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Review
. 2018 Mar 8;172(6):1319-1334.
doi: 10.1016/j.cell.2018.02.054.

Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines

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Free PMC article
Review

Common Features of Enveloped Viruses and Implications for Immunogen Design for Next-Generation Vaccines

Felix A Rey et al. Cell. .
Free PMC article

Abstract

Enveloped viruses enter cells by inducing fusion of viral and cellular membranes, a process catalyzed by a specialized membrane-fusion protein expressed on their surface. This review focuses on recent structural studies of viral fusion proteins with an emphasis on their metastable prefusion form and on interactions with neutralizing antibodies. The fusion glycoproteins have been difficult to study because they are present in a labile, metastable form at the surface of infectious virions. Such metastability is a functional requirement, allowing these proteins to refold into a lower energy conformation while transferring the difference in energy to catalyze the membrane fusion reaction. Structural studies have shown that stable immunogens presenting the same antigenic sites as the labile wild-type proteins efficiently elicit potently neutralizing antibodies, providing a framework with which to engineer the antigens for stability, as well as identifying key vulnerability sites that can be used in next-generation subunit vaccine design.

Figures

Figure 1
Figure 1
Class I Viral Fusion Proteins (A) Linear diagram illustrating the organization of the class I fusion protein polypeptide chain. A precursor glycoprotein is co-translationally translocated into the ER of the infected cell, where it is co- and post-translationally modified by glycosylation (Y symbols) and formation of disulfide bonds (not shown). An ER-resident signal peptidase generates the N terminus of the precursor upon cleavage (left vertical arrow) of the signal sequence (SS), while the precursor remains membrane-anchored through a C-terminal trans-membrane (TM) segment. The precursor folds as a trimer (represented by the three horizontal bars), which undergoes a subsequent proteolytic step (vertical arrow at the center) that generates two mature subunits NSU (N-terminal subunit, blue, red or yellow bars; the colors match the 3D diagrams in (B) and CSU (C-terminal subunit, white, gray or black bars). The two subunits remain associated in the mature trimer, which becomes metastable and primed for the irreversible conformational change that drives membrane fusion for infection of a new cell. The NSU is usually the receptor binding subunit, although in some viruses a separate protein carries this function. The CSU is the viral fusion protein, with a fusion peptide at its N-terminal end (symbolized by a dashed region) and an external membrane-proximal region (MPR) near the TM domain (symbolized by short dashes). (B) Two representative class I fusion proteins in their prefusion conformation shown in surface representation (left panels), with NSU and CSU colored according to panel A. The glycan chains are shown as sticks with carbon atoms cyan and oxygen atoms red. The viral membrane is diagrammed to scale, with the aliphatic moiety in dark beige at the center, dingfa out as it enters the hydrophilic lipid head-group moiety. Left, top panel, structure of a fully-glycosylated, clade B native HIV Env trimer determined by cryo-EM to 4.4Å resolution (Lee et al., 2016) in the presence of the TM segment (empty arrow) and in complex with bnAb PGT151, which is displayed with the variable domains as green ribbons. The MPR segment and TM region appeared mostly disordered. The bottom left panel shows the cryo-EM structure of the NL63 α-coronavirus NL63 spike protein ectodomain, determined to 3.4Å resolution, and displaying clear density for many of the N-linked glycans (Walls et al., 2016b). The prefusion forms display the NSU subunit (S1 in coronaviruses and gp120 in HIV) making a crown (red/yellow/blue) around a spring-loaded CSU (black/gray/white; S2 in coronaviruses and gp41 in HIV). Interactions with the target cell trigger the release of the NSU crown, allowing the CSU to undergo a fusogenic conformational change going through an extended intermediate that bridges the two membranes (represented in the top-middle panel). The hairpin conformation adopted by the CSU (diagrammed at the top right) has the fusion peptide inserted into the membrane next to its TM region. A schematic fused membrane is shown to scale. Immediately below the hairpin is a ribbon representation of the postfusion CSU from SIV (gp41), forming a 6-helix bundle (Yang et al., 1999). The N and C-terminal ends of the gp41 subunit in black are marked. The two lower panels show a representative structure of the coronavirus postfusion CSU trimers displayed as ribbons (middle) and as surface representation (bottom) (Walls et al., 2017) with the N-and C-terminal ends visible in the structure of the ectodomain indicated in blue and red, respectively. A schematic on the ribbon diagram shows the expected location of the missing segments in the intact postfusion protein on membranes (not present in the structure; blue at the N-terminal end, connecting to the fusion peptide inserted superficially on the outer lipid leaflet of the fused membranes, represented as a full blue rectangle) and the C-terminal end in red, connecting to the TM helices and the cytosolic tail.
Figure 2
Figure 2
Antibody Recognition of Class I Fusion Proteins Representative structures of enveloped viruses with class I fusion proteins in complex with neutralizing antibodies (drawn at the same scale and aligned by height on the viral membrane): from left to right: Arenaviridae (Lassa virus GP); Filoviridae (Ebola virus GP), Retroviridae (HIV-1 Env), Pneumoviridae (Respiratory syncytial virus F), Orthomyxoviridae (Influenza virus HA), Coronaviridae (MERS CoV S). In the case of the Ebola virus GP, the mucin-like domain missing from the structure was diagrammed in gray with multiple glycans drawn as sticks, as a guide. The color coding is the same as in Figure 1; the top panel shows a top view down the 3-fold axis of the trimer, and the bottom panel displays a side view with the viral membrane represented to scale. Note that RSV F is different in the sense that the CSU is the bulkiest subunit, in contrast to the others, which have a large NSU crown. The PDB accession code of each of these structure is indicated in between parenthesis in each case.
Figure 3
Figure 3
Class II Viruses: Cryo-EM Structures of Flavivirus, Alphavirus and Phlebovirus Particles (A) The top row shows the organization of an immature flavivirus particle (left, icosahedral non quasi-equivalent lattice with 3 protomers per asymmetric unit, 180 UGP/DGP heterodimers) (PDB 5u4w), the mature alphavirus (middle, icosahedral T = 4 quasi equivalent lattice, 240 UGP/DGP heterodimers)(PDB 5vU2) and phlebovirus particles (icosahedral T = 12 quasi-equivalent lattice, i.e., 720 UGP/DGP heterodimers) (PDB 6f9b) The three structures represented display a similar organization in which the DGP (shown in white, gray and black) makes the lateral contacts between spikes (trimeric in flaviviruses and alphaviruses, hexameric and pentameric in phleboviruses), and the UGP (in various colors) caps the fusion loop of the DGP at the spike apices. The viral membrane is shown in steel-grey (arrows). Left column: Flavivirus particle maturation: Immature flavivirus particles undergo a conformational change in response to exposure to low pH in the Golgi, in which the 60 (prM/E)3 spikes (top panel) reorganize into 90 (prM/E)2 dimers (middle panel) (PDB 3c6R). In this conformation, prM exposes a cleavage site specific for the cellular furin proteinase. Upon cleavage, the peripheral “pr” domain (red, blue and yellow) stays bound as long as the pH is acidic, but is shed from the particle in the neutral pH external environment. Mature flavivirus particles (bottom left) expose the E protein in a herringbone arrangement, completely covering the viral membrane (PDB 5iz7). All structures are shown at the same scale (bar bottom left, 10nm). (B) Flavivirus particle heterogeneity (left panel) arising from incomplete furin maturation. In the case of dengue viruses, the conformational change between the top left and middle left panels in (A) is reversible with pH, whereas the one from the middle to the bottom panel is irreversible. Because furin is membrane bound, the particles are often processed only on the side that is closest to the TGN membrane with the opposite side uncleaved. Upon release into the neutral external environment, the non-processed side returns to the immature conformation, whereas the processed side adopts the mature conformation, giving rise to mosaic particles exposing the fusion loop in the immature patches. A representation of a “breathing” mature particle (right) shows an expanded size and exposure of the membrane underneath. These particles also expose epitopes normally buried by dimer contacts on virions. (C) Organization of the flavivirus E dimer as present on mature virions as a representative class II DGP (PDB code 5lbv). One subunit is rainbow colored along the polypeptide chain (from N to C terminus, as indicated in the color key bar below), the other is gray. The three structured domains DI, DII and DIII are indicated as well as the fusion loop (FL). Sites where disulfide bridges have been engineered to stabilize the dimer in the prefusion conformation are marked (star and arrow). Scale bar (bottom, right): 1 nm.
Figure 4
Figure 4
Postfusion Class II Fusion Proteins The viruses are listed in the left with the corresponding PDB accession code indicated in parenthesis. Each postfusion trimer is displayed with two protomers in surface representation colored light and dark gray, and with the third protomer (in the foreground) shown in ribbons ramp colored from N to C terminus (compare to the pre-fusion form in Figure 3C), highlighting the common fold of the polyeptide chains. The membrane is represented on the right, roughly to scale, and the N and C-terminal ends of the crystallized ectodomain are labeled in blue and red, respectively. Domain III (red) swaps protomers in the non-arboviruses packing against the bottom one (dark gray) instead of the top one (light gray) in the others. The fusion loop in the front protomer is shown as thicker tubes and marked by green/cyan arrows in the schematized fused membrane represented to the right (note that the Rubella virus protomer has two fusion loops). In the case of the phlebovirus protein, a glycerophospholipid head group present in the structure is shown in magenta spheres and marked by magenta arrows. In the cellular proteins, C. elegans EFF-1 does not have a non-polar fusion loop, but the three C-terminal ends converge toward the 3-fold axis at the level of the aliphatic moiety of the membrane (marked by a red “C”, the C-terminal end of the ectodomain). In C. rheinhadtii HAP2, the fusion loops were disordered in the crystal structure (indicated by the broken arrow).

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