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1.
Capsid region involved in hepatitis A virus binding to glycophorin A of the erythrocyte membrane
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Sánchez G Aragonès L Costafreda MI Ribes E Bosch A Pintó RM 《Journal of virology》2004,78(18):9807-9813
Hepatitis A virus (HAV) has previously been reported to agglutinate human red blood cells at acidic pHs. Treatment of erythrocytes with different enzymes and chemical reagents indicated that HAV attachment is mediated through an interaction with sialylglycoproteins. HAV hemagglutination could be blocked by incubating the virus with glycophorin A, indicating that this sialylglycoprotein is the erythrocyte receptor. The number of receptors used was estimated to be around 500 per cell. At the same time, HAV-induced hemagglutination could also be blocked by either monoclonal antibody H7C27 or an anti-VP3(102-121) ascitic fluid, indicating that lysine 221 of VP1 and the surrounding VP3 residues lining the capsid pit are involved in HAV binding to erythrocytes. 相似文献
2.
Novel type of hepatitis B virus mutation: replacement mutation involving a hepatocyte nuclear factor 1 binding site tandem repeat in chronic hepatitis B virus genotype E
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Fujiwara K Tanaka Y Paulon E Orito E Sugiyama M Ito K Ueda R Mizokami M Naoumov NV 《Journal of virology》2005,79(22):14404-14410
3.
A variant of glycophorin A resulting from the deletion of exon 4 总被引:1,自引:0,他引:1
We have isolated a variant form of glycophorin A which has a 39 bp deletion corresponding to nucleotides 233 to 270 of the coding sequence, which is exon 4 of the glycophorin A gene. The remainder of the sequence is identical to that of the M phenotype of glycophorin A. 相似文献
4.
A M LeVine J A Whitsett K L Hartshorn E C Crouch T R Korfhagen 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(10):5868-5873
Mice lacking surfactant protein surfactant protein D (SP-D(-/-)) and wild-type mice (SP-D(+/+)) were infected with influenza A virus (IAV) by intranasal instillation. IAV infection increased the endogenous SP-D concentration in wild-type mice. SP-D-deficient mice showed decreased viral clearance of the Phil/82 strain of IAV and increased production of inflammatory cytokines in response to viral challenge. However, the less glycosylated strain of IAV, Mem/71, which is relatively resistant to SP-D in vitro, was cleared efficiently from the lungs of SP-D(-/-) mice. Viral clearance of the Phil/82 strain of IAV and the cytokine response were both normalized by the coadministration of recombinant SP-D. Since the airway is the usual portal of entry for influenza A virus and other respiratory pathogens, SP-D is likely to play an important role in innate defense responses to IAV. 相似文献
5.
Steinkühler C Biasiol G Cerretani M Di Renzo L Brunetti M Ingallinella P De Francesco R Altamura S 《Analytical biochemistry》2002,307(1):99-104
A binding assay suitable for the identification of active site-directed inhibitors of the hepatitis C virus serine protease NS3 was developed. A C-terminal extension of 13 residues that is specifically recognized by the Escherichia coli biotin holoenzyme synthetase (Bir A) was fused to a truncated NS3 protease domain, allowing the efficient production of in vivo biotinylated protease. This enzyme was purified and shown to have the same properties as its wild-type counterpart concerning substrate binding and turnover, interaction with a cofactor peptide, and inhibition by three different classes of inhibitors. Immobilization of the biotinylated protease, using streptavidin-coated scintillation proximity beads, allowed detection, by scintillation counting, of its interaction with a tritiated active site ligand spanning the whole substrate binding site of the protease from P6 to P4('). Immobilization did not measurably affect accessibility to either the active site or the cofactor binding site of the protease as judged by the unchanged affinities for a cofactor peptide and for two active site binders. Using the displacement of the radioligand as readout, we were able to set up a rapid, robust, and fully automated assay, suitable for the selective identification of novel active site ligands of the NS3 protease. 相似文献
6.
Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus 总被引:71,自引:0,他引:71
Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences. 相似文献
7.
A single mutation in the activation site of bovine trypsinogen enhances its accumulation in the fermentation broth of the yeast Pichia pastoris 总被引:4,自引:0,他引:4
Hanquier J Sorlet Y Desplancq D Baroche L Ebtinger M Lefèvre JF Pattus F Hershberger CL Vertès AA 《Applied and environmental microbiology》2003,69(2):1108-1113
We produced bovine trypsinogen in the yeast Pichia pastoris. Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells. We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin. This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors. Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation. Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism. This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation. 相似文献
8.
Haines DC Hegde A Chen B Zhao W Bondlela M Humphreys JM Mullin DA Tomchick DR Machius M Peterson JA 《Biochemistry》2011,50(39):8333-8341
Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure. 相似文献
9.
Ingallinella P Fattori D Altamura S Steinkühler C Koch U Cicero D Bazzo R Cortese R Bianchi E Pessi A 《Biochemistry》2002,41(17):5483-5492
Serine proteases are the most studied class of proteolytic enzymes and a primary target for drug discovery. Despite the large number of inhibitors developed so far, very few make contact with the prime site of the enzyme, which constitutes an almost untapped opportunity for drug design. In the course of our studies on the serine protease NS3/4A of hepatitis C virus (HCV), we found that this enzyme is an excellent example of both the opportunities and the challenges of such design. We had previously reported on two classes of peptide inhibitors of the enzyme: (a) product inhibitors, which include the P(6)-P(1) region of the substrate and derive much of their binding energy from binding of their C-terminal carboxylate in the active site, and (b) decapeptide inhibitors, which span the S(6)-S(4)' subsites of the enzyme, whose P(2)'-P(4)' tripeptide fragment crucially contributes to potency. Here we report on further work, which combined the key binding elements of the two series and led to the development of inhibitors binding exclusively to the prime site of NS3/4A. We prepared a small combinatorial library of tripeptides, capped with a variety of constrained and unconstrained diacids. The SAR was derived from multiple analogues of the initial micromolar lead. Binding of the inhibitor(s) to the enzyme was further characterized by circular dichroism, site-directed mutagenesis, a probe displacement assay, and NMR to unequivocally prove that, according to our design, the bound inhibitor(s) occupies (occupy) the S' subsite and the active site of the protease. In addition, on the basis of the information collected, the tripeptide series was evolved toward reduced peptide character, reduced molecular weight, and higher potency. Beyond their interest as HCV antivirals, these compounds represent the first example of prime site inhibitors of a serine protease. We further suggest that the design of an inhibitor with an analogous binding mode may be possible for other serine proteases. 相似文献
10.
The membranes from Miltenberger Class I (Mi I) and II (Mi II) erythrocytes, two rare variants at the blood group MNSs locus, exhibited an abnormal glycoprotein of 32 kDa apparent molecular mass sharply stained by the periodic acid/Schiff procedure and a decreased content of glycoprotein alpha (synonym glycophorin A, glycoprotein MN) as seen on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Purified 125I-labelled Vicia graminea lectin binds to the unusual 32 kDa glycoprotein separated from Mi I and Mi II erythrocyte membrane of blood group NN or MN, but no significant labelling of this band was observed with Mi samples typed MM. On the basis of such lectin-labelling experiments we have described two heterozygous MN, Mi I individuals that carry one copy of an M gene producing a normal alpha-glycoprotein with M-specificity and one copy of a MiI gene producing a 32 kDa glycoprotein with N-specificity. Further investigations have shown that the 32 kDa glycoprotein was immunoprecipitated by two mouse monoclonal antibodies (R18 and R10) reacting specifically with the external domain of glycoprotein alpha. These results demonstrate that Mi I and Mi II erythrocytes carry an unusual variant of glycoprotein alpha. 相似文献
11.
Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase
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Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 A away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3' untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo. 相似文献
12.
Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity 总被引:2,自引:0,他引:2
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Cellular cathepsins are required for Ebola virus infection and are believed to proteolytically process the Ebola virus glycoprotein (GP) during entry. However, the significance of cathepsin cleavage during infection remains unclear. Here we demonstrate a role for cathepsin L (CatL) cleavage of Ebola virus GP in the generation of a stable 18-kDa GP1 viral intermediate that exhibits increased binding to and infectivity for susceptible cell targets. Cell binding to a lymphocyte line was increased when CatL-proteolysed pseudovirions were used, but lymphocytes remained resistant to Ebola virus GP-mediated infection. Genetic removal of the highly glycosylated mucin domain in Ebola virus GP resulted in cell binding similar to that observed with CatL-treated full-length GP, and no overall enhancement of binding or infectivity was observed when mucin-deleted virions were treated with CatL. These results suggest that cathepsin cleavage of Ebola virus GP facilitates an interaction with a cellular receptor(s) and that removal of the mucin domain may facilitate receptor binding. The influence of CatL in Ebola virus GP receptor binding should be useful in future studies characterizing the mechanism of Ebola virus entry. 相似文献
13.
Uebelhoer L Han JH Callendret B Mateu G Shoukry NH Hanson HL Rice CM Walker CM Grakoui A 《PLoS pathogens》2008,4(9):e1000143
Mechanisms by which hepatitis C virus (HCV) evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA) class I-restricted epitopes targeted by CD8(+) T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637), displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC) anchor and T cell receptor (TCR) contact residues. Only one of these amino acid substitutions at position 9 (P9) of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7) TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily stable, but are eventually replaced with variants that achieve a balance between immune evasion and fitness for replication. 相似文献
14.
15.
A single mutation in the E2 glycoprotein important for neurovirulence influences binding of sindbis virus to neuroblastoma cells
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The amino acid at position 55 of the E2 glycoprotein (E2(55)) of Sindbis virus (SV) is a critical determinant of SV neurovirulence in mice. Recombinant virus strain TE (E2(55) = histidine) differs only at this position from virus strain 633 (E2(55)= glutamine), yet TE is considerably more neurovirulent than 633. TE replicates better than 633 in a neuroblastoma cell line (N18), but similarly in BHK cells. Immunofluorescence staining showed that most N18 cells were infected by TE at a multiplicity of infection (MOI) of 50 to 500 and by 633 only at an MOI of 5,000, while both viruses infected essentially 100% of BHK cells at an MOI of 5. When exposed to pH 5, TE and 633 viruses fused to similar extents with liposomes derived from BHK or N18 cell lipids, but fusion with N18-derived liposomes was less extensive (15 to 20%) than fusion with BHK-derived liposomes ( approximately 50%). Binding of TE and 633 to N18, but not BHK, cells was dependent on the medium used for virus binding. Differences between TE and 633 binding to N18 cells were evident in Dulbecco's modified Eagle medium (DMEM), but not in RPMI. In DMEM, the binding efficiency of 633 decreased significantly as the pH was raised from 6.5 to 8.0, while that of TE did not change. The same pattern was observed with RPMI when the ionic strength of RPMI was increased to that of DMEM. TE bound better to heparin-Sepharose than 633, but this difference was not pH dependent. Growth of N18 and BHK cells in sodium chlorate to eliminate all sulfation decreased virus-cell binding, suggesting the involvement of sulfated molecules on the cell surface. Taken together, the presence of glutamine at E2(55) impairs SV binding to neural cells under conditions characteristic of interstitial fluid. We conclude that mutation to histidine participates in or stabilizes the interaction between the virus and the surface of neural cells, contributing to greater neurovirulence. 相似文献
16.
T-cell-mediated clearance of mouse hepatitis virus strain JHM from the central nervous system 总被引:12,自引:10,他引:2
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Clearance of the neurotropic JHM strain of mouse hepatitis virus from the central nervous system was examined by the transfer of spleen cells from immunized donors. A T cell with the surface phenotype of Thy1.2+ CD4+ CD8- asialo-GM1+ Mac-1- was found to be necessary for viral clearance. The surface phenotype and adherence to nylon wool suggest that these cells are activated helper-inducer T cells. Adoptive transfer to congenic histocompatibility strains demonstrated the necessity for compatibility at the D locus of the major histocompatibility complex. The expression of the CD4 surface marker and the requirement for major histocompatibility complex class I were further studied by the transfer of cells to recipients treated with anti-CD4 or anti-CD8 monoclonal antibodies. Treatment of recipients with either the anti-CD8 or the anti-CD4 antibodies inhibited virus clearance from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system. This suggests that the CD4+ cell acts as a helper and that virus is cleared from the central nervous system by CD8+ cells that recognize viral antigen in the context of the H-2Db gene product. 相似文献
17.
A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity 总被引:10,自引:0,他引:10
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Faber M Faber ML Papaneri A Bette M Weihe E Dietzschold B Schnell MJ 《Journal of virology》2005,79(22):14141-14148
Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs. 相似文献
18.
Substitution in the murine nectin1 receptor of a single conserved amino acid at a position distal from the herpes simplex virus gD binding site confers high-affinity binding to gD
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Menotti L Casadio R Bertucci C Lopez M Campadelli-Fiume G 《Journal of virology》2002,76(11):5463-5471
By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC'C" surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of beta-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD. 相似文献
19.
Monné M Miniero DV Daddabbo L Robinson AJ Kunji ER Palmieri F 《The Journal of biological chemistry》2012,287(11):7925-7934
Mitochondrial carriers are a large family of proteins that transport specific metabolites across the inner mitochondrial membrane. Sequence and structure analysis has indicated that these transporters have substrate binding sites in a similar location of the central cavity consisting of three major contact points. Here we have characterized mutations of the proposed substrate binding site in the human ornithine carriers ORC1 and ORC2 by carrying out transport assays with a set of different substrates. The different substrate specificities of the two isoforms, which share 87% identical amino acids, were essentially swapped by exchanging a single residue located at position 179 that is arginine in ORC1 and glutamine in ORC2. Altogether the substrate specificity changes demonstrate that Arg-179 and Glu-180 of contact point II bind the C(α) carboxylate and amino group of the substrates, respectively. Residue Glu-77 of contact point I most likely interacts with the terminal amino group of the substrate side chain. Furthermore, it is likely that all three contact points are involved in the substrate-induced conformational changes required for substrate translocation because Arg-179 is probably connected with Arg-275 of contact point III through Trp-224 by cation-π interactions. Mutations at position 179 also affected the turnover number of the ornithine carrier severely, implying that substrate binding to residue 179 is a rate-limiting step of the catalytic transport cycle. Given that Arg-179 is located in the vicinity of the matrix gate, it is concluded that it is a key residue in the opening of the carrier to the matrix side. 相似文献
20.
Suzuki M Ohki G Ishibashi K Imai M 《Biochemical and biophysical research communications》2002,291(2):278-285
Epithelial Ca(2+) channel (ECaC1 and 2 = CAT1) molecules are characterized by properties including inward rectification and Ca(2+)-dependent fast and slow inactivation. To elucidate the electrophysiological differences based on the amino acid residues, we compared human and rodent ECaC1, and ECaC2 alignments, made mutants, and investigated their function in Xenopus and mammalian cells. Expression of the ECaC1 mutant Q579H and a H587Q mutation in ECaC2 in Xenopus oocytes resulted in a possible change in the rate of fast decay. Currents of H587C and H587N were not detected, and the H587R diminished the rate of rapid decay. Treatment of the oocytes with BAPTA magnified the amplitude of the current and abolished the decay. The expressions of mutants, therefore, implied that H587 in ECaC2 is a position related to the mechanism of the rapid decay rather than the magnitude of the current or the slow decay. Decay measurements were carefully performed in mammalian cells by tight-seal patch clamping. The rapid decay was exaggerated in H587C and H587N mutants but was undetectable in the H587R mutant. The results indicate that the amino acid 579Q of ECaC1, corresponding to 587H of ECaC2, is of primary importance in the structure for the fast inactivation by intracellular Ca(2+). 相似文献