FIG. 1.

Isolation of three viral forms from Bunyamwera virus-infected BHK-21 cells. (A) At 8 h postinfection, confocal microscopy shows the large perinuclear structure (arrow), known as the viral factory (vf), where viral proteins (here Gc is labeled in red) and mitochondria (green) are recruited. Labeling was performed as described in Materials and Methods. Small red dots seen on the cell periphery correspond to secretory vesicles filled with viruses. (B) Electron microscopy shows the viral factory around the nucleus (N) as associations of mitochondria (mi) and membranes (arrows). In these factories viral particles are assembled by recruitment of the structural components represented in C: an envelope with spikes (made of Gc and Gn glycoproteins) and an internal core containing three ribonucleoproteins (RNPs) of RNA, nucleocapsid protein (N), and RNA polymerase (L). (D) Viral particles with three different morphologies are seen in thin sections of infected cells: intracellular viruses on the left correspond either to type I (annular structures that correspond to immature precursors) or type II (an intermediate dense form thatassembles in a trans-Golgi-dependent manner) morphology. Extracellular virions (E) on the right are also dense particles but with a more defined coat of spikes (arrowheads). Higher-magnification fields on the bottom show the structural characteristics of the viral particles in more detail. (E) Isolation of these three viral forms gave homogeneous populations of type I (left), type II (middle), and extracellular (right) viruses. Viral structural proteins were separated by SDS-PAGE and analyzed by Coomassie blue staining (CB) and Western blotting (WB). The asterisk on the gel for extracellular viruses marks a band of seralbumin identified by MALDI peptide mass fingerprinting. Thin sections of purified viruses (high-magnification EM fields on the bottom) show the internal structure of the three purified viral forms. Bars: 1 μm in B, 100 nm in D and E.