Crystal structure of Schmallenberg orthobunyavirus nucleoprotein-RNA complex reveals a novel RNA sequestration mechanism.

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus (family Bunyaviridae) that has caused severe disease in the offspring of farm animals across Europe. Like all orthobunyaviruses, SBV contains a tripartite negative-sense RNA genome that is encapsidated by the viral nucleocapsid (N) protein in the form of a ribonucleoprotein complex (RNP). We recently reported the three-dimensional structure of SBV N that revealed a novel fold. Here we report the crystal structure of the SBV N protein in complex with a 42-nt-long RNA to 2.16 Å resolution. The complex comprises a tetramer of N that encapsidates the RNA as a cross-shape inside the protein ring structure, with each protomer bound to 11 ribonucleotides. Eight bases are bound in the positively charged cleft between the N- and C-terminal domains of N, and three bases are shielded by the extended N-terminal arm. SBV N appears to sequester RNA using a different mechanism compared with the nucleoproteins of other negative-sense RNA viruses. Furthermore, the structure suggests that RNA binding results in conformational changes of some residues in the RNA-binding cleft and the N- and C-terminal arms. Our results provide new insights into the novel mechanism of RNA encapsidation by orthobunyaviruses.

Shuni virus as cause of neurologic disease in horses.

To determine which agents cause neurologic disease in horses, we conducted reverse transcription PCR on isolates from of a horse with encephalitis and 111 other horses with acute disease. Shuni virus was found in 7 horses, 5 of which had neurologic signs. Testing for lesser known viruses should be considered for horses with unexplained illness.

Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification.

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.

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.

Turlock-like bunyavirus associated with encephalomyelitis and myocarditis in an ostrich chick.

In the fall of 1995, a 20-day-old female ostrich chick, 1 of a group of 20, was presented live with clinical signs of 2 days duration characterized by unsteady gait, circling to the left, and walking backward. Another bird with similar clinical signs had died and another had recovered. The bird was euthanized and examined at necropsy. Twenty-five milliliters of serous fluid was in the abdominal cavity and there was increased pericardial fluid. Histopathology of the brain revealed mild to moderate nonsuppurative encephalitis characterized by mild multifocal malacia, perivascular cuffing by lymphocytes, and gliosis. The heart had multifocal infiltrations of lymphocytes mixed with macrophages and a few plasma cells throughout the myocardium. Cytopathic effects were observed in primary chicken embryo liver cells following inoculation with a tissue homogenate prepared from the brain of the affected ostrich. Virus particles the size and morphology of the family Bunyaviridae were observed in cell culture lysate by negative-stain electron microscopy. Viral characterization demonstrated that the virus isolate is a previously unknown serotypic variant (subtype) of Turlock virus. Twelve of 65 sera collected over a 3-year period from ostriches aged from 1 month to 4 years were positive for neutralizing antibody to both the Turlock prototype strain and the new subtype of Turlock virus described in this report.

Cell biology of viruses that assemble along the biosynthetic pathway.

In this review we discuss five groups of viruses that bud into, or assemble from, different compartments along the biosynthetic pathway. These are herpes-, rota-, corona-, bunya- and pox-viruses. Our main emphasis will be on the virally-encoded membrane glycoproteins that are responsible for determining the site of virus assembly. In a number of cases these proteins have been well characterized and appear to serve as resident markers of the budding compartments. The assembly and dissemination of these viruses raises many questions of cell biological interest.