Structural studies of Hantaan virus

Abstract

Hantaan virus is the prototypic member of the Hantavirus genus within the family Bunyaviridae and is a causative agent of the potentially fatal hemorrhagic fever with renal syndrome. The Bunyaviridae are a family of negative-sense RNA viruses with three-part segmented genomes. Virions are enveloped and decorated with spikes derived from a pair of glycoproteins (Gn and Gc). Here, we present cryo-electron tomography and single-particle cryo-electron microscopy studies of Hantaan virus virions. We have determined the structure of the tetrameric Gn-Gc spike complex to a resolution of 2.5 nm and show that spikes are ordered in lattices on the virion surface. Large cytoplasmic extensions associated with each Gn-Gc spike also form a lattice on the inner surface of the viral membrane. Rod-shaped ribonucleoprotein complexes are arranged into nearly parallel pairs and triplets within virions. Our results differ from the T=12 icosahedral organization found for some bunyaviruses. However, a comparison of our results with the previous tomographic studies of the nonpathogenic Tula hantavirus indicates a common structural organization for hantaviruses.

FIG. 1.

Virion diameter. Histogram showing the diameter ranges from 120 to 155 nm. The distribution is approximately normal with the largest numbers of virions having a diameter of between 130 and 134 nm.

FIG. 2.

Cryo-EM projection images. (A) A digital micrograph of several Hantaan virions embedded in vitreous ice. Strong density is black. Bar, 100 nm. (B) The periphery of a Hantaan virus virion with apparently ordered, membrane-embedded spikes. The black asterisks indicate what appears to be a pair of ectodomain density lobes separated by ∼7 nm. The white triangles indicate two adjacent sets of ectodomain lobes, separated by ∼14 nm. Each set of paired lobes likely represents a single Gn-Gc spike complex in projection. The spikes extend ∼10 nm above the membrane. Strong density is black. Bar, 10 nm.

FIG. 3.

Tomographic sections. (A to D) Rod-like densities within the boundaries of the viral membrane are presumed to be RNP complexes. These rods have a diameter of ∼10 nm and cluster into sets of nearly parallel densities with a spacing of ∼18 nm between the rods. (E) Occasionally, thinner rod-like densities with a narrower spacing were seen. (F) White triangles indicate RNP complexes, viewed end-on, located near the viral membrane. The Gn-Gc spikes are indicated by the black triangle. Strong density is black. Bar, 50 nm.

FIG. 4.

The asymmetric and 2-fold averaged reconstructions. (A) A section through the asymmetric reconstruction generated from Hantaan virus subparticle images. The section is parallel to the plane of the membrane and cuts through densities presumed to be the Gn-Gc ectodomains. The ectodomain lobes form a nearly 4-fold lattice and are separated by ∼7 nm. (B) The section is rendered in the same orientation as that in panel A, illustrating the 2-fold averaged Gn-Gc ectodomains. Strong density is black. Bar, 5 nm.

FIG. 5.

Sections from the 2-fold averaged subparticle reconstruction. Left-hand images (A, C, and E) are parallel to the plane of the membrane but are at different heights along an axis perpendicular to the membrane. Right-hand images (B, D, and F) are sections perpendicular to the plane of the membrane. (A) The section cuts through the 2-fold averaged Gn-Gc ectodomains. The white, gray, and black arrows indicate the positions of the planes from which panels B, D, and F are derived, respectively. (B) The section cuts through the center of the ectodomain lobes. Here, the white, gray, and black arrows indicate the positions of the planes from which panels A, C, and E are derived, respectively. (C) The section cuts through the array of Gn-Gc cytoplasmic tail densities located on the inner surface of the viral membrane. (D) The section cuts through the center of two adjacent Gn-Gc spikes. White triangles indicate the location of densities below the center of the Gn-Gc spikes, presumed to be the glycoprotein cytoplasmic tails. (E and F) The sections show density presumed to be an RNP complex (black triangle) in lengthwise (E) and end-on (F) orientations. Strong density is black. Bar, 5 nm.

FIG. 6.

A three-dimensional isosurface rendering of the 2-fold averaged reconstruction. The map was contoured at 1.5σ and tilted to show the cytoplasmic densities. Densities were colored according to radius, so that the Gn-Gc ectodomains are green, the membrane and associated cytoplasmic tails are yellow, and the RNP densities are red. Note that the internal ceiling of the yellow density represents the boundary between the cytoplasmic components of the spikes and the lower-density interior of the virus. Bar, 5 nm.

FIG. 7.

An isosurface rendering of the 4-fold averaged reconstruction. The map was contoured at 1.1σ and is shown down an axis normal to the membrane. The color scheme is the same as in Fig. 6. A system of ridges and valleys at the membrane surface (yellow) was found beneath the four ectodomain lobes (green). The ectodomains of the central spike complex nearly make contact with the neighboring spikes at this contour level. Bar, 5 nm.

FIG. 8.

Sections through the 4-fold averaged reconstruction. (A to C) Sections parallel to the plane of the membrane show the glycoprotein ectodomains (A), ridges on the virion surface in the shape of an X (B), and the Gn-Gc cytoplasmic tails on the inner viral membrane (C). (D to F) Sections normal to the plane of the membrane. The white, gray, and black arrows in panel A indicate the positions of the planes from which panels D, E, and F are derived, respectively. In panel D, the white, gray, and black arrows indicate the positions of the planes from which panels A, B, and C are derived, respectively. Bar, 5 nm. Strong densities are black.

FIG. 9.

Comparison of the Hantaan and Tula hantavirus spike complex maps. Left-hand images are sections through the 4-fold averaged Hantaan virus spike complex map. Right-hand images are corresponding sections from the Tula virus map. For both maps, positive density is black. All sections are parallel to the plane of the viral membrane but at different levels along an axis perpendicular to the plane of the membrane. (A and B) Ectodomains. The two maps show similar arrangements of the glycoprotein ectodomains. (C and D) Stalks. Each of the four Hantaan ectodomain lobes is tethered to the membrane via a stalk, making a total of four stalk densities. However, the Tula virus spike complex has a single stalk, which is central to the four ectodomain lobes, as well as peripheral stalks which link adjacent spikes. These peripheral stalks are not apparent in the Hantaan virus map. (E and F) Ridges. Ridges form an X on the virion surface, or outer membrane, in both maps. (G and H) Protrusion into virion. In both maps a large cytoplasmic protrusion is seen on the inner surface of the virion, located beneath the center of the four ectodomain lobes.