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Insights Into Bunyavirus Architecture From Electron Cryotomography Of Uukuniemi Virus

Bunyaviridae is a large family of viruses that have gained attention as “emerging viruses” because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or “spikes,” were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry.

Bunyaviridae
envelope glycoproteins
ribonucleoprotein particles
virus assembly
virus structure

The family Bunyaviridae consists of a diverse group of >350 viruses and is divided into five genera: Hantavirus, Nairovirus, Orthobunyavirus, Phlebovirus, and Tospovirus. Most members of the Bunyaviridae family are spread by hematophagous arthopods, such as mosquitoes and ticks. However, members of the genus Hantavirus are spread by persistently infected rodents (1). Bunyaviruses are distributed worldwide, and many of them are considered “emerging viruses,” with increasing numbers of outbreaks. In addition, some members of the family, such as La Crosse virus (Orthobunyavirus), Rift Valley fever virus (Phlebovirus), Crimean-Congo hemorrhagic fever virus (Nairovirus), and Sin Nombre virus (Hantavirus), cause severe illness in humans, including hemorrhagic fever and encephalitis (2).

Bunyaviruses are enveloped, spherical viruses (≈100 nm in diameter), with a segmented negative-sense RNA genome. The three genome segments encode four structural proteins. The L segment encodes the RNA-dependent RNA polymerase; the M segment encodes the glycoprotein precursor, which is cleaved into two glycoproteins (GN and GC); and the S segment encodes the nucleoprotein (N protein). The N protein is associated with the RNA genome and, together with the polymerase, forms the ribonucleoproteins (RNPs) (3). The two glycoproteins are situated in the envelope and form surface protrusions called “spikes.”

Bunyaviruses likely enter the cell via receptor-mediated endocytosis, as has been shown for many other animal viruses. Slightly acidic conditions of the endosome are required for the fusion between the viral and cellular membrane and for subsequent release of the RNPs into the cytoplasm (4-7). Nascent glycoproteins are inserted into the endoplasmic reticulum membrane during translation and mature to form heterodimers. The glycosylated dimers are transported to the Golgi, were they are retained until budding occurs (8). Most enveloped viruses bud from the plasma membrane into the extracellular space; however, bunyavirus virions form by budding into the Golgi (9). Bunyaviruses do not have a matrix protein facilitating the interaction between the membrane and RNPs, as is seen in most negative-sense RNA viruses (10). Instead, the RNPs interact directly with the cytoplasmic tails of GN or GC. The GN cytoplasmic tail has been shown to interact with RNPs and to mediate genome packaging (11), and the cytoplasmic tails of both GN and GC contain motifs essential for virus assembly (12, 13). From the Golgi, large vesicles filled with numerous viruses are transported to the plasma membrane, and the viruses are released (14).

Uukuniemi virus (UUKV; genus Phlebovirus) is a particularly convenient model virus for studying bunyaviral packaging, budding, and structure. Firstly, UUKV is not a human pathogen (15), and virions can be grown to high titers in cell culture and studied in low-biosafety-level laboratories. In addition, virus-like particles can be produced in different compositions, for example, with variable amounts of genome segments, for structural studies (16). This tick-borne virus was isolated in Uukuniemi, Finland, in 1964 (17). The UUKV glycoproteins, GN (75 kDa) and GC (65 kDa) (18), are responsible for the structural stability of the virus and have been shown to be sufficient for formation of virus-like particles that are morphologically similar to wild-type UUKV (11). The UUKV glycoproteins have been depicted clustered as hollow cylindrical morphological units (19). The lateral organization of the spikes has been postulated to be icosahedrally symmetric, with hexameric and pentameric clusters creating a specific so-called “T = 12” arrangement (19), but the exact organization and structure of the spikes have remained obscure. In general, bunyavirus virion structure constitutes largely uncharted territory because no crystal structure of the two glycoproteins-and no 3D structures of the virions-exist for any of the 350 members of the family. Many family members have pleomorphic virion structures (20) and thus have not been amenable to structural characterization using the averaging techniques commonly used in the study of icosahedrally symmetric virions (21).

In this study, we analyzed the structure of extracellular, purified, unstained UUKV particles by using electron cryotomography. The technique allows the 3D density distribution for a biological object to be resolved in a near-native, vitreous state (22, 23). The 3D structure of the UUK virion was solved to 5.9 nm resolution, providing a structural model for a bunyavirus and insight into conformational changes in the glycoproteins during entry.

UUKV Particles Display a Variable Degree of Pleiomorphy.

We collected tomographic tilt series of vitrified UUKV samples, and from these image series we calculated 3D density maps (tomograms). Three types of samples, which differed in their pH and fixation by glutaraldehyde (19), were used for tomography: (i) pH6, a virus suspension buffered to pH 6 and fixed with glutaraldehyde, (ii) pH7, a virus suspension buffered to pH 7 and fixed with glutaraldehyde, and (iii) pH7*, a virus suspension without buffering, fixed with glutaraldehyde (Table 1). Although particles in the pH7 (Fig.