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1.
B C Barnett D S Burt C M Graham A P Warren J J Skehel D B Thomas 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(8):2663-2669
In a recent study, we reported extensive diversity in the Iak-restricted T cell repertoire for the hemagglutinin molecule (HA) of influenza A viruses (H3 subtype). Synthetic peptides identified six nonoverlapping epitopes on the HA1 subunit, and CD4+ T cell clones, specific for these regions, discriminated between natural variant viruses that had accumulated amino acid substitutions during antigenic drift. Here, we demonstrate similar specificity and diversity for the Iad haplotype and have identified multiple T cell epitopes within the sequences HA1 56-76, 71-91, 81-97, 177-199, 186-205, and 206-227. These also include recognition sites for neutralizing antibodies and correlations can be made between antigenic drift substitutions in H3 subtype viruses and the specificity of individual CD4+ clones for mutant HA. Moreover, these peptides appear not to exhibit structural homology and fail to compete for Ag presentation, indicating heterogeneity in peptide-Ia interaction. To explain the observation that CD4+ T cells, from two major haplotypes, recognize antibody binding regions of the HA molecule, we propose that surface Ig receptors of the Ag-specific B memory cell exert a direct effect on the processing of HA peptides and subsequent selection of the class II-restricted T cell memory repertoire after natural infection. 相似文献
2.
K H Mills D S Burt J J Skehel D B Thomas 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(12):4083-4090
We have previously demonstrated diversity in the specificity of murine, H-2k class II-restricted, T cell clones for the hemagglutinin (HA) molecule of H3N2 influenza viruses and have mapped two T cell determinants, defined by synthetic peptides, to residues 48-68 and 118-138 of HA1. In this study we examine the nature of the determinant recognized by six distinct P48-68-specific T cell clones by using a panel of truncated synthetic peptides and substituted peptide analogs. From the peptides tested, the shortest recognized were the decapeptides, P53-62 and P54-63, which suggests that the determinant was formed from the 9 amino acids within the sequence 54-62. Asn54 was critical for recognition since P49-68 (54S) was not recognized by the T cell clones. Furthermore this peptide analog was capable of competing with P48-68 for Ag presentation, thereby suggesting that residue 54 is not involved in Ia interaction and may therefore be important for TCR interaction. Residue substitutions at position 63 also affected T cell recognition, but in a more heterogeneous fashion. Peptide analogs or mutant viruses with a single amino acid substitution at position 63 (Asp to Asn or Tyr) reduced the responses of the T cell clones to variable extents, suggesting that Asp63 may form part of overlapping T cell determinants. However since the truncated peptide P53-62 was weakly recognized, then Asp63 may not form part of the TCR or Ia interaction site, but may affect recognition through a steric or charge effect when substituted by Asn or Tyr. Ag competition experiments with the two unrelated HA peptides, P48-68 and P118-138, recognized by distinct T cell clones in the context of the same restriction element (I-Ak), showed that the peptides did not compete for Ag presentation to the relevant T cell clones, whereas a structural analog of P48-68 was a potent inhibitor. This finding is discussed in relation to the nature of the binding site for peptide Ag on the class II molecule. 相似文献
3.
C J Hackett J L Hurwitz B Dietzschold W Gerhard 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(2):1391-1394
The immunogenicity of an isolated murine helper T cell determinant was studied. Mice were immunized with a synthetic peptide corresponding to amino acid residues 111-120 of the influenza PR8 hemagglutinin (HA) heavy chain, a region previously identified as a major target of the helper T cell response to the HA molecule in virus-primed BALB/c mice. Lymph node T cells from these mice were fused with BW 5147 cells to produce T hybrids for clonal analysis of their recognition specificities. Three T cell hybridoma clones, obtained from two different mice, responded to the immunizing peptide when presented by syngeneic antigen-presenting cells. All of these clones responded also to antigen provided as intact wild-type PR8 virus. The fine specificity of the peptide-induced T cell hybridomas, in response to a panel of mutant and variant influenza viruses, was indistinguishable from the fine specificities of T cells to the corresponding region of the HA1 chain of the HA molecule which had been generated by priming of mice with intact wild-type virus. These results suggest that an immunogenic determinant is contained within the 111-120 sequence that is able to elicit anti-influenza virus T cells with a similar repertoire to those elicited by immunization with whole virus. 相似文献
4.
D B Thomas J Hodgson P F Riska C M Graham 《Journal of immunology (Baltimore, Md. : 1950)》1990,144(7):2789-2794
Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis. 相似文献
5.
To define the relative contributions of HLA and peptide contacts with TCR complementarity-determining region (CDR) 3 residues in T cell recognition, systematic mutagenesis and domain swapping was conducted on two highly similar TCRs that both respond to the influenza hemagglutinin (HA) peptide, HA307-319, but with different HLA restrictions. Despite the primary sequence similarity of these TCRs, exchange of as little as two CDR3 residues between them completely abrogated responsiveness. At position 95 within CDR3alpha, various substitutions still allowed for some degree of recognition. One modest substitution, alanine for glycine (essentially the addition of a methyl group), significantly broadened the specificity of the TCR. Transfectants expressing this mutant TCR responded strongly in the context of multiple HLA-DR alleles and to HA peptide variants with substitutions at each TCR contact residue. These results suggest that the conformations of CDR3 loops are crucial to TCR specificity and that it may not be reliable to extrapolate from primary sequence similarities in TCRs to similarities in specificity. The ease with which a broad specificity is induced in this mutant TCR has implications for the mechanisms and frequency of alloreactivity and promiscuity in T cell responses. 相似文献
6.
H Quill R H Schwartz L H Glimcher 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(9):3351-3359
Antigen-presenting cells (APC) expressing mutant Ek beta and Ak alpha proteins were isolated after chemical mutagenesis of TA3 cells and negative immunoselection for altered Ek beta molecules. Mutant clones were analyzed for biosynthesis, assembly, and cell surface expression of altered Ia molecules, and were assayed for antigen-presenting function by using a variety of T cell clones. Three types of mutants were detected: type 1, which had lost expression of the Ek beta chain and produced altered Ak alpha chains; type 2, which also expressed altered Ak alpha chains, and which expressed Ek beta proteins that had lost reactivity to the 17.3.3 and 74D monoclonal antibodies (mAb), but retained reactivity to other anti-Ek beta mAb; and type 3, which had lost expression of both Ek beta and Ak beta: Ak alpha surface molecules. Thus, all of the mutant clones that produced modified Ak alpha proteins also displayed either total loss or serologic modification of the Ek beta molecule. Ek beta:E alpha-reactive T cell clones were not stimulated when type 1 or type 3 cells were used as APC, but all such T cells were fully reactive with type 2 mutant APC. Most Ak beta:Ak alpha-reactive T cell clones could respond to type 1 and 2 APC, and none were responsive to type 3 APC. However, two autoreactive Ak beta:Ak alpha-specific T cell hybridomas were stimulated only very weakly by type 1 and type 2 cells expressing modified Ak alpha proteins. These results demonstrate that Ia mutations can have highly selective effects on antigen presentation to T cells as well as on mAb binding, and thus suggest that individual Ia molecules may be composed of many different functional subsites. 相似文献
7.
G G Schlauder M P Bell B N Beck A Nilson D J McKean 《Journal of immunology (Baltimore, Md. : 1950)》1985,135(3):1945-1954
Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides. 相似文献
8.
R W Karr P Panina-Bordignon W Y Yu A Lanzavecchia 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(12):4242-4247
The contributions of the amino acids at 13 polymorphic positions in the HLA-DR7 beta 1 chain to T cell recognition of two antigenic peptides of tetanus toxin (p2 and p30) were assessed using transfectants expressing mutant DR7 beta 1 chains as APC for six toxin-specific T cell clones with two different restriction patterns: monogamous (restricted by DR7 only) or promiscuous (restricted by DR7; DR1; DR2, Dw21; and DR4, Dw4). Each of the 13 substitutions significantly decreased or eliminated the ability of the DR7 molecule to present a peptide to one or more of the T cell clones, but none of the substitutions abolished recognition by all clones. Interestingly, substitutions at positions 4 and 25, which are predicted in the class II model to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to present Ag to some clones but not to others. Each of the four clones specific for the p2 peptide and the two clones specific for peptide p30 had a different reactivity pattern to the panel of DR7 beta 1 mutants, indicating that the TCR of each clone has a different view of the p2/DR7 or p30/DR7 complex. These data emphasize the complexity of the interactions of multiple residues in DR7 beta 1 chains in Ag-specific T cell recognition. 相似文献
9.
R R Olson M T De Magistris A Di Tommaso R W Karr 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(9):2703-2708
We have examined the role of 12 polymorphic residues of the beta-chain of the HLA-DR1 class II molecule in T cell recognition of an epitope of pertussis toxin. Murine L cell transfectants expressing wild-type or mutant DR1 molecules (containing single amino acid substitutions in DR(beta 1*0101)) were used as APC in proliferation assays involving nine DR1-restricted T cell clones specific for peptide 30-42 of pertussis toxin. Four different patterns of recognition of the mutants were found among the pertussis-specific clones. Residues in the third hypervariable region (HVR) of DR(beta 1*0101) are critically important for all the T cell clones; amino acid substitutions at positions 70 and 74 abrogated recognition by all of the T cell clones, and substitutions at positions 67 and 71 eliminated recognition by most of the clones. In contrast, most single amino acid substitutions in the first and second HVR, predicted to be located in the floor of the peptide binding groove, had little or no effect on the proliferative responses of these clones. However, the involvement of beta-chain first and second HVR residues was demonstrated by the inability of transfectants expressing wild-type DR(beta 1*0404) (DR4Dw14) or DR(beta 1*1402) (DR6Dw16) to present peptide to these clones. These beta-chains have completely different first and second HVR compared with DR(alpha,beta 1*0101) although the third HVR are identical. These results illustrate the functional importance of third HVR residues of DR(beta 1*0101) and allow definition of the molecular interactions of the DR1 molecule with the 30-42 peptide. 相似文献
10.
S Rosen-Bronson W Y Yu R W Karr 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(12):4264-4270
The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule. 相似文献
11.
Evidence that multiple residues on both the alpha-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor 总被引:8,自引:0,他引:8
P Ajitkumar S S Geier K V Kesari F Borriello M Nakagawa J A Bluestone M A Saper D C Wiley S G Nathenson 《Cell》1988,54(1):47-56
Single amino acid substitutions at nine different positions on the H-2Kb molecules from in vitro-mutagenized, immunologically altered, somatic cell variants were correlated with their patterns of recognition by monoclonal antibodies (MAbs) and allogeneic cytotoxic T lymphocyte (CTL) clones. While MAbs were found to detect spatially discrete, domain-specific sites, CTLs interacted simultaneously with multiple residues on the alpha 1 and alpha 2 domains of the Kb molecule. The computer graphic three-dimensional Kb model structure showed that, of the seven CTL-specific residues analyzed, six residues were located on the alpha-helical regions of the two domains. Every CTL clone was found to interact with a distinct pattern of residues composed of a specific subset of the CTL-specific residues. 相似文献
12.
Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin. 总被引:3,自引:2,他引:1
L E Brown R A Ffrench J M Gawler D C Jackson M L Dyall-Smith E M Anders G W Tregear L Duncan P A Underwood D O White 《Journal of virology》1988,62(1):305-312
A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA. 相似文献
13.
.174xCEM.T2 (T2) is a human cell hybrid that has a large homozygous deletion within the MHC, including all of the functional class II genes. We have generated stable HLA-DR3 and H-2 I-Ak transfectants of T2 that express parental levels of class II molecules at the cell surface. T2.Ak transfectants fail to stimulate a hen egg lysozyme (HEL)-specific, I-Ak-restricted T cell when incubated with intact HEL. However, stimulation occurs if the appropriate HEL peptide is provided. The T2 cell line therefore has a defect in class II-restricted Ag processing. Biosynthetic studies demonstrate that the kinetics of I-Ak transport in T2.Ak are similar to the parental rates of transport, although the percentage of I-Ak molecules transported appears somewhat lower. I-Ak glycoproteins in T2.Ak associate normally with the I-chain, which appears to be proteolytically cleaved after transport through the Golgi apparatus in a similar fashion to that in the parent cell line, .174xCEM.T1 (T1). The DR alpha beta heterodimers in T2 differ from the parental phenotype in two ways. First, HLA-DR3 expressed in T2 does not have the epitope recognized by the DR3-specific mAb 16.23, although DR3 expressed in the parent does have the epitope. Second, the alpha beta subunits in the parent remain associated when exposed to SDS at room temperature, although those in T2 dissociate. 相似文献
14.
Functional constraints of influenza A virus epitopes limit escape from cytotoxic T lymphocytes
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Berkhoff EG de Wit E Geelhoed-Mieras MM Boon AC Symons J Fouchier RA Osterhaus AD Rimmelzwaan GF 《Journal of virology》2005,79(17):11239-11246
Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes. 相似文献
15.
C B Davis D J Mitchell D C Wraith J A Todd S S Zamvil H O McDevitt L Steinman P P Jones 《Journal of immunology (Baltimore, Md. : 1950)》1989,143(7):2083-2093
The effect of polymorphic residues on the A alpha A beta molecule on T cell recognition of the N-terminal nonapeptide of myelin basic protein (R1-9) was determined. Ak-restricted T cell clones recognizing R1-9 were isolated. The peptide-Ia specificities of these clones were determined by testing the response to 1) a panel of peptide analogs of R1-11, 2) splenic APC from mice expressing MHC molecules from serologically distinct haplotypes, and 3) L cell transfectants expressing mutant/recombinant A beta cDNA containing combinations of polymorphic nucleotide sequences from the k and u alleles. Comparisons were made between the Ak-restricted clones and a previously characterized panel of Au-restricted clones. Certain Ak-restricted clones were able to recognize MBP peptide analogs that were not recognized by any of the Au-restricted clones. The Au-restricted T cell clones did not cross-react with R1-9 presented in the context of Ak, whereas the majority of the Ak-restricted clones responded to R1-9 presented in the context of Au. This nonreciprocal cross-reactivity was also reflected in the relative responses of the two sets of T cell clones to the interchange of u- and k-derived residues in the A beta chain. Residues in regions corresponding both the alpha-helical or beta-sheet portions of the hypothetical Ia three-dimensional structure were involved. The results suggest that overall specificity of the T cell clones is the summation of numerous distinct subspecificities for different regions of the peptide-Ia ligand. These results indicate that there can be striking differences in T cell specificity for an autoantigenic epitope, even in the context of A alpha A beta molecules from very closely related haplotypes. 相似文献
16.
A M Haberman C Moller D McCreedy W U Gerhard 《Journal of immunology (Baltimore, Md. : 1950)》1990,145(9):3087-3094
The site-1 determinant of the hemagglutinin molecule of influenza virus (A/PR/8/34) is one of several immunodominant sites in the BALB/c Th cell response to Ha. A synthetic peptide comprising this T cell site (HA110-120), a panel of analogs containing single substitutions in this determinant, and homologs truncated at the amino- or carboxyl-terminal were used to examine the fine specificities of 15 T cells specific for site-1 in the context of I-Ed. The results indicate that every residue within the minimal determinant plays a role in the T cell recognition process, as single substitutions at any of these positions affected the ability of the peptide to stimulate at least some site 1-specific T cells. For the majority of the residues examined, substitutions had dissimilar effects on distinct T cells, indicating that the substituted residues were affecting recognition in a receptor-specific manner. Each of the 15 T cells examined had a distinct fine specificity pattern, suggesting that the BALB/c T cell repertoire for this site is likely to exceed 100 distinct clonotypes. 相似文献
17.
Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin. 总被引:2,自引:0,他引:2
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Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy-terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes. 相似文献
18.
Wang M Tscherne DM McCullough C Caffrey M García-Sastre A Rong L 《Journal of virology》2012,86(8):4455-4462
Influenza A virus glycoprotein hemagglutinin (HA) binds to host cell surface sialic acid (SA)-terminated sugars in glycoproteins to initiate viral entry. It is thought that avian influenza viruses preferentially bind to N-acetylneuraminic acid α3 (NeuAcα3) sugars, while human influenza viruses exhibit a preference for NeuAcα6-containing sugars. Thus, species-specific SA(s) is one of the determinants in viral host tropism. The SA binding pocket of the HA1 subunit has been extensively studied, and a number of residues important for receptor binding have been identified. In this study, we examined the potential roles of seven highly conserved HA surface-located amino acid residues in receptor binding and viral entry using an H5 subtype. Among them, mutant Y161A showed cell-type-dependent viral entry without obvious defects in HA protein expression or viral incorporation. This mutant also displayed dramatically different ability in agglutinating different animal erythrocytes. Oligosaccharide binding analysis showed that substituting alanine at Y161 of HA changed the SA binding preference from NeuAc to N-glycolylneuraminic acid (NeuGc). Rescued mutant Y161A viruses demonstrated a 5- to 10-fold growth defect, but they were robust in viral replication and plaque forming ability. Our results demonstrate that Y161 is a critical residue involved in recognition of different SA species. This residue may play a role in determining influenza virus host tropism. 相似文献
19.
Single amino acid changes in DR and antigen define residues critical for peptide-MHC binding and T cell recognition. 总被引:13,自引:0,他引:13
J I Krieger R W Karr H M Grey W Y Yu D O'Sullivan L Batovsky Z L Zheng S M Colón F C Gaeta J Sidney 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(7):2331-2340
Single amino acid substitutions of Ag and MHC were used to analyze the fine structure of the influenza hemagglutinin (HA)-derived epitope (HA 307-319) recognized in the context of DR7 molecules by a T cell clone. Putative T cell (HA 308, 310, 311, 313, and 316) and DR (HA 309, 312, and 317) contact residues of the Ag were identified by the use of single amino acid-substituted analogs that were tested for their T cell-activating and DR-binding capacities. The peptide-DR7-T cell interaction was further characterized by the use of a panel of 13 site-directed DR7 mutant transfectants analyzed for their capacity to present Ag to T cells, and for their purified mutant DR7 molecules to bind HA 307-319 or its single amino acid-substituted analogs. Eight mutants lost their Ag-presenting function, whereas only one had any decrease in peptide binding. Finally, for three of the mutants it was possible to correct the deleterious effects of mutation by using a particular single amino acid-substituted analog of the peptide molecule. The observed pattern of complementation led to a model that predicts that the Ag assumes an extended conformation, with a turn, in the binding groove, such that the following residues are in close proximity: DR 86-HA 309, DR 71-HA 312, DR 30-HA 314, and 315. 相似文献
20.
Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. 总被引:14,自引:4,他引:10
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To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses. 相似文献