Ebola Virus Mechanism of Infection

Ebola Virus Mechanism of InfectionThe Ebola virus (EBOV) is an enveloped, non-segmented, negative- mountain range RNA virus, whichtogether with Marburg virus, makes up the filoviridae family. The virus causes severehemorrhagic fever associated with 50-90% human mortality1. Four species of the virus (Zaire,Sudan, Cte dIvoire, and Reston ebolavirus) cod thus far been place, with Zaire typic whollyyassociated with the highest human lethality2. A fifth part EBOV species is confirmed in a 2007 forbiddenbreak in Bundibugyo, Uganda3,4. Infection with EBOV results in uncontrolled viralreplication and multiple organ failure with death occurring 6-9 old age after onset ofsymptoms5. Fatal cases are associated with high viremia and defective insubordinate responses,while survival is associated with early and vigorous humoral and cellular immuneresponses6-9. Although preliminary vaccine trials in primates have been highlysuccessful10-13, no vaccines, specific immunotherapeutics, or post-exposu re treatments arecurrently approved for human use. Since 1994, EBOV outbreaks have change magnitude more than quaternion-fold, thus necessitating the urgent development of vaccines and therapeutics for use in theevent of an intentional, accidental or natural EBOV release.The EBOV genome contains seven genes, which direct the synthesis of eight proteins.Transcriptional modify of the fourth gene (GP) results in expression of a 676-residue transmembrane- think glycoprotein termed GP, as well as a 364-residue secreted glycoproteintermed sGP14,15. EBOV GP is the main target for the design of vaccines and entry inhibitors.GP is post-translationally cleaved by furin16 to yield disulfide-linked GP1 and GP2subunits17. GP1 effects supplement to host cells, while GP2 mediates fusion of viral and hostmembranes16,18-20. EBOV is thought to enter host cells through receptor-mediatedendocytosis via clathrin-coated pits and caveolae21, followed by actin and microtubuledependent convey to the endo some21, where GP is further processed by endosomalcathepsins22-24. Essential cellular receptor(s) have not yet been identified, but DC-SIGN/LSIGN25,hMGL26, -integrins27, folate receptor-28 and Tyro3 family receptors29 have allbeen involve as cellular factors in entry. Here, we report the crystal structure of EBOV GP,at 3.4 resolution, in its trimeric, pre-fusion conformation in complex with neutralizingantibody Fab KZ52. GP1 is responsible for cell rebel attachment, which is probably mediated by a regionincluding residues 54-20132. GP1 is composed of a single farming (65 30 30 ),arranged in the topology shown in Fig. 2a, and can be further subdivided into the (I) pedestal, (II)head and (III) glycan cap regions (Fig. 2a and Supplemental Fig. S3). The imbruted (I) subdomainis composed of two sets of sheets, forming a semi-circular fold up which clamps the internalfusion kink and a helix of GP2 through hydrophobic interactions (Fig. 2b). Moreover, thissubdomain contains Cy s53, which is proposed to form an intermolecular disulfide bridge toCys609 of the GP2 subunit17. Cys53 resides near GP2 in the 2-3 loop at the viral membraneproximalend of the miserly subdomain (Fig. 2a-b). Our EBOV GP contains an intact GP1-GP2disulfide bridge, based on reducing and non-reducing SDS-PAGE analysis. However, the region containing the counterpart GP2 cysteine is disordered, which may reflect functionallyimportant mobility in the region. The head (II) is located between the base and glycan capregions towards the host membrane surface. Two intramolecular disulfide bonds stabilize thehead subdomain and confirm the biochemically determined disulfide bridge assignments17.Cys108-Cys135 connects a surface- unfastened loop (8-9 loop) to strand 7, while Cys121-Cys147 bridges the 8-9 and 9-10 loops (Fig. 2a). The glycan cap (III) contains fourpredicted N-linked glycans (at N228, N238, N257 and N268) in an / dome over the GP1head subdomain (Fig. 1b and 2a). This subdomain does not form any monomer-monomercontacts and is fully exposed on the upper and outer surface of the chalice. The central sheetsfrom the head and glycan cap together form a fairly flat surface and, in the scope of the GPtrimer, form the three inner sides of the chalice field.Ebola virus GP2GP2 is responsible for fusion of viral and host cell membranes and contains the internal fusionloop and the seven repeat regions, HR1 and HR2. Many viral glycoproteins have fusionpeptides, located at the N terminus of their fusion subunit, which are released upon cleavageof the precursor glycoprotein. By contrast, pattern II and class III fusion proteins, as well as classI glycoproteins from Ebola, Marburg, Lassa and avian sarcoma leukosis viruses, containinternal fusion loops deficient a free N terminus. The crystal structure reveals that the EBOVGP internal fusion loop, which encompasses residues 511-556, utilizes an antiparallel stranded scaffold to display a partially turbinate hydrophobic f usion peptide (L529, W531, I532,P533, Y534 and F535) (Fig. 2c). The side chains of these hydrophobic residues pack into aregion on the GP1 head of a neighboring subunit in the trimer, resonating of the fusion peptidepacking in the pre-fusion parainfluenza virus 5 F structure33. A disulfide bond between Cys511at the base of 19 and Cys556 in the HR1 helix covalently link the antiparallel sheet. Thisdisulfide bond between the internal fusion loop and HR1 is conserved among all filoviruses,and is analogous to a pair of critical cysteines flanking the internal fusion loop in avian sarcomaleukosis virus34,35. Interestingly, the EBOV internal fusion loop has features more similar tothose observed in class II and III viral glycoproteins (in particular to flaviviruses) than those previously observed for class I glycoproteins (Supplemental Fig. S4). It thus appears thatregardless of viral protein class, internal fusion loops share a common architecture for theirfusion function.EBOV GP2 con tains two heptad repeat regions (HR1 and HR2), connected by a 25-residuelinker containing a CX6CC motif and the internal fusion loop. The crystal structures of postfusionGP2 dispels30,31 have revealed that the two heptad repeat regions form antiparallel helices and that a CX6CC motif forms an intrasubunit disulfide bond between Cys601 andCys608 (Supplemental Fig. S5). In the pre-fusion EBOV GP, HR2 and the CX6CC motif aredisordered. By contrast, the HR1 region is well ordered and can be divided into four segmentsHR1A, HR1B, HR1C and HR1D (Fig. 2c), which together assemble the cradle encircling GP1.Similarly, heptad repeat regions in influenza and parainfluenza viruses also contain multiplesegments in their pre-fusion helices that substantially rearrange in their post-fusionconformations33,36,37.The first two segments, HR1A and HR1B (residues 554-575), together form an helix with an40 kink at T565, which delineates HR1A from HR1B. Interestingly, the bend betweenHR1A and HR1B conta ins an unusual 3-4-4-3 stutter, which may act as a conformationalswitch31, rather than the typical 3-4 periodicity of heptad repeats (Supplemental Fig. S6). Asimilar stutter has also been noted in parainfluenza virus 5 F33. The Ebola virus HR1C (residues576-582) forms an extended lock linking HR1B to the 14-residue helix of HR1D (residues583-598). HR1D forms an amphipathic helix and the hydrophobic faces of each HR1D join toform a three-helix bundle at the trimer interface. Although the breakpoint maps directly to aLee et al. Page 3Nature. causality manuscript available in PMC 2009 June 22.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptchloride ion binding site in the post-fusion conformation of GP230,31 and at least two otherviruses38,39, no chloride ion is observed here as HR1 and HR2 do not come together to formthe six-helix bundle. Instead, the pre-fusion GP2 adopts a novel conformation, intimately curling around GP1 (Fig. 1c).Ebola virus GP-KZ52 int erfaceKZ52 is an antibody isolated from a human survivor of a 1995 outbreak in Kikwit, DemocraticRepublic of the Congo (formerly Zaire)40. This antibody neutralizes Zaire ebolavirus invitro40 and offers protection from lethal EBOV challenge in rodent sit downs41, but has minimaleffects on viral pathogenicity in non-human primates42. KZ52 is directed towards a vulnerable,non-glycosylated epitope at the base of the GP chalice, where it engages three discontinuoussegments of EBOV GP residues 42-43 at the N terminus of GP1, and 505-514 and 549-556at the N terminus of GP2 (Fig. 3 and Supplemental Fig. S7). Although the majority of the GPsurface buried by KZ52 belongs to GP2, the presence of some(prenominal) GP1 and GP2 are critical forKZ52 recognition43. It is likely that GP1 is needed to maintain the proper pre-fusionconformation of GP2 for KZ52 binding. Indeed, KZ52 is the only antibody known to bridgeboth attachment (GP1) and fusion (GP2) subunits of any viral glycoprotein. dispos ed(p) that KZ52requires a conformational epitope seen only in the GP2 pre-fusion conformation and that theKZ52 epitope is distant from the putative(prenominal) receptor-binding site (RBS), KZ52 likely neutralizesby preventing rearrangement of the GP2 HR1A/HR1B segments and pulley host membraneinsertion of the internal fusion loop. Alternatively, IgG KZ52 may sterically hinder access tothe RBS or to a separate binding site of another(prenominal) cellular factor, especially if multiple attachmentevents are required for entry.The KZ52 epitope of GP is convex and does not have a high shape complementarity to theantibody (Sc index of 0.63), although 1600 2 of each GP monomer are occluded upon KZ52binding. The antibody contacts a total of 15 GP residues by van der Waals interactions and 8direct hydrogen bonds (Supplemental Fig. S7). Ten out of 15 residues in the structurally definedKZ52 epitope are unique to Zaire ebolavirus (Supplemental Fig. S6), thus explaining the Zairespecificity of KZ52.Ebola virus GP glycosylationWe generated a fully glycosylated molecular model of EBOV GP to illustrate the native GPtrimer as it exists on the viral surface (Fig. 4). The majority of N-linked glycosylation sites areconcentrated in the mucin-like domain and glycan cap of GP1. Given that the mucin-likedomain is 75 kDa in mass (protein and oligosaccharide), the volume of this domain ispredicted to be similar to each GP monomer observed here. The crystal structure suggests thatthe mucin-like domain is linked to the side of each monomer and may further build up the wallsof the chalice, forming a deeper bowl (Fig. 4). Although a mixture of complex, oligomannoseand hybrid-type glycans are found on intact, mucin-containing GP144, those glycans outsidethe mucin-like domain are likely to be complex in nature the mucin-deleted GP used forcrystallization is sensitive to PNGaseF, but not to EndoH treatment (Supplemental Fig. S8).Modeling of complex-type oligosaccharides on the EBOV GP indi cates that the majority ofthe GP trimer is cloaked by a thick layer of oligosaccharide, even without the mucin-likedomain (Fig. 4). The 19 additional oligosaccharides on the full-length GP (17 on the mucinlikedomain and 2 more on GP1, disordered here) further conceal the sides and top of thechalice. The KZ52 binding site and, presumably, the flexible regions of HR2 and themembrane-proximal external region (MPER) go along exposed and perhaps vulnerable tobinding of antibodies and inhibitors.Lee The development of neutralizing antibodies is limited in natural Ebola virus infection. Manysurvivors have low or unimportant titres1,7, and those antibodies that are elicited preferentiallyrecognize a secreted version of the viral glycoprotein that features an alternate quaternarystructure and lacks the mucin-like domain43. The glycocalyx surrounding EBOV GP likelyforms a harbour that protects it from humoral immune responses and/or confers stability insideor outside a host. The mucin-like domain and glycan cap sit together as an external domain tothe viral attachment and fusion subunits, reminiscent of the glycan shields of HIV-1gp12045,46 and Epstein-Barr virus gp35047, perhaps pointing to a common theme for immuneevasion. Alignment of filoviral sequences indicate that regions involved in immune evasionhave a low degree of sequence conservation i.e. GP1 glycan cap (5%) and mucin-like domain(0%), but the N-glycosylation sites in the glycan cap are mostly conserved among all EBOVsubtypes (Supplemental Fig. S6), indicating the functional importance of these posttranslationalmodifications.Sites of receptor binding and cathepsin cleavageAlthough a definitive receptor for EBOV remains to be identified, previous studies32,48,49have determined that residues 54-201, which map to the base and head subdomains of GP1,form a putative receptor-binding site (RBS) for attachment to host cells. Additionalexperimental studies have identified at least 19 GP1 residues, depute into fo ur groups basedon the location in the structure, that are critical for viral entry48-50 (Fig. 5). Many of theseresidues are apolar or aromatic and are involved in maintaining the structural integrity of GP1for receptor binding or fusion. However, six residues (K114, K115, K140, G143, P146 andC147) cluster within a 20 15 surface in the inner bowl of the chalice and may thusrepresent important receptor contact sites. All residues in the putative RBS are highly conservedamong Ebola virus species (Supplemental Fig. S6).Importantly, this putative RBS is recessed beneath the glycan cap and perhaps further maskedby the mucin-like domain (Fig. 4), suggesting that additional conformational change or removalof the mucin-like domain could reveal additional surfaces required for receptor or cofactorbinding. It has been demonstrated that endosomal proteolysis of EBOV GP by cathepsin Land/or B charters the mucin-like domain to produce a stable 18 kDa GP1 intermediate whichhas enhance viral bin ding and infectivity22-24. The precise site of cathepsin cleavage isunknown and the role of cathepsins in natural infection is as yet unclear. However, formationof an 18 kDa GP1 fragment implies that cathepsin may cleave near the GP1 13-14 loop(residues 190-213). Indeed, this loop is unresolved in the pre-fusion structure, suggestingenhanced mobility and accessibility to enzymatic cleavage. Cleavage within this loop wouldremove the entire mucin-like domain and glycan cap region (Fig. 5). As a result, 7 to 9strands and their associated loops would become exposed. These regions of GP are in proximityto the previously identified residues critical for viral entry. The fold, location andphysicochemical properties of this site should now provide new leads in the search for theelusive filoviral receptor(s).A summary of the Ebola virus mechanics of infection, including the events of cathepsincleavage and conformational changes to GP2 during fusion, is presented.