-terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C-terminus are important for regulating current amplitude. 相似文献
11.
Two chemically similar triazine dyes, coupled to chromatographic gels, have been investigated for their ability to bind and retain hepatitis B surface antigen marker of the hepatitis B virus. Both dyes, Procion Blue-HB and Cibacron Blue 3GA, were effective in removing viral particles from plasma protein solutions. A number of combinations of dye and gel support have been tried, to obtain optimal binding. A Cibacron Blue-Trisacryl adsorbent achieved almost complete retention (greater than 99.5%) of HBsAg before breakthrough occurred. Binding was better at reduced flow rates and on large pore gel matrices, suggesting that steric restrictions limit the interaction. The interaction is directly between immobilised dye and the viral particle, rather than through plasma albumin, which also binds to these affinity adsorbents. These findings are important in the use of immobilised triazine dyes for plasma protein purification. Furthermore, the interaction may be utilised either for the removal of HB viral particles from plasma or as a rapid method of their purification. 相似文献
12.
Purification and properties of virus particles, infectious subviral particles, and cores of bluetongue virus serotypes 1 and 4 总被引:13,自引:0,他引:13
Effective purification methods have been developed for virus particles, infectious subviral particles (ISVP), and virus cores of bluetongue virus (BTV) serotypes 1 and 4. The purified particles were analysed by indirect ELISA or PAGE using either silver staining, or fluorography of [35S]methionine-labelled preparations. No significant contamination with host cell proteins, or with the majority of BTV nonstructural proteins was detectable in any of the particle preparations. In addition to the two major outer capsid and five core proteins previously described, the purified virus particles of both serotypes were consistently found to contain small amounts of BTV protein NS2, previously regarded as exclusively nonstructural. This protein could be removed from the particle surface by treatment with a combination of chymotrypsin and sodium N-lauroyl sarcosinate, which also resulted in the cleavage of the larger of the two major outer capsid components (protein VP2). Two of the cleavage products of VP2 and the whole of the other major outer capsid component (protein VP5) formed a modified outer capsid layer in the resultant ISVP. These subviral particles were as or more infectious than the intact virus particles but had lost haemagglutinating activity. The core-associated RNA polymerase remained inactive in ISVP. 相似文献
13.
The structure and function of the C-terminal domain of the murine leukemia virus Surface protein (MuLV SU) is not well defined. Passage of chimeric ecotropic-amphotropic MuLV viruses with junctions within the SU C-terminus results in the selection of specific point mutations which improve virus viability and Env function. Point mutations were characterized that alter the conformation of the SU/TM heterodimers on the viral particles. Mutation of position E311 within the Moloney MuLV SU protein alters the conformation of the TM protein and its recognition by antibody 42-114 in immunoprecipitation reactions. Mutation of either G541R in the amphotropic 4070A TM, V421M in the 4070A SU, or deletion of S39 and P40 at the N-terminus of the M-MuLV SU results in an irreversible cold-sensitive phenotype at 4 degrees C. This loss of viral titer can be restored by incorporating V421M plus G541R or del S39 P40 plus G541R in cis within the SU/TM. 相似文献
14.
The yeast retrovirus-like element Ty3 GAG3 gene encodes a Gag3 polyprotein analogous to retroviral Gag. Gag3 lacks matrix, but contains capsid, spacer, and nucleocapsid domains. Expression of a Ty3 Gag3 or capsid domain optimized for expression in Escherichia coli was sufficient for Ty3 particle assembly. Virus-like ordered particles assembled from Gag3 were similar in size to immature particles from yeast and contained nucleic acid. However, particles assembled from the CA domain were variable in size and displayed much less organization than native particles. These results indicate that assembly can be driven through interactions among capsid subunits in the particle, but that the nucleocapsid domain, likely in association with RNA, confers order upon this process. 相似文献
15.
16.
Anke Maaßen Annett Rehfeldt S. Kießig A. Ladhoff W. E. Höhne Helga Meisel 《Archives of virology》1994,135(1-2):131-142
Summary The properties of three different recombinant hepatitis B virus core proteins expressed inEscherichia coli were compared: an N-terminal fusion protein, a C-terminally truncated protein and a sequence-authentic protein. All three proteins assembled into capsid-like particles with typical HBc-antigenicity, sedimentation behavior and distinctive electron microscopical images. Apart from this, however, variant HBc proteins displayed properties different from sequence-authentic HBc protein p21.4. Unlike p21.4, the particles of the N-terminal fusion protein p22.2 were sensitive to proteolytic attack by trypsin at variable sites within its arginine-rich C-terminus but not in its extended N-terminus. We therefore conclude that the C-terminal region is located on the surface of the p22.2 particle. These particles also showed increased HBe-antigenicity, as did the C-terminally truncated core particles p17.6, and to an even greater extent p18* particles which were derived from p22.2 by tryptic digestion. This might be interpreted as evidence for an — albeit minor — structural change. All variant core particles were less stable and contained less RNA. Electron microscopic indication for DNA binding of C-terminal deleted p17.6 particles was obtained using an aqueous spreading technique. 相似文献
17.
Vogel M Voigt C Knabe C Radlanski RJ Gross UM Müller-Mai CM 《Journal of biomedical materials research. Part A》2004,70(3):370-379
Bioglass particles of the compositions 45s5, 52s, and 55s were implanted in the distal femoral epiphysis of rabbits. Animals were sacrificed at 7, 28, and 84 days postoperatively and specimens investigated using electron microscopy and electron dispersive X-ray analysis. The intention was to correlate the finding of different types of multinuclear giant cells (MNGC) in the center of the implantation bed with earlier hypothesized accumulated particle eluates and changed particle compositions. The distribution of Si, Na, Ca, P, O, S, and Cl throughout the implantation bed was analyzed. Bioglass particles degraded either in Si-rich remnants or in CaP-shells. MNGC of foreign body giant cell type in high numbers as well as of osteoclast-like type at later time intervals in small numbers were found on the surface of Si-rich as well as on Ca- and P-rich particle remnants. Osteoclast-like cells were detected on the particles after transformation in CaP-shells. It is concluded that the formation of different types of MNGC is determined by the composition of the substrate, that is, osteoclast-like cells develop exclusively on resorbable substrates. The absolute number of MNGC depended on the time after implantation and the solubility of the implant. Bone bonding, however, only occurred on Ca- and P-rich surfaces. 相似文献
18.
K Mabrouk A Benjouad J C Gluckman H Rochat J Van Rietschoten E Bahraoui 《Molecular immunology》1992,29(11):1309-1318
Specificity of anti-p25 antibodies produced against either whole Human Immunodeficiency Virus type 1 (HIV-1) particles in humans and chimpanzees, or against soluble forms of the protein in chimpanzees and rabbits was analyzed by ELISA using a panel of 37 long (> or = 30 residues) or shorter (9-21 residues) overlapping peptides covering the entire p25 sequence. Antibodies elicited by intact virions presented similar reactivity patterns in HIV-1-infected humans and in HIV-1-infected or immunized chimpanzees and recognized only a limited region mostly the C-terminus of the molecule. Moreover, 8 of the human sera (36%), which nonetheless reacted with high titers and avidity with native p25, did not bind to any long or short peptide. These results suggest that the majority of antibodies elicited by viral particles are presumably directed to conformational epitopes. In contrast, antibodies raised against soluble forms of p25 could react against all long peptides but one (residues 211-245) and against some short peptides, indicating that most of p25 sequence may be immunogenic under these conditions. These results suggest that the reactivity spectrum of anti p25 antibodies is rather different if they are produced against intact HIV-1 particles or the soluble protein. They also indicate that it may be possible to manipulate the specificity of the humoral immune response by using either intact virions or purified proteins. 相似文献
19.
Virus-like particles (VLPs) of numerous viruses have been considered as possible candidates for vaccine development. We have
constructed HIV chimeric genes by coupling the gag gene of HIV-2 with the V3 domain of the gp120 gene of either HIV-1 or HIV-2 and expressed the chimeric genes in SF21 cells using the recombinant baculovirus expression
system. Although the level of expression of the chimeric HIV-2 gag gene with the V3 domain of either HIV-1 gp120 (gagC-1V3) or HIV-2 gp120 (gagC-2V3) was high, the VLP assembly and extracellular release of GagC-1V3 was very poor. In contrast, GagC-2V3 chimeric proteins
formed VLPs and released efficiently. We have constructed substitution mutants to investigate the effects of the hydrophobic
region of the V3 domain of HIV-1 Gp120 (1V3) in VLP assembly and release. The substitution mutant analyses revealed that in
replacing the hydrophobic region of the 1V3 in GagC-1V3 with the hydrophilic sequence of the V3 domain of HIV-2 Gp120 (2V3)
enhanced the extracellular VLP. We demonstrate here that disruption of the hydrophobic character of the C-terminus of the
chimeric protein improves assembly and release of the VLPs. Our results suggest that the poor GagC-1V3 VLP release was attributed
to the hydrophobic region in the V3 sequence of the chimeric protein, and that not only the N-terminal myristylation and positively
charged domain of the Gag protein functioned as a targeting signal to direct membrane binding, but also that the C-terminal
hydrophobic region affected release of chimeric VLPs. 相似文献
20.
Chronic hepatitis C virus (HCV) infection results in a progressive disease that may end in cirrhosis and, eventually, in hepatocellular carcinoma. In the last several years, tremendous progress has been made in understanding the HCV life cycle and in the development of small molecule compounds for the treatment of chronic hepatitis C. Nevertheless, the complete understanding of HCV assembly and particle release as well as the detailed characterization and structure of HCV particles is still missing. One of the most important events in the HCV assembly is the nucleocapsid formation which is driven by the core protein, that can oligomerize upon interaction with viral RNA, and is orchestrated by viral and host proteins. Despite a growing number of new factors involved in HCV assembly process, we do not know the three-dimensional structure of the core protein or its topology in the nucleocapsid. Since the core protein contains a hydrophobic C-terminal domain responsible for the binding to cellular membranes, the assembly pathway of HCV virions might proceed via coassembly at endoplasmic reticulum membranes. Recently, new mechanisms involving viral proteins and host factors in HCV particle formation and egress have been described. The present review aims to summarize the advances in our understanding of HCV assembly with an emphasis on the core protein as a structural component of virus particles that possesses the ability to interact with a variety of cellular components and is potentially an attractive target for the development of a novel class of anti-HCV agents. 相似文献