【病毒外文文獻(xiàn)】1992 Bovine coronavirus peplomer glycoproteins_ detailed antigenic analyses of S1, S2 and HE
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Journal of General Virology 1992 73 1725 1737 Printed in Great Britain 1725 Bovine coronavirus peplomer glycoproteins detailed antigenic analyses of SI 2 and HE Jean Francois Vautherot Marie Fran oise Madelaine Pascal Boireau and Jacques Laporte LN R A Unit de Virologic et Immunologie Molkculaires C R J J Domaine de Vilvert F78352 Jouy en Josas Cedex France Forty four monoclonal antibodies MAbs to the G11o isolate of bovine enteric coronavirus were used for the characterization of the peplomer proteins S and HE Fourteen of these MAbs reacted with HE and the remaining 30 with the products of the S gene S1 19 MAbs 2 six MAbs and gp200 five MAbs S1 and HE were found to carry major neutralization determi nants and S 1 appeared to elicit the production of the MAbs displaying the highest neutralizing activity The topography of the epitopes was assessed by means of a competitive binding assay the 44 MAbs defined four independent antigenic domains on S1 two on 2 one on gp200 and two on HE All the neutralizing anti S1 MAbs mapped in antigenic sites A and B and all the neutralizing anti HE MAbs in HE B Antigenic site S1 B was further subdivided into four subsites Func tional mapping was performed by testing a library of neutralization resistant mutants against the neutraliz ing MAbs Analysis of their reactivity in a neutral ization test confirmed the overall distribution of epitopes in S1 B and HE B Introduction Bovine coronavirus BCV is an enteric virus which multiplies in the differentiated enterocytes of the small intestine and colon causing acute enteritis in newborn calves Stair et aL 1972 Babiuk et al 1985 and chronic shedding Crouch et al 1985 or diarrhoea Takahashi et aL 1980 in adult cows BCV particles consist of large single stranded positive RNA 30 kb associated with a basic phosphonucleoprotein N Mr 52K forming a helical ribonucleoprotein protected by a viral envelope containing three glycoproteins M S and HE Laporte King Deregt et al 1987 The transmembrane glycoprotein M is deeply embed ded in the lipid bilayer and migrates as a heterogeneous set of polypeptides 23K to 28K reflecting various degrees of glycosylation Deregt et al 1987 The other two glycoproteins S and HE form the two fringes of spikes which are characteristic of BCV and other haemagglutinating coronaviruses Dea King et al 1985 Kienzle et al 1990 The role of HE in the agglutination of murine red blood cells was demonstrated by biochemical investigations King et al 1985 and by the inhibiting activity of monoclonal antibodies MAbs to HE Parker et al 1989 HE also has a receptor binding and a receptor destroying activity RDE similar to that of influenza C virus Vlasak et al 1988a b Schultze et al 1991 the RDE is due to acetylesterase AE activity which appeared to be essential for viral infectivity Vlasak et al 1988b Peplomer proteins have been shown to play a major role in the interaction between the coronaviruses and their target cells or the host immune system for a review see Spaan et al 1988 Consequently considerable work has been done on the characterization of B cell epitopes on the S protein of mouse hepatis virus MHV Wege et al 1984 transmissible gastroenteritis virus TGEV Laude et aL 1986 Delmas et al 1986 1990 and infectious bronchitis virus IBV Niesters et aL 1987 Koch et aL 1990 and their possible association with a biological function The S and HE glycoproteins of BCV were shown to elicit the production of neutralizing MAbs Vautherot Deregt et al 1987 which for some Deregt et al 1987 were used in a topographi cal study of S1 and HE and tested for their protective 0001 0723 1992 SGM 1726 J F Vautherot and others activity against BCV infections in calves Deregt et al 1989 To characterize the peplomer glycoproteins further and in particular the cleavage products of S we selected a large panel of MAbs which allowed us i to characterize S1 and 2 on the basis of their reactivity with MAbs ii to analyse the functional inhibitions exerted by MAbs on the virus and iii to study the distribution of B cell epitopes on each glycoprotein Methods Viruses and cell lines All BCV isolates Laporte et al 1979 Vautherot Sigma Isotopic labelling of intracellular viral polypeptides HRT 18 mono layers were infected as described Vautherot et al 1984 except that the m o i was raised to 20 p f u cell and depending on the experiment 1 85 MBq ml of each of 3H valine and 3H leucine CEA or 0 185 MBq ml of a 14C labelled amino acid mixture CEA were used At the end of the labelling period the cells were rinsed with cold PBS lysed in radioimmunoprecipitation assay RIPA buffer 20mM Tris HC1 pH 7 5 0 5 M NaCI 2 Triton X 100 0 2 mM PMSF and 2ktg ml Aprotinin Sigma The lysates were then prepared as described Delmas et al 1986 Isotopic labelling of virus proteins HRT 18 cells were infected with BCV at an m o i of 0 05 p f u cell and incubated for 24 h Infected monolayers were placed in MM lacking the labelling amino acid for 1 h after which 1 48 MBq ml of each of 3H valine and 3H leucine Amersham were added Infection was stopped 48 to 50 h post infection and the labelled virus was harvested and purified according to standard procedures Spike proteins were solubilized by incubating the virus pellet for 30 min at 25 C in a 60 mM octylglucoside Boehringer Mannheim solution made in Tris HCl buffer pH 7 5 containing 0 15 M NaCl and 2 tg ml Aprotinin octyl buffer Prior to immuno precipitation the detergent disrupted virus was ultracentrifuged for 1 h at 45000 r p m in a Beckman Ti 45 rotor Radioimmunoprecipitation Immunoprecipitations were carried out as described Vautherot et al 1984 Delmas et al 1986 Immune complexes were washed four times with RIPA buffer or octyl buffer once in 20 mM Tris HCl buffer pH 7 5 dissolved in electrophoresis sample buffer Laemmli 1970 and heated at 65 C for 5 rain Viral polypeptides were separated by electrophoresis on polyacrylamide gels using the discontinuous buffer gel system of Laemmli 1970 Gels were dried fluorographed and exposed to Fuji X ray films RX films Fuji at 80 C Western blotting Viral proteins separated by electrophoresis on 10 SDS polyacrylamide gels were transferred to a nitrocellulose sheet BAS 85 Schleicher Calbiochem at 6 tM in HEPES buffer 50 mM HEPES pH 7 4 110 mM NaCI 5 mM KCI and 1 mM MgClz Absorbances were read on a multichannel spectrophoto meter Titertek Multiskan MCC 340 Flow at 405 nm for pNPA or 492 nm for 5 CFDA Purification and conjugation of MAbs MAbs were purified from ascites fluids by ammonium sulphate precipitation and gel filtration on a Sephacryl S 300 Pharmacia column equilibrated in PBS Purified MAbs I7 I9 B5 A20 J18a A9 Z17 and PI1 were conjugated to horseradish peroxidase HRPO by the two step method of Avrameas none of these MAbs was able to bind to the SDS denatured HE data not shown MAbs to HE identified epitopes which were common to all BCV isolates tested as revealed by IIF MAbs to S glycoproteins all of which immunoprecipi tated the S precursor pS or gpl70 were classified as anti S gpl05 anti S gp95 and anti S gp200 according to the polypeptide s they recognized MAbs to S gpl05 pre cipitated a glycoprotein with an Mr of 105K together with pS from infected cell lysates or the 105K species alone when immunoprecipitations were done with radiolabelled detergent disrupted virus Fig 1 lanes 2 Table 1 Characterization of MAbs to HE MAb Isotype ELISA Neutralization HI designation species titre t titret titre Y 16 IgM R 43 700 10900 20 J 18c IgG2b 88 000 17 500 5120 J 17b IgG 1 58 000 3950 80 B4 IgG1 55000 400 2560 B7 IgG2a 275000 700 20480 F 13 IgG 3 37 500 300 5120 A 12 IgG 3 131000 300 20 480 G20 IgG2a 300000 750 640 J 21 IgG 66000 300 160 J l0 IgG2a 71000 300 640 J 18b IgG2a 64 800 300 1280 HI7 IgG2a 101000 300 2560 BI 3 IgG2a 8600 300 0 B22 IgM 5450 200K from infected cell lysates and S gp95 together with a 200K moiety from purified virus preparations Fig 1 lanes 3 and 7 Four of these MAbs reacted strongly with a 95K protein and faintly with a high Mr species 200K on Western blots Fig 2 lane 3 These four MAbs recognized a linear epitope conserved in two recombi nant fusion proteins which were expressed in Escherichia coil transformed by pUEX plasmids containing two overlapping restriction DNA fragments corresponding to the N terminus of 2 J F Vautherot unpublished results From these results and by comparing the Mr deduced from the sequence with the M r observed by PAGE we infer that anti S gpl05 MAbs recognize S1 the amino terminal part of the S molecule Five additional MAbs reacted only with pS and gp240 from infected cell lysates 1728 J F Vautherot and others Table 2 Characterization of MAbs to S glycoproteins Antigenic cross reactivity Polypeptide specificity BCV MAb Isotype ELISA Neutralization G110 F15 designation species titter titret WB RIPA Mebus MHV HEV OC43 C 13 G 1 M 100 6400 S gpl05 S gpl05 P11 G R 130000 170 S gpl05 S gpl05 I11 G2a M 8600 17000 S gpl05 S gpl05 17 G2a M 14700 97900 S gp 105 S gp 105 19 G2a M 15000 8800 S gpl05 J 17a G2a M 11000 5400 S gp 105 GI9 G2a M 900 1600 S gpl05 ZI7 G R 470000 23200 S gpl05 B5 G2a M 211000 46300 S gpl05 A20 G2a M 100000 13000 S gpl05 J18a G2a M 72000 64300 S gpl05 Ell G2a M 150 350 S gpl05 El9 G2a M 27000 100 S gpl05 HI3 G2a M 21000 100 S gpl05 El3 G2a M 300000 100 S gpl05 A9 G2a M 50250 10000 S gpl05 S gpl05 F7 G1 M 1300 100 S gpl05 S gpl05 ct8 G R 60 100 S gpl05 116 G1 M 25 100 S gpl05 S gpl05 I 1 G2a M 2600 100 S gp95 S gp95 I22 G2a M 640 100 S gp95 S gp95 J22 G2a M 1260 100 S gp95 S gp95 G4 G2a M 50 100 S gp95 S gp95 H3 A M 1400 100 S gp95 H14 G2a M ND 100 S gp95 B23 G2a M 500 100 gp200 E5 G2a M 19000 100 gp200 H7 G2a M 4530 100 gp200 HI9 G2a M r o 100 gp200 I 12 G2a M 1600 100 gp200 Immunoglobulin isotype murine species immunized M mouse and R rat MAbs t As in Table 1 Polypeptide specificities were assessed by reacting MAbs either in immunoprecipitation with radiolabelled viral proteins RIPA or on Western blots WB Antigenic cross reactivities of MAbs were tested in an IIF assay as described Vautherot p200 gp2 200K SI 1 2 HE IE q 97 4K 92 5K M 20 lK Fig I Immunopreci pitation of viral proteins from BCV strain G o Viral polypeptides were precipitated from infected cell lysates labelled with 14C lanes 1 to 3 or 3H labelled amino acids lane 9 or from octylglucoside disrupted 3H labelled virus lanes 4 to 8 by a rabbit polyclonal antiserum lane 8 or MAbs to HE MAb Al2 lanes 1 and 5 S1 gpl05 MAb I7 lanes 2 and 4 2 gp95 MAb I1 lanes 3 and 7 and gp200 MAb E5 lanes 6 and 9 After dissociation immunoprecipitates were separated on 10 polyacrylamide gels lanes 1 to 3 and 9 or 5 to 15 polyacrylamide gradient gels lanes 4 to 8 Positions of the Mr markers are shown on the right side for lanes l to 3 and left side for lane 9 1 23 4 5 67 S1 s2 a N i ii 7 I i i i Fig 2 Reactivity of representative MAbs to denatured BCV proteins Purified BCV G lo isolate was dissolved in sample buffer with lanes 5 and 7 or without lanes 1 to 4 and 6 2 ME subjected to electrophoresis on 10 polyacrylamide gels and transferred to nitrocellulose sheets Individual nitrocellulose strips were reacted with a rabbit polyclonal antiserum lane 1 MAbs to S1 gpl05 MAb P11 in lanes 2 6 and 7 MAb 116 in lanes 4 and 5 or MAb 122 to 2 gp95 lane 3 The arrows point to the high Mr products identified by anti S2 MAbs when pNPA Mr 181 2 was used as the substrate When 5 CFDA Mr 460 4 was used instead of pNPA anti HE MAbs G20 B13 and A12 were able to inhibit the viral AE Fig 4 MAb G20 was the only one which displayed a significant neutralizing activity Table 1 Delineation of epitopes on S and HE by competitive antibody binding assays ABAs Two of 44 MAbs G4 and B23 showed a major loss in their binding ability after biotinylation as measured in the direct binding assay By comparing the neutralizing titres before and after conjugation we also found that the coupling reaction reduced the neutralizing activity of all anti HE and six anti S1 MAbs however anti S1 MAbs 17 I11 Jl8a Z17 and Pll displayed an enhanced neutralizing power after enzyme four of five or hapten conjugation one of five data not shown MAbs were first assayed for self competition in a sequential test two step assay those which were poor competitors were further tested using the simultaneous assay one step assay as described by Delmas et al 1986 On S1 four independent sites were delineated using a panel of 19 MAbs in the sequential sites B C and D and or the simultaneous assay sites A and B Fig 5 The MAbs binding to unrelated epitopes did not compete for fixation as illustrated by the competition curves of MAbs C13 F7 and 116 for antigenic sites A C and D respectively Fig 5 Twelve neutralizing and three non neutralizing MAbs mapped in site B which was further subdivided into subsites defined by MAbs showing a similar profile of reactivity MAbs to S1 B1 enhanced the binding of labelled MAb Ell whereas MAbs to S1 B2 competed with MAb Ell Table 3 MAbs to S1 B1 and B2 did not compete with MAb A9 1730 J F Vautherot and others a 10 1 0 E 0 1 I I 0 20 I Time rain I I I 40 6O Fig 3 Comparison of kinetics of BCV neutralization by hyperimmune mouse serum MAbs to S1 gpl05 and to HE All MAbs used in these experiments were purified from ascites fluid by ammonium sulphate precipitation and their concentration was adjusted to 0 1 mg ml Kinetic experiments were performed as described by incubating dilutions of antibodies with an equal volume of culture medium containing 10000 p f u ml Surviving virus was titrated at 10 15 30 and 60 min of incubation by plating on HRT 18 cells The following symbols are used polyclonat antisera from a hyperimmunized or a control A mouse MAbs A9 O J18a N A20 O C13 11 17 Fq Z 17 A I 11 x to S 1 gp 105 in a and MAbs B7 Y 16 IEI B4 O G20 11 J18c F l and A12 A to HE in b defining S1 B4 unlike MAb J18a S1 B3 which com peted with all MAbs to site B except P11 Fig 5 and Table 3 Competitive ABAs with weakly J17a and G19 or non neutralizing El9 and H13 anti S1 MAbs showed that they competed non reciprocally with neutralizing MAbs However analysis of the interactions between these MAbs and MAbs A9 and El3 strongly suggests that MAbs J17a and G19 bind to S1 B1 B2 and MAbs El9 and H13 to S1 B3 Table 4 Competitive ABAs between MAbs H 13 and E 13 were strong and reciprocal 0 800 I I I I I I I I I I I I I I I I I I I 0 600 0 400 0 200 0 000 I I I I 1 2 3 4 5 6 7 8 9 MAb dilution log2 Fig 4 Inhibition of AE activity by MAbs to HE After contact with MAbs AI2 O B7 B13 Fq G20 O or HI7 11 residual AE activity of purified BCV was monitored by the conversion of the non fluorescent 5 CFDA to fluorescent 5 CF by AE Absorbances were read on a multichannel spectrophotometer at 492 nm Table 4 whereas neutralizing MAbs mapping in S 1 B 1 and S l B2 which strongly inhibited the fixation of H13 did not compete for fixation with El3 These results illustrate the specificity of the competi tive ABAs in delineating different sites and subdivisions independent from the respective avidity of MAbs and show that within an epitope differences in antibody avidity could be recognized as all MAbs were tested in a reciprocal fashion Table 3 and Fig 5 We also verified that the size of the molecule did not influence the results of competitive ABAs by comparing the competitive binding curves of MAbs J18a Ill and Pll which were conjugated successively to HRPO and biotin data not shown The delineation of epitopes by competitive ABAs was confirmed by the results on the stability of the binding sites in the presence of denaturing agents as MAb I16 defined a unique epitope which was not affected by SDS and 2 ME Fig 2 and MAb P11 reacted strongly with the SDS denatured S1 gpl05 when all other MAbs to site B reacted weakly S1 B1 B3 and B4 or did not bind the viral protein S1 B2 Another indication of the accuracy of our epitope map was indirectly obtained through the analysis of the reactivity of MAbs with BCV isolates which revealed that subsite B4 MAbs A9 and El3 was not conserved in the BCV Mebus strain Vautherot 50 to 70 binding inhibition 40 to 50 binding inhibition blank binding inhibition below 40 The binding of the conjugated MAb was facilitated by the unlabelled MAb 1732 J F Vautherot and others Table 4 Binding characteristics of weakly or non neutralizing MAbs mapping in antigenic site B simultaneous assay Labelled MAb MAb Epitope competitor site J17a G19 El9 H13 El3 A9 J17a B1 B2 GI9 El9 B2 B3 HI3 El3 B4 A9 The results of competition reactions are as presented in Table 3 differed from the latter in its reactivity with the denatured 2 or with coronaviruses OC43 and HEV Table 2 All anti S gp200 MAbs mapped in a unique site Table 5 and reacted similarly with all coronaviruses tested Table 2 Identical experiments were performed using 14 MAbs against HE which mapped in two separate antigenic sites Table 6 Epitope HE A was delineated by MAb B22 and the remaining 13 MAbs apparently all interacted with one another thus defining antigenic site HE B However considering the results obtained using anti HE B MAbs in competitive ABAs three subgroups of MAbs were tentatively identified In each subgroup Table 5 Results of competitive ABAs between anti S2 and anti gp200 MAbs Labelled MAb MAb Antigenic competitor site I1 I22 J22 H3 HI4 E5 H7 HI9 I12 I1 122 S2 A J22 H3 S2 A HI4 S2 B E5 H7 gp200 H19 112 I F t L L I The results of competition reactions are as presented in Table 3 t The binding of the conjugated MAb was facilitated by the unlabelled MAb Table 6 Epitope mapping of HE by competitive ABA Labelled MAb MAb Epitope competitor site B22 Y16 J18c Jl7b B4 B7 F13 G20 A12 J21 J10 J18b H17 B13 B22 A YI6 J18c J17b B1 B4 B7 F13 G20 B2 A12 J21 J10 J18b B3 H17 B13 F The t The The MAbs were tested in the simultaneous one step assay results of the competition reactions are as presented in Table 3 binding of the conjugated MAb was facilitated by the unlabelled MAb BCV spike proteins and epitopes 1733 Table 7 Resistance to neutralization of escape mutants selected by anti S1 MAbs Antigenic mutants Neutralizing MAb 17 I11 19 J17a GI9 JlSa B5 A20 A9 Z17 C13 I7 Ill I9 Jl7a G19 Jl8a B5 A20 A9 ZI7 C13 1 I I t l q I L i i 1 i 1 I 1 I The resistance to neutralizing MAbs was rated as total when all mutants assayed were not neutralized by the selecting or related MAbs or as partial when mutants showed a decreased sensitivity to the neutralizing MAb compared to the original BCV stock MAbs competed strongly and reciprocally In addition MAbs to HE B1 prevented the binding of all the other MAbs to HE B whereas MAbs to HE B2 could only prevent the binding of HE B2 and B3 MAbs Strongly neutralizing MAbs were found directed against the HE B 1 five of five and B2 one of four epitopes inhibition of the AE activity was associated with MAbs at the same sites HE B1 one of five B2 two of four Selection and functional analysis of mutants resistant to neutralization by MAbs to S1 and HE A library of mutants was selected these were resistant to neutralization by 11 of 13 anti S1 MAbs and two of six anti HE MAbs Mutant resistant to anti S1 MAbs spontaneously arose from BCV stocks at frequencies ranging from 10 5 to 10 6 8 whereas the selection of escape mutants with anti HE MAbs needed the use of a mutagenized virus stock Delmas et al 1986 and yielded only two sets of mutants Two to four mutants selected by each of the 11 anti Sl MAbs were tested in a neutralization assay against all the selecting MAbs Table 7 All MAbs mapping in subsite S1 B1 17 I11 19 and J17a selected mutants displaying a homogeneous pattern of reactivity to the four selecting MAbs and to MAb G19 with the exception of J17a mutants which were sensitive to neutralization by MAb 17 Table 7 Resistance to neutralization by MAb J18a was a common feature of mutants selected by MAbs G19 J18a B5 or A20 these mutants progressively lost their resistance to neutralization by MAbs 17 and Ill G19 and J18a mutants MAb 19 B5 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- 病毒,外文文獻(xiàn) 【病毒,外文文獻(xiàn)】1992 Bovine coronavirus peplomer glycoproteins_ detailed antigenic analyses of S1 S2 病毒
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