The structure of a putative scaffolding protein of immature poxvirus particles as determined by electron microscopy suggests similarity with capsid proteins of large icosahedral DNA viruses

Abstract

Orf virus, the prototype parapoxvirus, is responsible for contagious ecthyma in sheep and goats. The central region of the viral genome codes for proteins highly conserved among vertebrate poxviruses and which are frequently essential for viral proliferation. Analysis of the recently published genome sequence of orf virus revealed that among such essential proteins, the protein orfv075 is an orthologue of D13, the rifampin resistance gene product critical for vaccinia virus morphogenesis. Previous studies showed that D13, arranged as "spicules," is necessary for the formation of vaccinia virus immature virions, a mandatory intermediate in viral maturation. We have determined the three-dimensional structure of recombinant orfv075 at approximately 25-A resolution by electron microscopy of two-dimensional crystals. orfv075 organizes as trimers with a tripod-like main body and a propeller-like smaller domain. The molecular envelope of orfv075 shows unexpectedly good agreement to that of a distant homologue, VP54, the major capsid protein of Paramecium bursaria Chlorella virus type 1. Our structural analysis suggests that orfv075 belongs in the double-barreled capsid protein family found in many double-stranded DNA icosahedral viruses and supports the hypothesis that the nonicosahedral poxviruses and the large icosahedral DNA viruses are evolutionarily related.

FIG. 1.

SEC of orfv075 with or without a His tag and estimation of molecular mass of His tag-cleaved orfv075. (A) The majority of the His-tagged orfv075 (chromatogram with dashed line), purified by immobilized metal affinity chromatography, exists as aggregates eluting in the void volume. Upon cleavage of the His tag by rTEV protease, the majority of protein sample (chromatogram with solid line) dissociates into smaller oligomers. In both cases, the protein is essentially pure as indicated by the ∼65-kDa (+His tag) or ∼60-kDa (−His tag) bands in SDS-PAGE (inset). (B) Log values of molecular mass of standards are plotted as a function of distribution coefficients, K, in order to estimate the molecular mass of the eluent from gel filtration. Based on the elution volume of His tag-cleaved orfv075 in the gel filtration chromatogram, the molecular mass was estimated to be ∼160 kDa, indicated by a circle. Assuming that a nonglobular shape of oligomeric orfv075 is hampering precise measurement, the estimated molecular mass would correspond to homotrimers of orfv075.

FIG. 2.

The purified His-tagged orfv075 oligomers exhibit variable morphologies as visualized from specimens preserved in negative stain. Aggregates of honeycomb-shaped crystalline patches (A) and tubular objects with heterogeneous length (B) are observed. The background consists of smaller protein oligomers. The images were recorded using a BIOSCAN charge-coupled device camera. Bars, 100 nm.

FIG. 3.

Negatively stained 2-D crystals of recombinant His-tagged orfv075. (A) Crystals produced in situ (type I crystals). (B) Crystals produced on lipid monolayer (type II crystals). Relatively large aggregates overlay both crystal types. The type II crystals often showed discontinuity, possibly due to mechanical stress upon transfer from the air-water interface. The insets show computed Fourier transforms of sections of crystals that exhibit hexagonal symmetry. Reflections up to resolutions of 18 Å and 20 Å are visible for type I and type II crystals, respectively. Bars, 200 nm in the micrographs and 200 Å⁻¹ in the computed diffractograms.

FIG. 4.

Projection density maps calculated from images of nominally untilted recombinant orfv075 2-D crystals. No symmetry was imposed, and one unit cell is demarcated. Positive and negative densities, represented by continuous and dashed contour lines, denote, respectively, regions of protein and stain. (A) Type I crystal. Compact trimeric subunits arranged with p3 symmetry are clearly visible. Regions between the trimers are extensively permeated by stain. The unit cell edge is a = b = 140 Å. (B) Type II crystal. Compact trimeric subunits arranged with p6 symmetry exhibit denser crystal packing. Six adjacent trimers surround large stain-filled channels in the crystal. The unit cell dimensions are a = b = 154 Å.

FIG. 5.

Phase and amplitude variations as a function of reciprocal lattice coordinate z* generated from tilted type I crystals and analyzed with the p3 plane group of symmetry. Phase and amplitude profiles for two example lattice lines (2,2 and 4,0) are shown. The least-squares-fitted profiles were regularly sampled at intervals of 0.005 Å⁻¹ to yield 3-D structure factors. Data to a maximum resolution of ∼25 Å were used in the reconstruction.

FIG. 6.

A montage of different views of the surface-rendered 3-D density volume of the recombinant orfv075 trimer generated from analysis of images of type I crystals. The 3-D reconstruction at a resolution of ∼25 Å was computed using images of tilted negatively stained crystals. The top row shows views of the model rotated around the axis of threefold symmetry, and the bottom row shows views of the model rotated around an arbitrary axis perpendicular to threefold symmetry and includes views along the threefold from the “top” (far left) and from the “bottom” (far right) of the orfv075 trimer. The trimer (∼90 Å in height by ∼70 Å wide) is composed of a small “propeller” domain connected to a larger “tripod” base, designated in orange (propeller domain) and in red (tripod base) apportioned arbitrarily for one monomer. The “legs” of the tripod base surround a deep cavity that appears to be bounded by a small protrusion in the propeller domain. The density threshold (= 1.3σ) is appropriate for the expected volume of the trimer.

FIG. 7.

Docking of VP54 of PBCV-1 into the 3-D model of orfv075 trimer. The top and bottom panels represent views, respectively, down and normal to the threefold axis of the trimer of PBCV-1 VP54 (A and D). The atomic structure of VP54 is represented as a cartoon with the N- and C-terminal jelly roll domains in blue and red, respectively. (B and E) The fit of the VP54 trimer into the 3-D model of orfv075 is shown as a composite of the docked X-ray structure and a surface-rendered (1.3 σ) view of orfv075 cutting through approximately the center of the model. (C and F) Similar views of the whole model.

FIG. 8.

View of thin section of cytoplasm of an ORFV-infected cell at 14 h postinfection. The cells were fixed and embedded in Quetol 651 resin prior to ultrathin sectioning. The arrowhead indicates a spherical structure that resembles IV of vaccinia virus and encloses dense regions possibly representing condensed viral proteins and DNA. The arrow indicates an incomplete spherical object similar to the crescent-shaped precursor of IV. An extra layer on the outer surface of these structures is apparent, suggestive of orfv075 scaffolding analogous to D13 lattice on vaccinia virus IV. Bar, 0.5 μm.