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1.
The E protein is a multifunctional membrane protein of SARS-CoV 总被引:1,自引:0,他引:1
Wu Q Zhang Y Lü H Wang J He X Liu Y Ye C Lin W Hu J Ji J Xu J Ye J Hu Y Chen W Li S Wang J Wang J Bi S Yang H 《基因组蛋白质组与生物信息学报(英文版)》2003,1(2):131-144
The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin. 相似文献
2.
It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed. 相似文献
3.
Recent work has shown that efficient di- or trimerization of hydrophobic transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a non-aqueous subcompartment within the translocon. 相似文献
4.
SARS冠状病毒E、M基因序列比较及B细胞抗原表位预测 总被引:2,自引:0,他引:2
为了比较SARS冠状病毒分离株E、M基因序列及氨基酸序列之间的差异,分析E、M蛋白的可能B细胞抗原表位。利用Lasergene软件包中的Editseq将E、M基因从SARS-CoV全基因序列中截取出,再翻译成氨基酸序列,用Clustal X软件分析它们之间的异同,然后利用Protean软件进行氨基酸序列分析,预测E、M蛋白的B细胞抗原表位。结果证明SAPS-CoV的E、M基因序列相当保守,变异甚少,并分别预测出E、M蛋白有2段和7段可能为B细胞抗原表位。 相似文献
5.
Hu Y Wen J Tang L Zhang H Zhang X Li Y Wang J Han Y Li G Shi J Tian X Jiang F Zhao X Wang J Liu S Zeng C Wang J Yang H 《基因组蛋白质组与生物信息学报(英文版)》2003,1(2):118-130
We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To inve 相似文献
6.
Bowie JU 《Protein science : a publication of the Protein Society》1999,8(12):2711-2719
In the fold recognition approach to structure prediction, a sequence is tested for compatibility with an already known fold. For membrane proteins, however, few folds have been determined experimentally. Here the feasibility of computing the vast majority of likely membrane protein folds is tested. The results indicate that conformation space can be effectively sampled for small numbers of helices. The vast majority of potential monomeric membrane protein structures can be represented by about 30-folds for three helices, but increases exponentially to about 1,500,000 folds for seven helices. The generated folds could serve as templates for fold recognition or as starting points for conformational searches that are well distributed throughout conformation space. 相似文献
7.
SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity. 相似文献
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9.
Coronavirus envelope (E) protein is a small integral membrane protein with multi-functions in virion assembly, morphogenesis and virus-host interaction. Different coronavirus E proteins share striking similarities in biochemical properties and biological functions, but seem to adopt distinct membrane topology. In this report, we study the membrane topology of the SARS-CoV E protein by immunofluorescent staining of cells differentially permeabilized with detergents and proteinase K protection assay. It was revealed that both the N- and C-termini of the SARS-CoV E protein are exposed to the cytoplasmic side of the membranes (N(cyto)C(cyto)). In contrast, parallel experiments showed that the E protein from infectious bronchitis virus (IBV) spanned the membranes once, with the N-terminus exposed luminally and the C-terminus exposed cytoplasmically (N(exo(lum)-)C(cyto)). Intriguingly, a minor proportion of the SARS-CoV E protein was found to be modified by N-linked glycosylation on Asn 66 and inserted into the membranes once with the C-terminus exposed to the luminal side. The presence of two distinct membrane topologies of the SARS-CoV E protein may provide a useful clue to the pathogenesis of SARS-CoV. 相似文献
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11.
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) structural proteins (S, E, M, and NC) localize in different
subcellular positions when expressed individually. However, SARS-CoV M protein is co-localized almost entirely with S, E,
or NC protein when co-expressed in the cells. On the other hand, only partial co-localization was observed when S and E, S
and NC, or E and NC were co-expressed in the cells. Interactions between SARS-CoV M and other structural proteins but not
interactions between S and E, S and NC, or E and NC were further demonstrated by co-immunoprecipitation assay. These results
indicate that SARS-CoV M protein, similar to the M proteins of other coronaviruses, plays a pivotal role in virus assembly.
The cytoplasmic C-terminus domain of SARS-CoV M protein was responsible for binding to NC protein. Multiple regions of M protein
interacted with E and S proteins. A model for the interactions between SARS-CoV M protein and other structural proteins is
proposed. This study helps us better understand protein-protein interactions during viral assembly of SARS-CoV.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
Induction of Th1 type response by DNA vaccinations with N, M, and E genes against SARS-CoV in mice 总被引:6,自引:0,他引:6
Jin H Xiao C Chen Z Kang Y Ma Y Zhu K Xie Q Tu Y Yu Y Wang B 《Biochemical and biophysical research communications》2005,328(4):979-986
Vaccination against the SARS-CoV infection is an attractive means to control the spread of viruses in public. In this study, we employed a DNA vaccine technology with the levamisole, our newly discovered chemical adjuvant, to generate Th1 type of response. To avoid the enhancement antibody issue, genes encoding the nucleocapsid, membrane, and envelope protein of SARS-CoV were cloned and their expressions in mammalian cells were determined. After the intramuscular introduction into animals, we observed that the constructs of the E, M, and N genes could induce high levels of specific antibodies, T cell proliferations, IFN-gamma, DTH responses, and in vivo cytotoxic T cells activities specifically against SARS-CoV antigens. The highest immune responses were generated by the construct encoding the nucleocapsid protein. The results suggest that the N, M, and E genes could be used as the targets to prevent SARS-CoV infection in the DNA vaccine development. 相似文献
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SARS-CoV N蛋白与人冠状病毒HCoV-OC43和HCoV-229E的交叉反应表位及特异表位的确定 总被引:1,自引:0,他引:1
为确定SARS-CoV N蛋白的特异抗原表位,对3种人冠状病毒SARS-CoV、HCoV-OC43和HCoV-229E N蛋白之间的交叉免疫反应进行了系统研究。构建了分别表达SARS-CoV、HCoV-OC43和HCoV-229E N蛋白的重组痘苗病毒,并制备了相应的小鼠免疫血清。用间接免疫荧光方法,检测了3种N蛋白的表达及其与3种冠状病毒免疫动物血清和SARS病人恢复期血清之间的反应。与此同时,用Western blot方法分析了原核表达的39个不同区段的SARS-CoV N蛋白与3种冠状病毒动物免疫血清和SARS病人恢复期血清之间的交叉反应性。免疫荧光检测结果表明,SARS-CoV、HCoV-OC43和HCoV-229E3种病毒的N蛋白在重组痘苗病毒感染的HeLa细胞中均可以特异表达;3种N蛋白之间存在明显交叉免疫反应。Western blot结果显示,SARS-CoV N蛋白的表位主要位于30~60aa、170~184aa、301~320aa和360~422aa;与HCoV-OC43的交叉反应表位主要位于30~60aa、90~120aa、204~214aa和320~360aa;与HCoV-229E的交叉反应表位主要位于30~60aa、150~160aa和301~360aa。含SARS-CoV N蛋白特异表位的重组肽N155b(60~214aa)和N185(30~214aa)只与SARS病人恢复期血清和灭活SARS-CoV免疫小鼠的血清反应,而不与灭活HCoV-OC43和HCoV-229E免疫的山羊血清产生交叉反应。上述结果为使用SARS-CoV N蛋白抗原进行特异诊断试剂的研究,提供了重要的实验依据。 相似文献
15.
Susan Schlegel Anna Hjelm Thomas Baumgarten David Vikström Jan-Willem de Gier 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. 相似文献
16.
为了表达SARS-CoV的S蛋白的受体结合区并对其免疫原性进行分析,用PCR方法扩增S蛋白的受体结合区基因片段,克隆至原核表达质粒pET-F32a+并在大肠杆菌中表达,应用Western—blot鉴定表达的目的蛋白,而后以该蛋白作为诊断抗原包被酶联卡反来检测20份SARS病人血清和28份健康人血清,结果原核表达的S蛋白能够和所用的SARS病人血清反应。这提示表达的S重组蛋白具有良好的抗原性。将变性纯化的重组蛋白和复性蛋白分别皮下免疫小鼠,第三次免疫一周后收集抗血清,用ELISA测定抗体和同时测定中和抗体活性。用变性的抗原免疫的小鼠血清均无中和活性;而用复性的蛋白免疫的小鼠产生了中和抗体。实验表明,S蛋白受体结合区无线性中和表位,中和抗体的产生是由构象表位诱导的。提示该蛋白有可能应用于亚单位疫苗的研究。 相似文献
17.
In mycobacteria, probing the association of cytoplasmic proteins with the membrane itself, as well as with integral or peripheral membrane proteins, is limited by the difficulty in extracting intact sealed membrane vesicles due to the complex cell wall structure. Here we tested the association of Mycobacterium tuberculosis SecA1 and SecA2 proteins with intact membrane vesicles by a flotation assay using iodixanol density gradients. These protocols have wide applications for studying the association of other mycobacterial cytoplasmic proteins with the membrane and membrane-associated proteins. 相似文献
18.
Patrik Björkholm Andreas M. Ernst Moritz Hacke Felix Wieland Britta Brügger Gunnar von Heijne 《生物化学与生物物理学报:生物膜》2014
Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function. 相似文献
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20.
Yi Zhang Wei Wang Jin-rong Gao Li Ye Xiao-nan Fang Ying-chun Zeng Zheng-hui Wu Ying-long She Lin-bai Ye 《中国病毒学》2007,22(1):1-7
SARS-CoV is a newly discovery pathogen causing severe acute respiratory problems. It has been established that the S protein
in this pathogen plays an important rule in the adsorption and penetration of SARS-CoV into the host cell by interaction with
the ACE2 receptor. To determinant which functional motif of the S protein was involved in the interaction with ACE2, seven
truncated S proteins deleted from the N or C terminal were obtained by an E.coli expression system and purified by column
chromatography to homogeneity. Each truncated S protein was fixed on to the well of an ELISA plate and an interaction was
initiated with the ACE2 protein. The adsorption were quantified by ELISA, and the results indicated that amino acids from
388 to 496 of the S protein was responsible for the interaction with the ACE2 receptor, and the interaction could be completely
disrupted by an antibody specific to these amino acids. Deletions adjacent to this domain did not appear to have a significant
impact on the interaction with ACE2, suggesting that the S protein of SARS-CoV could be developed as a vaccine to prevent
the spread of SARS-CoV. 相似文献