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Localization of Membrane Permeabilization and Receptor Binding Sites on the VP4 Hemagglutinin of Rotavirus: Implications for Cell Entry

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Localization of Membrane Permeabilization and Receptor Binding Sites on the VP4 Hemagglutinin of Rotavirus: Implications for Cell Entry doi:10.1006/jmbi.2001.5238 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 314, 985±992 Localization of Membrane Permeabilization and Receptor Binding Sites on the VP4 Hemagglutinin of Rotavirus: Implications for Cell Entry Mariana Tihova1, Kelly A. Dryden1, A. Richard Bellamy2 Harry B. Greenberg3 and Mark Yeager1,4* 1The Scripps Research Institute The surface of rotavirus is decorated with 60 spike-like projections, each Departments of Cell and composed of a dimer of VP4, the viral hemagglutinin. Trypsin cleavage Molecular Biology, 10550 N. of VP4 generates two fragments, VP8*, which binds sialic acid (SA), and Torrey Pines Rd., La Jolla VP5*, containing an integrin binding motif and a hydrophobic region CA 92037, USA that permeabilizes membranes and is homologous to fusion domains. Although the mechanism for cell entry by this non-enveloped virus is 2Biochemistry and Molecular unclear, it is known that trypsin cleavage enhances viral infectivity and Biology, School of Biological facilitates viral entry. We used electron cryo-microscopy and difference Sciences, University of map analysis to localize the binding sites for two neutralizing mono- Auckland, Private Bag clonal antibodies, 7A12 and 2G4, which are directed against the SA-bind- Auckland, New Zealand ing site within VP8* and the membrane permeabilization domain within 3Departments of Medicine and VP5*, respectively. Fab 7A12 binds at the tips of the dimeric heads of Microbiology and Immunology VP4, and 2G4 binds in the cleft between the two heads of the spike. Stanford University Medical When these binding results are combined with secondary structure anal- Center, 300 Pasteur Drive ysis, we predict that the VP4 heads are composed primarily of b-sheets Stanford, CA 94305, USA in VP8* and that VP5* forms the body and base primarily in b-structure 4 and a-helical conformations, respectively. Based on these results and Scripps Clinic, Division of those of others, a model is proposed for cell entry in which VP8* and Cardiovascular Diseases, 10666 VP5* mediate receptor binding and membrane permeabilization, and North Torrey Pines Road uncoating occurs during transfer across the lipid bilayer, thereby generat- La Jolla, CA 92037, USA ing the transcriptionally active particle. # 2001 Academic Press Keywords: rotavirus; electron cryo-microscopy; virus neutralization; virus *Corresponding author entry; membrane receptors Introduction and outer capsid protein layers are formed by 260 trimers of VP6 and 780 monomers of the VP7 gly- Rotavirus causes severe gastroenteritis in infants coprotein, respectively.3,4 Sixty dimers of the VP4 and young animals and is responsible for the death hemagglutinin extend 110 AÊ from the viral of 700,000 children per year in developing surface.5,6 Each multi-domained VP4 spike has two 1 countries. Rotavirus is a non-enveloped icosahe- globular heads attached to a square-shaped body Ê dral virus (diameter 1000 A) with three protein that is connected to a rod-like domain, which layers that encapsidate 11 segments of double- 2 merges with a globular base. The VP4 spike pene- stranded (ds) RNA genome (reviewed by Estes ). trates 90 AÊ beneath the outer surface and inter- VP1, VP2 and VP3 are protein components associ- acts with both the VP7 and VP6 layers. ated with the innermost core layer. The middle Infection requires attachment of the virus to membrane receptors of the epithelial cells of the M.T. and K.A.D. contributed equally to this work. small intestine (reviewed by Estes2). There is evi- Abbreviations used: SA, sialic acid; ds, double- dence that rotaviruses have multiple plasma mem- stranded; Mab, monoclonal antibodies; cryo-EM, brane receptors, including sialic acid (SA),7 electron cryo-microscopy; Fab, antigen-binding 8±10 integrins such as a2b1, a4b1 and aVb3 or other fragment; DLP, double-layered particle; ER, 11 endoplasmic reticulum; IgG, immunoglobulin G. membrane proteins. The attachment of the virus E-mail address of the corresponding author: to cell surface receptors is mediated by VP4 [email protected] (88 kDa).12 Trypsin cleavage of VP4 generates two 0022-2836/01/50985±8 $35.00/0 # 2001 Academic Press 986 Membrane-binding Sites on Rotavirus VP4 virion-associated polypeptides: VP5* (60 kDa) and acts with one Fab fragment (Figure 1(b)). The 2G4 VP8* (28 kDa). This event greatly enhances viral Fab fragments bind on both sides of the dimeric infectivity, induces membrane permeability, and is spike in the cleft between the heads, associated necessary for maximal viral growth.13± 15 Some ani- with a slight ¯attening of the head domains mal rotaviruses can bind to the cell either through (Figure 1(c); arrows), consistent with an induced ®t interactions mediated by VP8* or VP5* via SA-con- mechanism for antigen-antibody recognition.27 taining and SA-independent cell surface receptors, Hence, neutralization by these antibodies is due to respectively.16 Human strains appear to use an direct steric interference as well as possible confor- 17 SA-independent route, and an a2b1 integrin bind- mational changes that alter cell receptor inter- ing motif (DGE) at residues 308-310 may function actions. as the receptor-binding site.18 In agreement with neutralization assays, the het- Rotavirus serotypes are designated as G or P erotypic antibody 2G4 was able to bind to rhesus, depending on whether the antibody response is bovine and SA11 rotavirus, whereas 7A12 only directed against VP7 or VP4, respectively.2 VP4 bound to rhesus rotavirus (Figure 1). In particular, has been directly implicated as a target for sero- the 3D reconstruction of 2G4-decorated bovine type-speci®c neutralization in vitro and protection rotavirus (Figure 1(c)) was indistinguishable from 19,20 in vivo. Homotypic and heterotypic monoclonal reconstructions of 2G4-rhesus rotavirus (data not antibodies (Mabs) directed against VP4 neutralize shown) and 2G4-SA11 rotavirus (data not shown) both trypsin-activated and non-trypsin treated at 25 AÊ resolution. Three-dimensional reconstruc- virus, as well as viral hemagglutination. An effec- tions of bovine and SA11 rotavirus incubated with tive vaccine for preventing rotavirus infection in 7A12 were indistinguishable from native viruses 21 infants and neonates was unfortunately associ- (data not shown), whereas the additional Fab den- 22 ated with signi®cant side effects. Understanding sity on 7A12-rhesus rotavirus was clearly visible the antigenic properties of the virus is essential to (Figure 1(b)). the design of rational preventive or therapeutic The density level of the maps was selected strategies. according to the expected volume of the capsid Libraries of neutralizing Mabs have been ana- shells, and the same level was used for contouring lyzed either by competitive inhibition studies or by the Fab density. The constant and variable serologic analyses of Mab mutants.23,24 Monoclonal domains of each Fab were especially well de®ned antibody 7A12 is homotypic and reacts only with in the particles decorated with 2G4 (Figure 2(c)), P serotype 5, which includes rhesus rotavirus. The consistent with full occupancy of binding sites. The epitope for antibody-binding fragment (Fab) 7A12 density of the 7A12 Fab was somewhat less includes residue 188 and lies within the SA-bind- ing site of rhesus rotavirus.25,26 Monoclonal anti- de®ned at maximal radii (Figure 2(b)), suggesting body 2G4 is heterotypic and reacts with P that this Fab may have had less than full occu- serotypes 5, 6 and 7, which includes bovine, rhesus pancy or more conformational ¯exibility compared and SA11 rotaviruses. The epitope for Fab 2G4 with 2G4. Since the Fab molecules were so well includes residue 393 and is within a membrane de®ned, atomic models for canonical Fab struc- permeabilization domain that is homologous to tures could be docked within the cryo-EM density fusion domains (residues 384-401).19,26 maps (Figure 2). Different Fab molecules display Here, we used electron cryo-microscopy (cryo- elbow angles ranging from a tight 125 to an 28 EM), 3D image reconstruction and difference map almost straight 178 angle. The Fab we used for analysis to localize the neutralizing Fab molecules docking has a comparatively tight elbow angle of 29 7A12 and 2G4, and thereby the SA-binding site 125 , which ®t quite well into our 3D maps. A and membrane permeabilization domain on poly- top, angled view of the structures with the red peptides VP8* and VP5*, respectively. (7A12) and green (2G4) Fab densities superim- posed on a single VP4 spike revealed that the two Fab molecules bind at 90 with respect to each Results and Discussion other (Figure 2(a)). Side views of the individual Fab densities showed that 7A12 Fab molecules Localization of Fab fragments bind to the head of the spike angled about 10 Surface-shaded 3D maps of the Fab-decorated from the plane of the viral surface (Figure 2(b)) particles show striking similarity to native rota- and 2G4 Fab molecules bind lower in the cleft virus in icosahedral symmetry, the number and between the heads angled 60 from the viral sur- distribution of surface holes and the rippled outer face (Figure 2(c)). These results con®rm an earlier capsid surface (Figure 1). In addition, the position analysis, which showed that 2G4 binds to the VP4 and the left-handed helical twist of the square- spikes.30 However, in contrast to that study, our shaped body of VP4 and the rod-like domain have maps at higher resolution do not reveal an appreci- a similar appearance in native SA11,5,6 bovine able difference in the binding orientation on the (Figure 1(a)) and rhesus4 rotavirus. However, there two sides of the VP4 spike for either 2G4 or 7A12. is obvious additional density attached to the VP4 The side views also demonstrate that the 7A12 and spikes, which we attribute to the bound Fab mol- 2G4 Fab orientations are rotated by 90 along ecules.
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