Membrane remodeling by the double-barrel scaffolding protein of poxvirus

Abstract

In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors.

Figure 1. The scaffolding protein D13 of poxvirus is a trimeric double-barrel capsid protein.

(A) The three structural domains of D13 are two successive jellyrolls (J1 and J2 colored in blue and red respectively), and a “head” domain (H colored in yellow) with a novel β-sandwich fold partially wrapped around a helix projecting away from the domain J2. Expanded details of these domains are shown as insets with a blue-to-red gradient from the N- to C-terminus of the protein. (B, C, E) Cartoon representation of the D13 trimer with each monomer colored as in A. The trimer is viewed in profile oriented on the viral membrane (B) and normal to the membrane as seen from the outside of the immature virion at different heights in the trimer centered on the head (C) or the J1/J2 domains (E). The arrow points to the portion of the His₆-tag (residues −11 to 1) visible in one of the three subunits. (D). Domain swapping of the N-terminal loop. The molecular surface of the three subunits of a D13 trimer is represented. This surface is semi-transparent for residues 1–31 of one subunit. The cartoon representation of the N-terminal loop reveals the packing of the first helix (arrow) between the J1 and J2 domains of the subunits colored in blue and green respectively.

Figure 2. D13 is an atypical member of the double-barrel capsid family of viruses infecting all kingdoms of life.

(A) Schematic representations of virions containing double-barrel capsid proteins, grouped according to the viral hosts. The schematics are to scale except for the immature vaccinia virion that is represented both as a 1/8^th slice to scale and a reduced schematic of the complete particle. Poxvirus immature particles are by far the largest objects and the only one lacking infectivity and an icosahedral symmetry. Apart from adenovirus, all viruses contain an internal lipid membrane indicated in blue. The double-barrel capsid layers are shown for PM2 (pink), PRD-1 (green), STIV (red), PBCV-1 (magenta), vaccinia virus (blue) and adenovirus (yellow). Appendages are represented in grey for turreted viruses and orange for viral fibers. (B) Cartoon representations of the double-barrel capsid proteins characterized by the highly-conserved central double-barrel domain with the same coloring scheme for one of the subunit as in (A). The bottom panels represent the proteins viewed from the outside of the particle and the top panels include those for an orthogonal view. In contrast, when present, the “head” and “feet” regions highlighted in red and orange boxes respectively correspond to the most dissimilar domains amongst the capsid proteins.

Figure 3. Polymorphic assemblies of purified D13 visualized by electron microscopy.

(A) An electron micrograph of homogeneous purified D13 trimers visualized by uranyl acetate staining (negative staining). (B) Self-assembly of short tubular objects is observed in the absence of arginine and glutamate additives and when the His₆-tag is removed by proteolytic digest. When purified protein is dialyzed into low ionic strength buffers, assembly is observed in the form of small crystalline patches (negatively stained) (C) and irregular spherical assemblies as observed by cryoEM (D). Scale bars represent 100 nm (A) and 200 nm (B–D).

Figure 4. Immature virion-like particles formed by D13 on artificial membranes.

(A) D13 was incubated with a mixture of dissolved lipids (59% DOPC, 18% DOPE, 3% DOPS doped with 20% of nickel loaded DOGS-NTA) and detergent (4% n-βOG). After dialyzing away the detergent, assembly of large spherical shells is observed by cryoEM only when His₆-tagged D13 is present. The top left inset shows a blown up area where the D13 spikes are readily visible. (B–D) Electron micrographs of negatively stained samples of control experiments using no protein (B), D13 after His₆-tag removal by rTEV protease digest (C), and a non-relevant His₆-tagged protein (D). Scale bars represent 200 nm.

Figure 5. Honeycomb lattice formed by D13 on artificial membranes.

(A) When a lipid monolayer composed of egg PC and PG doped with nickel loaded DOGS-NTA was carefully formed to produce a flat membrane at the air-liquid interface, D13 trimers formed 2-D crystals. These crystals are relatively large and undistorted areas could be identified by optical diffraction. (B) Projection map computed from the negative stain images of the 2-D crystals without imposing any symmetry. Similarity is evident with the honeycomb lattices observed in vivo on the surface of immature particles (Figure 7 of reference [8]) and on flat sheets of the D13^D513G protein (Figure 4 of reference [9]). Scale bars represent 100 nm in panel (A) and 14 nm in panels (B).

Figure 6. Pseudo-atomic model of the honeycombed assembly of D13.

Docking of the atomic structure of D13 trimer into the ∼20 Å resolution 3-D density map generated by electron crystallography of 2-D crystals shown in Figure 5A. (A) View of a slab approximately through the middle of the trimers showing the p6 honeycombed assembly of D13 looking onto the membrane plane from ‘outside’. The inset represents a detailed view of the interface between two D13 trimers involving J1-J2 contacts. Residue G₅₁₃ is shown as a sphere colored in green. (B) Slab proximal to the lipid membrane indicating differences between the X-ray crystal structure and the EM structure. The inset shows an orthogonal view of the fit of the atomic model of a D13 trimer colored as in Figure 1 into an excised volume of density from the EM 3-D map. (C) Conserved domains in D13 mapped onto the molecular surface. The surface is colored in a cyan-white-magenta gradient from the least to the most conserved residues as estimated from an alignment of 16 unique sequences of poxviruses. Residue G₅₁₃ is highlighted in red (arrow). The two panels represent detailed views of the most highly conserved regions of D13. Side chains of strictly conserved residues are indicated as sticks within the semi-transparent surface and residues close to lattice contacts are labeled.

Figure 7. Mutations in response to rifampicin cluster at the base of the D13 trimer.

Surface rendered view of a D13 trimer indicating the three sites, defined by Charity and coll. , where mutations that produce resistance to the antiviral effect of rifampicin cluster. Site I (residues 17–33; cyan), II (residues 222–243; magenta) and III (residues 480–488; green) are highlighted with the rest of the molecular surface colored brown. The first and last residues of each cluster are indicated in (B) and (C) for one of the subunit. Panel (A) represents a hexameric ring of D13 trimers on the viral membrane (bottom, grey layer) and normal to the membrane viewed from the inside of the particle (top). Panel (B) represents a view from inside the particle through the membrane similar to those in (A) and (C) and represents a slice approximately through the centre of the double-barrel domain as viewed from the outside of the particle.

Figure 8. Model of the immature virion-like particle assembly.

The color scheme used for D13 trimers is the same as in Figure 1. The membrane is represented in blue and the nickel-lipids are represented as yellow crosses and spikes. (a) Attachment sites for the His₆-tag promote membrane association of D13 in vitro. This interaction appears to act as a surrogate of viral binding partner(s) embedded in the membrane such as the A17 protein. (b) Subsequently, D13 trimers dock onto the surface and form a honeycomb lattice. (c) The apparently continuous curvature of the honeycomb lattice results in a crescent-like formation. (d) This structure is eventually extended to form a spherical particle. Defects in the lattice are not depicted here but they are necessary to achieve a closed shell and were frequently observed on immature virions in previous studies . (e) Because D13 trimers do not interact directly with the membrane, cleavage of the attachment anchor, such as the N-terminal region of A17, may be sufficient to induce the detachment of either single trimers or assembled D13 sheets from the enveloped particle.

Figure 9. Comparison between the lattices and capsids of vaccinia virus and mimivirus.

(A, D) Honeycomb lattices formed by vaccinia virus D13 and mimivirus P1 respectively. The color scheme is the same as in Figure 5 and the mimivirus capsomer is schematized by a hexagonal base and a head domain colored in blue and yellow respectively . The p6 symmetry is only local and other members of the double-barrel lineage adopt a local p3 lattice with an additional trimer instead of a gap at the 6-fold axis. The positions of the 2- and 6-fold symmetry axes are indicated by ellipses and hexagons. Lattice parameters are indicated in red. (B, E) Analogous views of D13 and a model of the mimivirus P1 capsid protein determined by the Phyre2 server with a blue-red gradient from N- to C-terminus. The confidence is 99.83% with 457 aligned residues sharing 21% sequence identity. The head domain of mimivirus is predicted to be inserted in the J2_DE loop like its counterpart in D13 and to share a similar spatial arrangement at the top of the spike. (C, F) Topology diagrams for D13 and P1. J1 and J2 are represented in blue and red respectively, the head domains are shown in yellow. The head domain of P1 is only represented schematically as a box.