Ebola Virus Mechanism of Infection

Ebola Virus Mechanism of InfectionThe Ebola virus (EBOV) is an enveloped, non-segmented, negative-strand RNA virus, which together with Marburg virus, makes up the filoviridae family. The virus causes severehemorrhagic fever associated with 50-90% human mortality1. 4 species of the virus (Zaire,Sudan, Cte dIvoire, and Reston ebolavirus) give thus farthest been identified, with Zaire ordinarylyassociated with the highest human lethality2. A fifth EBOV species is confirmed in a 2007outbreak in Bundibugyo, Uganda3,4. Infection with EBOV results in uncontrolled viralreplication and quadruplex organ failure with death occurring 6-9 days afterwards onset ofsymptoms5. Fatal cases argon associated with high viremia and defective resistant responses,while survival is associated with early and vigorous humoral and cellular tolerantresponses6-9. Although preliminary vaccine trials in primates have been highlysuccessful10-13, no vaccines, specific immunotherapeutics, or post-exposure tre atments atomic number 18currently approved for human use. Since 1994, EBOV outbreaks have increased more thanfour-fold, thus neces stickating 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 trail the deductive reasoning of eight proteins.Transcriptional editing of the fourth gene (GP) results in expression of a 676-residue transmembrane-linked glycoprotein termed GP, as closely as a 364-residue secreted glycoproteintermed sGP14,15. EBOV GP is the main cigaret for the design of vaccines and entry inhibitors.GP is post-translationally cleaved by furin16 to yield disulfide-linked GP1 and GP2subunits17. GP1 effects fixing to host cells, while GP2 mediates jointure of viral and hostmembranes16,18-20. EBOV is thought to move in host cells through sense organ-mediated hold onocytosis via clathrin-coated pits and caveolae21, followed by actin and microtubuledepen denttransport to the endosome21, where GP is further elegant by endosomalcathepsins22-24. Essential cellular receptor(s) have not yet been identified, only if DC-SIGN/LSIGN25,hMGL26, -integrins27, folate receptor-28 and Tyro3 family receptors29 have allbeen implicated as cellular factors in entry. Here, we report the crystal complex body part of EBOV GP,at 3.4 resolution, in its trimeric, pre- coalescence figure in complex with neutralizingantibody Fab KZ52. GP1 is responsible for cell surface adhesion, which is believably mediated by a areaincluding residues 54-20132. GP1 is composed of a case-by-case demesne (65 30 30 ),arranged in the topology shown in Fig. 2a, and dismiss be further subdivided into the (I) sottish, (II) item and (III) glycan cap domains (Fig. 2a and Supplemental Fig. S3). The base (I) sub areais composed of deuce sets of sheets, conformitying a semi-circular surface which clamps the midlandfusion hand-build and a ringlet of GP2 through hyd rophobic interactions (Fig. 2b). Moreover, thissubdomain contains Cys53, which is proposed to form an intermolecular disulfide connect toCys609 of the GP2 subunit17. Cys53 re statuss near GP2 in the 2-3 cringle at the viral membraneproximalend of the base 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 whitethorn reflect functionallyimportant mobility in the region. The division (II) is located amongst the base and glycan capregions towards the host membrane surface. devil intramolecular disulfide trusss stabilize thehead subdomain and confirm the biochemically unflinching disulfide bridge assignments17.Cys108-Cys135 connects a surface-exposed 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, N25 7 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 f number and outer surface of the chalice. The central sheetsfrom the head and glycan cap together form a fairly flat surface and, in the context of the GPtrimer, form the three inner sides of the chalice trough.Ebola virus GP2GP2 is responsible for fusion of viral and host cell membranes and contains the internal fusionloop and the septette buy up regions, HR1 and HR2. umteen viral glycoproteins have fusionpeptides, located at the N goal of their fusion subunit, which are released upon cleavageof the precursor glycoprotein. By contrast, class II and class III fusion proteins, as well as classI glycoproteins from Ebola, Marburg, Lassa and avian sarcoma leukosis viruses, containinternal fusion loops lacking a free N terminus. The crystal social structure reveals that the EBOVGP internal fusion loop, which encompasses residu es 511-556, utilizes an antiparallel stranded scaffold to expose a partially helical hydrophobic fusion 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, reminiscent 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 links the antiparallel sheet. Thisdisulfide bond between the internal fusion loop and HR1 is keep among all filoviruses,and is kindred 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 notice 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 c lass, internal fusion loops share a common architecture for theirfusion function.EBOV GP2 contains two heptad repeat regions (HR1 and HR2), connected by a 25-residuelinker containing a CX6CC melodic theme and the internal fusion loop. The crystal structures of postfusionGP2 fragments30,31 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 circle GP1.Similarly, heptad repeat regions in influenza and parainfluenza viruses also contain multiplesegments in their pre-fusion helices that substantially rearrange in their post-fusioncon validations33,36,37.The first two segments, HR1A and HR1B (residues 554-575), together form an helix with a n40 kink at T565, which delineates HR1A from HR1B. Interestingly, the bend betweenHR1A and HR1B contains an unusual 3-4-4-3 stutter, which whitethorn act as a conformationalswitch31, rather than the typical 3-4 periodicity of heptad repeats (Supplemental Fig. S6). Asimilar stutter has also been storied in parainfluenza virus 5 F33. The Ebola virus HR1C (residues576-582) forms an extended coil linking HR1B to the 14-residue helix of HR1D (residues583-598). HR1D forms an amphipathic helix and the hydrophobic faces of each(prenominal) HR1D join toform a three-helix compile at the trimer interface. Although the breakpoint maps directly to aLee et al. Page 3Nature. write manuscript available in PMC 2009 June 22.NIH-PA Author hologram NIH-PA Author Manuscript NIH-PA Author Manuscriptchloride ion grooming target 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 bu ndle. Instead, the pre-fusion GP2 adopts a unexampled conformation, intimatelycurled around GP1 (Fig. 1c).Ebola virus GP-KZ52 interfaceKZ52 is an antibody isolated from a human survivor of a 1995 outbreak in Kikwit, Democratic republic of the Congo (formerly Zaire)40. This antibody neutralizes Zaire ebolavirus invitro40 and offers protection from lethal EBOV challenge in rodent models41, 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 noncontinuoussegments 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 both GP1 and GP2 are critical forKZ52 recognition43. It is likely that GP1 is postulate to curb the proper pre-fusionconformation of GP2 for KZ52 binding. Indeed, KZ52 is t he only antibody known to bridgeboth attachment (GP1) and fusion (GP2) subunits of any viral glycoprotein. Given that KZ52requires a conformational epitope seen only in the GP2 pre-fusion conformation and that theKZ52 epitope is distant from the putative receptor-binding localise (RBS), KZ52 likely neutralizesby preventing rearrangement of the GP2 HR1A/HR1B segments and blockage host membraneinsertion of the internal fusion loop. Alter indigenely, IgG KZ52 may sterically hinder access tothe RBS or to a separate binding site of another cellular factor, especially if multiple attachmentevents are required for entry.The KZ52 epitope of GP is convex and does not have a high fix 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 delineateKZ52 epitop e 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 Endo H treatment (Supplemental Fig. S8). stamp of complex-type oligosaccharides on the EBOV GP indicates 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) remain 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 insignificant 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 shield that protects it fro m humoral resistive 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 insubordinateevasion. 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, assigned into four groups basedon the berth 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 recess 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 r emoves the mucin-like domain to produce a stable 18 kDa GP1 mediocre whichhas enhanced viral binding and infectivity22-24. The precise site of cathepsin cleavage is unknown quantity 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. segmentation 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, status andphysicochemical properties of this site should now provide new leads in the seem for theelusive filoviral receptor(s).A summary of the Ebola virus mechanism of infection, including the events of cathepsincleavage and conformational changes to GP2 during fusion, is presented.