Published Ahead of Print 17 August 2011. 2011, 85(20):10509. DOI: 10.1128/JVI.00234-11. J. Virol. Greenberg, Philip R. Dormitzer and Stephen C. Harrison Scott T. Aoki, Shane D. Trask, Barbara S. Coulson, Harry B. Inhibits Viral Entry Protein VP7 by Antibodies or Disulfides Cross-Linking of Rotavirus Outer Capsid http://jvi.asm.org/content/85/20/10509 Updated information and services can be found at: These include: REFERENCES http://jvi.asm.org/content/85/20/10509#ref-list-1 at: This article cites 60 articles, 35 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://jvi.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on April 18, 2012 by Harvard Libraries http://jvi.asm.org/ Downloaded from
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Published Ahead of Print 17 August 2011. 2011, 85(20):10509. DOI: 10.1128/JVI.00234-11. J. Virol.
Greenberg, Philip R. Dormitzer and Stephen C. HarrisonScott T. Aoki, Shane D. Trask, Barbara S. Coulson, Harry B. Inhibits Viral EntryProtein VP7 by Antibodies or Disulfides Cross-Linking of Rotavirus Outer Capsid
http://jvi.asm.org/content/85/20/10509Updated information and services can be found at:
This article cites 60 articles, 35 of which can be accessed free
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Cross-Linking of Rotavirus Outer Capsid Protein VP7 by Antibodiesor Disulfides Inhibits Viral Entry�
Scott T. Aoki,1‡ Shane D. Trask,1† Barbara S. Coulson,2 Harry B. Greenberg,3
Philip R. Dormitzer,1§ and Stephen C. Harrison1,4*Laboratory of Molecular Medicine, Children’s Hospital, 3 Blackfan Circle, Boston, Massachusetts 021151; Department of
Microbiology and Immunology, The University of Melbourne, Victoria, Australia 30102; Department ofMicrobiology and Immunology and Department of Medicine, Stanford University School of
Medicine, Stanford, California 943053; and Howard Hughes Medical Institute,Harvard Medical School, Boston, Massachusetts 021154
Received 1 February 2011/Accepted 3 August 2011
Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry.The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444–1447, 2009) led us to reclassifyepitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. Itfurther led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virionouter layer. We have now tested representative antibodies for each of the defined structural epitope regions andfind that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers.Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at theintrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linkedVP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yieldsparticles defective in cell entry as determined both by lack of infectivity and by loss of �-sarcin toxicity in thepresence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viralpenetration into cells.
Rotaviruses cause a large proportion of the worldwide casesof dehydrating childhood diarrhea, accounting for over 400,000deaths annually (47). Their nonenveloped virions enclose an11-segment, double-stranded RNA genome (19). The infec-tious particles (triple-layered particles [TLPs]) have three ico-sahedrally organized protein shells (38, 48, 52, 60). The outer-most layer, which comprises two proteins, is an essentiallycontinuous shell of trimeric VP7 arranged in a T�13l icosa-hedral lattice with 60 spikelike projections of VP4. Neutraliz-ing antibodies recognize epitopes on either of these proteins.
VP4 is the viral attachment protein (23). Trypsin activates itby a cleavage that yields two fragments, VP8* and VP5* (4, 18,20). Uncleaved VP4 incorporates into assembling virions as atrimer. The trypsin-activated spike bears two lectinlike VP8*attachment domains at its tip, supported by three VP5*s; thespike appears to have lost the attachment domain of the thirdVP8* (52). The VP7 layer anchors the VP4 spikes onto theunderlying double-layered particle (DLP) and allows properproteolytic cleavage of VP4 to prime it for membrane disrup-tion (2, 56). During cell entry, the VP7-VP4 outer layer un-
coats, releasing the intact DLP into the cytosol. VP4 mediatescell binding (23) and membrane disruption (8, 35), but the finalsteps of DLP delivery require uncoating of VP7 (7, 39). Fulluncoating of the outer layer is also necessary to activate theDLP (5, 27, 36), which contains multiple copies of the viralpolymerase complex that synthesize and extrude mRNA tran-scribed from each of the 11 genome segments.
Protection against rotavirus infection is mediated primarilyby the adaptive immune system, and a heterotypic response isimportant for broad protective immunity (21, 25, 28). Antibod-ies directed against VP4 and VP7 define the 14 P and 14 Ggroup A rotavirus serotypes; reassortment generates a largenumber of P-G combinations (19, 51). Neutralizing antibodiesthat recognize epitopes on VP8* inhibit infection by blockingattachment (49). Some of those directed against epitopes onVP5* may have a similar mechanism (through steric interfer-ence); others probably prevent membrane disruption by inter-fering with a VP5* conformational rearrangement required forDLP penetration. We consider here the mechanism(s) of neu-tralization by antibodies directed against VP7. VP7 epitopesmap to the outer surface of the VP7 trimer (1).
We confirm here that VP7 antibodies reduce viral infectivityby cross-linking VP7 subunits on the outer layer of the virion,as initially demonstrated by Ludert et al. (40), thereby inter-fering with the required uncoating step (7, 39). We extendthese findings by demonstrating that cross-linking can be ef-fected either by a divalent antibody or by a monovalent Fabwith a footprint that extends across an intersubunit contact.We further show that the introduction of an intersubunit di-sulfide has a similar inhibitory effect and that VP7 uncoating isnecessary for the penetration activity of VP5*.
‡ Present address: Department of Biochemistry, University of Wis-consin—Madison, Madison, WI 53706.
† Present address: Laboratory of Infectious Diseases, National In-stitute of Allergy and Infectious Diseases, National Institutes ofHealth, Bethesda, MD 20892-8026.
§ Present address: Novartis Vaccines and Diagnostics, Inc., 350Massachusetts Ave., Cambridge, MA 02139.
Rotavirus and recombinant protein purification. MA104 cells were grown inM199 medium (Invitrogen) supplemented with 7.5% fetal bovine serum(FBS; HyClone Laboratories, Inc.), 10 mM HEPES, pH 7.3, 2 mM l-glu-tamine, and 100 units/ml penicillin. For rotavirus DLP/TLP production, cellswere grown in 10-stack cell culture chambers (Corning), and confluent mono-layers were infected with rhesus rotavirus (RRV; G3 serotype, P5B[3]) at amultiplicity of infection (MOI) of 0.1 focus-forming unit (FFU)/cell in M199medium supplemented with 1 �g/ml porcine pancreatic trypsin (WorthingtonBiochemical). Cell culture medium was collected after 24 to 36 h, when celladherence was less than 5%. TLPs or DLPs were purified by freeze-thawing,ultracentrifuge pelleting, freon-113 extraction, and a cesium chloride gradi-ent as previously described (56).
Recombinant RRV VP4 and VP7, including mutant VP7, were expressed andpurified from Sf9 cells infected with a baculovirus vector as previously described(11, 12). Briefly, wild-type RRV VP4 was purified from VP4-expressing baculo-virus-infected insect cell pellets by ion exchange and size exclusion chromatog-raphy. RRV wild-type VP7 and a disulfide mutant (VP7 S-S) were purified frominfected insect cell medium by concanavalin A lectin affinity and a monoclonalantibody (MAb) 159 antibody column, which is specific to the VP7 trimer.Elution of VP7 wild-type protein from the antibody column was achieved bycalcium chelation with EDTA. VP7 S-S was eluted by EDTA with similar effi-ciency to the elution of the wild-type protein. The ratio of trimer to dimer andmonomer species on nonreducing Coomassie blue SDS-PAGE gels could beincreased by prolonged incubation (�48 h) at 4°C or by long-term storage at�80°C.
Antibody and Fab production. Mouse hybridomas expressing MAb 159, 4C3,or 5H3 were adapted gradually over several weeks from RPMI supplementedwith 10% FBS, 2 mM l-glutamine, and 100 units/ml penicillin to serum-freehybridoma growth medium (Invitrogen). RV4:3 mouse ascites fluid was bound toa protein G column and eluted with 100 mM glycine, pH 3.0, into 1 M HEPES,pH 7.0. Protein-containing fractions were determined by Bradford assay (Bio-Rad),pooled, and dialyzed into 20 mM HEPES, pH 7.3, and 100 mM NaCl. Thesample was separated on a Sephacryl S200 gel filtration column (GE Health-care), and the antibody peak collected, concentrated, and stored at �80°C. Toproduce Fabs, purified monoclonal antibodies were incubated with papain (1�g/ml; Worthington Biochemical) for 2 to 4 h, and Fc fragments were removedwith protein A resin (Pierce). Uncleaved MAbs were separated from Fabs byS200 gel filtration. Final concentrations of MAbs and Fabs were determined byBradford assay (Bio-Rad).
Antibody neutralization infectious focus assay. TLPs purified on a CsCl gra-dient were primed with trypsin (5 �g/ml; Worthington Biochemical) for 1 h at37°C, followed by trypsin inactivation with 1 mM phenylmethylsulfonyl fluoride(PMSF; Sigma-Aldrich). Virus was 10-fold serially diluted (6 logs) and incubatedwith antibody or Fab for 1 h at 37°C. Medium without MAb or Fab was used asa negative control. For RV3:4 Fab inhibition rescue experiments, virus dilutionswere incubated for 1 h at 37°C in the presence or absence of RV3:4 Fab (10�g/ml), followed by a 1-h incubation at 37°C with an antibody generated tomouse Fabs (goat anti-mouse Fab; Kirkegaard & Perry Laboratories, Inc.).Other antibodies targeting the mouse antibody heavy chain or intact MAb weretested and also rescued inhibition, although not as effectively as the mouseFab-targeted antibody (data not shown).
Confluent MA104 cell monolayers on 96-well plates were washed twice withM199 supplemented with 10 mM HEPES, pH 7.3, 2 mM l-glutamine, and 100units/ml penicillin and inoculated with serial virus dilutions in triplicate. Afterincubation for 1 h at 37°C, wells were supplemented with serum-supplementedM199 containing 1 �g/ml MAb 159 to inhibit secondary infection of replicatingvirus. At 14 to 16 h postinfection, cells were washed with phosphate-bufferedsaline (PBS; Sigma-Aldrich) and fixed with methanol. Infectious foci were de-tected by immunoperoxidase staining using MAb 60, which recognizes VP7under nonreducing conditions, and a horseradish peroxidase-coupled goat anti-mouse secondary antibody (Kirkegaard & Perry Laboratories, Inc.). Stained cellswere counted, and the titers were averaged between triplicate wells. Standarddeviations were calculated from the results of three individual experiments.Structure images were created in PyMOL (The PyMOL Molecular GraphicsSystem, version 1.2r3pre; Schrodinger LLC).
Recombinant VP7 fluorescent labeling and agarose gel shift assays. For flu-orescent labeling of VP7, a solution of 1.5 mg/ml wild-type recombinant VP7 in2 mM Tris, pH 8.0, 10 mM NaCl, 0.1 mM CaCl2 was supplemented withNaHCO3, pH 8.6, to 100 mM and incubated with a final concentration of 0.04mg/ml dimethylformamide (DMF)-solubilized atto 488 N-hydroxysuccinimide(NHS) ester dye (ATTO-TEC, Germany) for 1.5 h at room temperature (22°C).
The reaction was quenched by adding 1 M Tris, pH 8.0, to 200 mM, and thesolution was dialyzed overnight against calcium-containing buffer (20 mMHEPES, pH 7.0, 100 mM NaCl, 0.1 mM CaCl2). The resulting VP7-488 wasincubated in a 2-fold molar excess with DLPs for at least 1 h at room temperaturebefore use.
The agarose gel shift assay was performed as previously described (24, 43, 50).To determine the effective calcium concentration necessary to maintain VP7 onthe particle, VP7-488 DLP recoated particles (100 ng/lane) were incubated for 20min with decreasing ratios of calcium-EGTA buffer (20 mM HEPES, pH 7.0, 100mM NaCl, 10 mM CaCl2, 10 mM EGTA) to EGTA buffer (20 mM HEPES, pH7.0, 100 mM NaCl, 10 mM EGTA). Effective free calcium was calculated basedupon final calcium and EGTA concentrations (26). To test the effect of antibod-ies on recoated particle disassembly, recoated particles were incubated withMAbs or Fabs to VP7 (4F8 at molar ratios of 3:1 to 1:27 and RV3:4 at molarratios of 9:1 to 1:9) for 1 h at room temperature before incubation in a buffer thatnormally promotes VP7 dissociation (21 nM free calcium). Recoated particleswere incubated with buffer alone as negative and positive controls. We electro-phoresed the samples at 80 V for 2 h at room temperature in 0.5% agarose gelsbuffered with 10 mM morpholinepropanesulfonic acid (MOPS)-Tris, pH 7.2.Gels were stained with ethidium bromide, washed, and imaged with a TyphoonFLA 9000 (GE Healthcare). All experiments were repeated at least twice withsimilar results.
Antibody capture ELISA. Antibodies 60 (53), 159 (45), 4F8 (45), and 7A12(42) were bound to 96-well plates at 1 �g/well (10 �g/ml) in PBS overnight andblocked with PBS, 0.5% Tween 20, 2% (wt/vol) bovine serum albumin (BSA;Sigma-Aldrich). Serial dilutions of either wild-type VP7 or the disulfide mutant(VP7 S-S) were added to each well at specified concentrations and incubated for1 h at room temperature (�22°C). Wells were also incubated with buffer aloneto serve as a background control. Bound VP7 was detected with a primary guineapig anti-RRV hyperimmune serum and secondary goat anti-guinea pig horse-radish peroxidase-conjugated antibody (Kirkegaard & Perry Laboratories, Inc.)diluted in PBS, 0.5% Tween 20, 2% BSA for 1 h at room temperature. Betweeneach incubation step, plates were washed with either PBS containing 0.5%Tween 20 or PBS containing 0.5% Tween 20 and 0.9 mM CaCl2, as performedpreviously (9, 10). Tetramethylbenzidine (TMB; Pierce) was used to assay for thepresence of peroxidase. The reactions were stopped with 2 M sulfuric acid, andwells read for the A450 using an enzyme-linked immunosorbent assay (ELISA)plate reader (Biotek). Data were normalized to background, and standard devi-ations (error bars) determined from triplicate wells. Experiments were repeatedat least three times with three different VP7 S-S preparations and showed similarresults.
Recoating reaction. Previous publications describe the reassembly of infec-tious virus particles in greater detail (56). Briefly, recombinant VP4 was added topurified DLPs in at least a 5-fold excess for complete occupancy in bufferadjusted to pH 5.2 with sodium acetate. After a 1-h incubation at room temper-ature (�25°C), mutant or wild-type VP7 was added in at least a 2-fold molarexcess for complete occupancy in buffer supplemented with calcium.
Titers of recoating reactions were determined by infectious focus assay asdescribed above. Proper assembly was also assessed by a protease protectionassay as previously described (56). Briefly, samples were incubated in the pres-ence or absence of trypsin (5 �g/ml; Worthington Biochemical) for 1 h at 37°C.Trypsinized samples were also treated with or without 5 mM EDTA. Sampleswere inactivated with 1 mM PMSF, separated by SDS-PAGE, and transferred toa polyvinylidene difluoride membrane (PVDF; Millipore) by electroblotting.VP4 was detected by MAb HS2 (46) under reducing conditions. Immunoblotswere developed by chemiluminescence after incubation with a horseradish per-oxidase-conjugated goat anti-mouse IgG secondary antibody (Kirkegaard &Perry Laboratories, Inc.).
�-Sarcin coentry. The �-sarcin coentry assay was performed as previouslydescribed (35). Briefly, MA104 cells in medium lacking cysteine or methioninewere infected with TLPs, DLPs, or recoated particles (�5 � 104 particles/cell) inthe presence or absence of �-sarcin (100 �g/ml; Sigma-Aldrich). Cells werewashed and transferred to Dulbecco’s modified Eagle’s medium (DMEM) con-taining 35S-labeled cysteine and methionine (1 �Ci/ml EasyTag EXPRE35S35Sprotein labeling mix; PerkinElmer). Samples were washed and precipitated bythe addition of 10% trichloroacetic acid to wells. Wells were then washed withethanol, air dried, and dissolved overnight with 0.5% SDS in 0.1 M NaOH.Sample lysates were analyzed by liquid scintillation counting in Opti-Fluor scin-tillation fluid (PerkinElmer). The results were normalized to the label incorpo-ration in uninfected cells not treated with �-sarcin.
VP7 neutralization epitopes. VP7 is a Ca2�-stabilized, tri-meric glycoprotein. The crystal structure of rhesus rotavirusVP7 (RRV, G3 serotype) complexed with the Fab of MAb 4F8(1) shows that two Ca2�-binding sites at the subunit interfacehold the trimer together. Positions of mutations that conferresistance to neutralization by various monoclonal antibodiesmap to two regions of the outward-facing surface of the protein(Fig. 1). One of these regions spans the Ca2�-stabilized, inter-subunit contact; the other spans an intrasubunit junction be-tween two compact domains. We have designated these re-gions 7-1 and 7-2, respectively, dividing the former into 7-1aand 7-1b, depending on which side of the subunit boundary thesurface residues fall (Fig. 1). Each of these regions includesseveral epitopes, mapped by neutralization escape mutationsand binding competition before the structure was known.
Neutralization by antibodies that bind in region 7-1. TheFab in the crystal structure contacts residues in both 7-1a and7-1b and should therefore stabilize the trimer. We postulatedthat neutralization is a consequence of this stabilization andsuggested that at suitable concentrations, the isolated Fabs ofMAbs that bind in this way would block infectivity. Indeed, theFab of MAb 4F8 did neutralize infectious virus, although athigher concentrations than does the intact antibody (1). Weattributed this difference to the divalency of the latter and itsconsequent higher avidity. We have extended our experimentaltest of this prediction, as shown in Fig. 2, for three additional7-1 antibodies, MAbs 159, 4C3, and 5H3, which all neutralizeG3 rotavirus strains (Fig. 2). Escape mutations to 159 and 4C3map to residue 94, in 7-1a (45) (Fig. 2A and B), and 5H3escape mutants map to residue 211, in 7-1b (41) (Fig. 2C). Wegenerated the corresponding Fab fragments by papain diges-tion and showed that they all neutralize virus (Fig. 2). Thedifference in effective concentration between intact antibodyand antibody fragment is approximately 3- to 70-fold, which weagain attribute to avidity differences between a divalent MAband monovalent Fab. Neutralization by the monovalent frag-ments implies that, like 4F8, these antibodies bridge the VP7subunit interface. We suggest that most neutralizing antibodiesthat map to region 7-1 will have this property.
Neutralization by antibodies that bind in region 7-2. Bindingof monovalent Fab to region 7-2 is not expected to stabilize the
VP7 trimer, as the sites are far from a subunit contact. MAbs thatmap to this region are less common than those that map to 7-1.We obtained frozen ascites fluid from mice producing antibodyRV3:4, for which escape mutations map to residues 148 and 264(37), purified the antibody by protein G binding and size exclusion
FIG. 1. Two views of VP7 (1). On the left, a view from the outside the virion, along the 3-fold axis. The three subunits are shown as molecularsurfaces, shaded different intensities of gray. Side chains in epitope regions 7-1a, 7-1b, and 7-2 are in red, pink, and blue, respectively. On the right,a view tangential to the virion.
FIG. 2. Neutralization curves for antibodies with escape mutationsin region 7-1. For each of the three monoclonal antibodies indicated,infectivity is shown for a concentration series of antibody or Fab.MAbs 159 (A) and 4C3 (B) select escape mutations at position 96 inregion 7-1a, and MAb 5H3 (C) at position 211 in 7-1b. Error bars showstandard deviations for three measurements from independent exper-iments. Infectivity below a 3-log reduction in titer is not reported.
chromatography, and prepared Fab by papain digestion. Unlikethe Fabs from 7-1 antibodies, the RV3:4 Fab did not block infec-tivity, except for a very small effect (43.5%) at very high concen-trations, which may be due to residual intact IgG, or steric effectsof a Fab-saturated particle on VP4-mediated virus binding andentry (Fig. 3A). This result supports the hypothesis that neutral-ization requires VP7 cross-linking, either by contact of a singleFab across a subunit interface or by divalent interaction of anintact antibody with two VP7 polypeptide chains. We tested thisidea by binding Fab RV3:4 and then adding an anti-Fab antibody(Fig. 3B)—an approach first used to show that neutralizing anti-bodies stabilize poliovirus capsids by cross-linking sites on theirsurface (17). The secondary antibody restored effective neutral-ization by Fab RV3:4. Samples without secondary antibody werenot affected by the presence of the Fab, and virus incubated withanti-Fab antibody alone was likewise unaffected. We concludethat, like antibodies directed against epitopes in region 7-1, thosedirected against 7-2 epitopes neutralize by preventing dissociationof the VP7 trimer. Divalency is necessary for antibodies in thelatter group. Because each Fab arm of antibodies in the formergroup makes contact across a subunit boundary, divalency is notessential, although it enhances potency.
Antibody stabilization of VP7-coated DLPs. RecombinantVP7 efficiently recoats DLPs in the presence of sufficient calciumand uncoats in the absence of sufficient calcium (56). We testedwhether VP7-targeted neutralizing antibodies stabilize the outercoat of viral particles at a low calcium concentration by assayinguncoating, using agarose gel electrophoresis to separate particle-associated and free VP7 (Fig. 4). We labeled recombinant VP7with a 488 fluorescent dye to allow observation of its position inthe gel; ethidium bromide staining detected coated or uncoated
DLPs. We adjusted the free calcium concentration in samplesbefore electrophoresis with Ca-EGTA buffers (24, 50). Fluores-cently labeled VP7 (VP7-488) assembled onto DLPs supports fullinfectivity (A. H. Abdelhakim and S. C. Harrison, unpublisheddata). The electrophoretic analysis showed that VP7 uncoatedfrom particles after incubation at free calcium concentrations of21 nM or lower (Fig. 4A). This threshold is similar to the con-centration previously reported for RRV uncoating, based onother techniques (40). VP7-coated DLPs migrate more rapidly inthe gel than uncoated DLPs, probably because of the negativecharge of VP7. Incubation of VP7-coated DLPs with region 7-1MAb 4F8 or with its Fab prevents uncoating in a dose-dependentmanner (Fig. 4B). 4F8 MAb-bound particles are retained in thesample wells, even at low calcium concentrations, probably be-cause of increased virus-particle radius and antibody-mediatedaggregation. 4F8 Fab-bound particles enter the gel, and DLP andVP7 fluorescence still migrate together in low calcium at thehigher Fab concentrations tested. Region 7-2 MAb RV3:4 alsoblocks VP7 dissociation at low free calcium concentrations, pre-venting complete conversion to DLPs at lower antibody dilutions.RV3:4 Fabs do not prevent dissociation of VP7 from DLPs,however, at any concentration tested (Fig. 4C). In the presence ofcalcium, MAb/Fab-treated viral particles migrated more slowly inthe gel than did untreated VP7-coated DLPs, showing that theconcentration and affinity of the Fab were adequate to achievehigh occupancy. These results support a general cross-linkingneutralization mechanism, in which both 7-1-specific and 7-2-specific MAbs prevent loss of the VP7 outer coat but only 7-1-specific Fabs can stabilize the outer capsid.
Disulfide cross-linking of VP7 trimers. Can a mutationallyintroduced cross-link mimic the effect of a neutralizing anti-body? Guided by the crystal structure, we introduced apposingcysteines at the subunit interface (Fig. 5A and B). Of the threeconstructs tested, the T276C-Q305C mutant gave the best re-sults, yielding soluble, trimeric protein secreted from recom-binant baculovirus-infected insect cells, as judged initially byreducing and nonreducing SDS-PAGE (Fig. 5C) and immuno-blot with a VP7 specific antibody (data not shown). We puri-fied this disulfide variant (VP7 S-S) with the same protocolused to prepare wild-type recombinant protein (12), conca-navalin A affinity chromatography followed by immunopurifi-cation with an immobilized, VP7 trimer-specific antibody(159). Elution of VP7 S-S from the antibody column could beaccomplished with similar efficiency by either calcium chela-tion (with EDTA) or a chaotropic concentration of magnesium(data not shown). Elution with EDTA shows that loss of Ca2�
reduces antibody affinity, probably because the single disulfidedoes not tether the interface as rigidly as do the two Ca2� ions,particularly for the trimeric species in which the disulfide hasformed at only two of the three interfaces. Wild-type VP7trimer and VP7 S-S eluted in essentially the same volume bysize exclusion chromatography (data not shown). Nonreducedsamples separated by SDS-PAGE and detected by Coomassieblue staining showed a predominant band at 100 kDa, whichwe interpret as cross-linked trimer (Fig. 5C). Minor bands at55 and 65 kDa were also observed and most likely represent asmall percentage of incorrectly formed, cross-linked dimericspecies. Samples boiled in the presence of reducing agent (-mercaptoethanol) migrated, like wild-type recombinant VP7,with an apparent molecular mass of �35 kDa (Fig. 5C).
FIG. 3. Neutralization curves for antibody RV3:4 with escape mu-tations at positions 148 and 264 in region 7-2. (A) Percent infectivityfor a concentration series of antibody or Fab. (B) Percent infectivity inthe presence of RV3:4 Fab (10 �g/ml) plus secondary, anti-Fab anti-body (2° Fab Ab; closed circles) or medium alone (open circles) at theconcentrations indicated on the abscissa. Error bars show standarddeviations for three measurements from independent experiments.Infectivity below a 3-log reduction in titer is not reported.
The VP7-directed, nonneutralizing MAb 60 binds the VP7subunit in monomers or trimers and does not require Ca2� todetect VP7 in an ELISA (Fig. 6A). The trimer-dependentMAbs 159 and 4F8, which map to region 7-1, will detect wild-type VP7 only when Ca2� is present in both the wash andincubation steps (9, 10) (Fig. 6A and B). VP7 S-S yields astrong ELISA signal with all three antibodies, regardless ofwhether Ca2� is included in the assay (Fig. 6C and D). Theneutralizing MAb 7A12, which binds VP4 (53), was used as anegative control in these experiments. Thus, the addition of anintersubunit disulfide link preserves 7-1 epitopes, even whenCa2� is depleted from the medium. Although binding of VP7S-S to MAb 159 is weaker under these conditions, as shown byEDTA elution of the disulfide-linked trimer from the 159antibody column, the affinity is sufficient for detection in theELISA format.
Recoating with VP7 S-S. Recombinant VP7 coats DLPscompletely and efficiently (2, 56). The incorporation of VP4 inits correct conformation requires a VP4 excess, the addition ofVP4 before VP7, and careful adjustment of conditions (2, 35,56). Selective trypsin sensitivity of VP4 is an indicator ofproper recoating, as VP7 protects VP4 from nonspecific trypticdigestion and restricts cleavage to specific sites between VP8*
and VP5* (2, 4, 18, 20, 35, 56). The data in Fig. 7A show thatVP7 S-S protects VP4 in this way. VP5* bands appear atcomparable levels following the addition of trypsin to particlesrecoated with wild-type VP7 and with VP7 S-S. The addition ofEDTA before trypsinization eliminates the VP5* band in bothsamples. These results imply that VP7 S-S recoats properlywith VP4 onto DLPs but that, despite covalent cross-linking,VP7 S-S depends on calcium for tight binding. Our prepara-tions of VP7 S-S were purified by elution with EDTA from theCa2�-dependent antibody, MAb 159, which binds region 7-1.While stably trimeric, even in the absence of Ca2� (Fig. 6),VP7 S-S retains enough flexibility at the subunit interface thatthe withdrawal of Ca2� loosens the contact, reducing affinityboth for 159 and for the DLP.
The levels of protection of VP4 from nonspecific proteolyticdigestion correlate with the infectious titers of recoated wild-type particles (35, 56). We prepared particles recoated withwild-type VP7 and VP7 S-S that gave comparable levels of VP4protection. Particles recoated with VP7 S-S had a 20-fold lowerinfectious titer than those recoated with wild-type VP7 (Fig.7B). We used an �-sarcin coentry assay (7, 35, 39) to determinethat the presence of the engineered disulfide indeed inhibitedpenetration (Fig. 7C). The toxin, which inactivates ribosomes,
FIG. 4. Analysis by agarose gel electrophoresis of VP7-DLP association at high and low free calcium concentrations and in the presence orabsence of VP7-binding MAbs or Fabs. The fluorescence of 488-labeled VP7 is green; that of ethidium bromide (EtBr)-stained DLPs is red.(A) Calcium dependence of uncoating in the absence of MAbs or Fabs. Free calcium concentrations vary from 147 to 2 nM. Controls are DLPsalone and 488-labeled VP7 alone (two lanes on the right). Note the separation of the EtBr signal and VP7 signal at low calcium concentrationsas DLP migration slows and VP7 migration accelerates. (B) Effects of 4F8 (region 7-1) MAb and Fab on uncoating. MAb and Fab concentrationsvary from 1.6 to 0.2 �M. Samples are in 147 nM free calcium (�) or 21 nM free calcium (�). Note the retention of both DLP and VP7 signalsin the wells in the presence of 4F8 MAbs, regardless of calcium concentration, and the associated DLP and VP7 signals in the presence of sufficient4F8 Fab and high or low free calcium concentrations. “VP7 � DLP” lanes contain recoated particles supplemented with buffer alone. (C) Effectof RV3:4 (region 7-2) MAb and Fab on uncoating. MAb and Fab concentrations vary from 4.8 to 0.6 �M. At low free calcium concentrations, thehighest RV3:4 MAb concentrations reduce the DLP signal from dissociated VP7. Even at the highest RV3:4 Fab concentrations, separation of theDLP and VP7 signals is essentially complete in low free calcium.
requires a “helper” (e.g., a virus) to gain access to the cytosol.As shown in Fig. 7C, the addition of �-sarcin to the incubationwith wild-type recoated particles led to prompt loss of 35Sincorporation; the use of VP7 S-S recoated particles or non-infectious DLPs yielded little to no reduction in label accumu-lation. Thus, introduction of the disulfide cross-link preventsthe particle from completing the sequence of conformationalevents that lead to membrane disruption and penetration. The
same approach has shown that antibodies directed against VP7act at a similar stage (7, 39).
DISCUSSION
Examining the high-resolution structure of VP7 led us togroup the various epitopes, identified by monoclonal antibodyescape mutation and other classical approaches, into two re-
FIG. 5. Disulfide cross-linking of VP7. (A) Positions of Ca2� ions (blue spheres) and location of introduced disulfide (red square) at the interfacebetween VP7 subunits within a trimer. View as in Fig. 1, right. (B) Detail of the region around the disulfide, with cysteines (T276C and Q305C) shownin stick representation. (C) Reducing (Red.) and nonreducing (Nonred.) SDS-PAGE of wild-type (wt) and disulfide cross-linked VP7 (VP7 S-S). Notethat because the intrasubunit disulfides have not been reduced, the VP7 monomer migrates more rapidly than the fully reduced monomer.
FIG. 6. Antigenicity of the wild-type and disulfide cross-linked VP7 trimer, measured by ELISA. x axis corresponds to the antibody concen-tration tested. (A) Wild-type VP7 (VP7 wt) in PBS. (B) Wild-type VP7 in PBS plus 0.9 mM CaCl2. (C) Disulfide-cross-linked VP7 (VP7 S-S) inPBS. (D) Disulfide cross-linked VP7 in PBS plus 0.9 mM CaCl2.
gions, designated 7-1 and 7-2, as shown in Fig. 1 (1). We alsofound that Fab 4F8, seen in the crystal structure to contactresidues on either side of the subunit boundary within a trimer,neutralized infectivity, presumably by stabilizing the trimer andpreventing uncoating (1). Neutralization by the Fab was lesspotent than neutralization by the intact antibody, as expectedfrom the contribution of divalency to overall IgG avidity. Oursuggestion that noncovalent cross-linking of VP7 is a generalmechanism for neutralization by antibodies that bind region7-1 appeared to be at odds with data in the literature, whichreported failure of neutralization by Fabs from certain anti-bodies in this class (40, 49). We have reinvestigated this pointfor MAbs 159, 4C3, and 4H3 and shown that, at suitableconcentrations, their Fab fragments indeed block infection.The concentrations required to neutralize are between 3- and70-fold higher than for the intact antibody, fully consistent withprobable differences in avidity.
Antibody valency also affects its serotype recognitionbreadth. Antibodies 159 and 4C3, both IgGs, neutralize a rel-atively narrow range of serotypes; both have escape mutationsat residue 94. Viruses with mutations at this position in region7-1 also escape neutralization by antibody 57-8 (41), an IgMwith a far broader neutralizing range than that of 159 or 4C3.IgMs have 10 antigen binding domains per molecule; IgGshave 2. The higher valency of IgM 57-8 might allow it to bindwith more than two contact points, leading to neutralization ofeven those G serotypes for which an individual antigen com-bining site has relatively weak affinity.
Region 7-2 lies at a domain boundary within one VP7 sub-unit. In principle, monovalent binding might be sufficient forneutralization, if the mechanism were to involve rigidificationof the VP7 subunit to an extent requiring expulsion of theantibody for VP7 to dissociate from the DLP. Fab RV3:4, froma region 7-2-directed MAb, did not, however, block infection.
The slight (43.5%) reduction observed at the highest concen-tration could be due to a trace of remaining intact IgG, non-specific interference with viral uptake when particles are fullydecorated with Fab, or steric interference with VP4 conforma-tional changes necessary for viral entry (14, 57, 61). Bridgingtwo RV3:4 Fabs with a Fab-directed secondary antibody re-stored neutralization. We conclude that cross-linking of siteson two subunits, either within a VP7 trimer or between two ofthem on the TLP surface, is the basis for neutralization byantibodies that recognize 7-2 epitopes. Both intra- and in-tertrimer cross-linking are possible, given the distances mod-eled on the cryoelectron microscopy reconstruction of the VP7recoated particle (3). Reconstructions of intact, divalent anti-bodies bound to virus particles indicate that flexibility of theFc-Fab hinge allows a generous distance range (60 Å to 140 Å)between Fab footprints (30, 54). Intertrimer binding would beacross a local dyad, matching the rough symmetry of the IgG.
Cross-linking of surface proteins is a mechanism by whichneutralizing antibodies block cell entry of a number of otherviruses. The classic example is poliovirus: a cross-linking mech-anism accounts for the relatively small number of antibodymolecules per virion needed to block infection, because theexpansion transition needed to initiate entry is highly cooper-ative (16, 17, 31). West Nile virus antibodies cross-link adjacentsubunit domains to prevent the dimer-to-trimer transition as-sociated with entry (34, 44). In other cases, Fabs neutralizewithout a second, bridging antibody. The mechanism appearsto involve interference with a conformational change in thetarget protein by stabilization of its pretransition conformer.Fabs targeting the base of the influenza hemagglutinin increasethe threshold necessary for pH-mediated conformationalchanges that permit entry from endosomes (15, 55).
To generate an efficient neutralizing antibody response thattargets VP7, the immunogen must contain VP7 in the form of
FIG. 7. Properties of virus DLPs recoated with VP4 and wild-type or disulfide cross-linked VP7. (A) Immunoblot showing trypsin cleavage ofVP4 to VP5* on wild-type (wt) and disulfide-linked (VP7 S-S) recoated particles, before (left, �T) or after (right, �E/T) the addition of EDTAto remove VP7. –, untreated samples. The VP4 antibody (46) used for detection recognizes an epitope protected from protease digestion whenVP4 is properly assembled on particles. (B) Infectivity of wild-type and disulfide-linked recoated particles. (C) Coentry of �-sarcin, measured by loss of35S incorporation, following exposure of cells to medium only, to DLPs recoated with wild-type or disulfide-linked VP7, or to unrecoated DLPs.
assembled oligomers, e.g., on authentic virions, inactivated vi-rions, virus-like particles, or the surface of cells expressing anengineered, membrane-anchored VP7 (6, 9, 13, 28, 32, 33, 59).Antisera generated from VP7 peptides, recombinant wild-typeVP7, or VP7 S-S have only modest effects on viral infectivity(22, 29; G. W. Both, personal communication, H. B. Green-berg, unpublished data, and N. Feng, S. T. Aoki, P. R. Dor-mitzer, and H. B. Greenberg, unpublished data). A high de-gree of polyvalency appears to be a useful attribute of manyeffective immunogens. It is also possible that obscuring non-neutralizing, immunodominant epitopes on VP7, by VP6 in-teractions or membrane tethering, enhances the selectivity ofB-cell stimulation.
The results reported here also provide some additional in-formation about VP7 uncoating during viral entry. Althoughthe disulfide does not prevent the release of VP7 when achelator is added in excess of calcium to virions in solution, itis likely to stabilize its attachment to DLPs when the virion isin the confines of an endocytic vesicle with a low (but nonzero)concentration of calcium. Previous work (35) has shown thatVP4 hydrophobic loops, at the tip of the VP5* -barrel do-main, interact with membranes and participate in the mem-brane disruption required for infectious entry, but the uptakevesicles relevant to rotavirus entry are just beginning to becharacterized (58). The effectiveness of the disulfide cross-linkfor blocking viral facilitation of �-sarcin entry suggests thatVP7 release must precede any membrane disruption that isextensive enough to admit a small protein. It is possible thatthe initial formation of a small pore, e.g., by insertion of theVP4 loops, causes Ca2� to leak from the entry vesicle, releas-ing VP7 and with it VP4, and that conformational rearrange-ment of released VP4 then perforates the vesicle more com-pletely. The VP7-directed antibodies and VP7 S-S will both beuseful probes of this process.
ACKNOWLEDGMENTS
We thank M. Babyonyshev for assistance with antibody preparation,J. Pan and A. Abdelhakim for assistance with rotavirus preparation,and Harrison Laboratory members and J. Patton for helpful discussionand advice.
This work was supported by NIH grants CA-13202 (to S.C.H.),AI-053174 (to P.R.D.), and AI-021362 and DK-56339 (to H.B.G.), byan Ellison Medical Foundation New Scholars in Global InfectiousDiseases Award to P.R.D., by a National Health and Medical Re-search Council of Australia Senior Research Fellowship to B.S.C., andby a VA Merit Award to H.B.G. S.C.H. is an Investigator in theHoward Hughes Medical Institute.
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