Structural maturation of rubella virus in the Golgi complex

Abstract

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.

Fig. 1

Associations of organelles in Vero cells infected with rubella virus. (A) Perinuclear areas in control, uninfected Vero cells, where mitochondria (mi) are randomly distributed. (B) Vero-infected cells at 16 h.p.i. exhibit cytopathic vacuoles (asterisks) surrounded by elongated, denser mitochondria (white arrows). (C) Higher magnification field showing a cytopathic vacuole (CV) attached to a mitochondrion (mi), rough endoplasmic reticulum (RER) cisternae, and a Golgi stack with viruses (V). (D) Cytopathic vacuole with vesicular structures containing dense spots (arrows). (E) Later in infection (48 h.p.i.) these associations of organelles mainly dissappear and in many cells both CVs (asterisks) and mitochondria (arrows) look empty. A, B, and D correspond to freeze-substituted cells, while C and E are conventionally processed samples. N, nucleus. Bars, 1 μm in A, B, and E; 200 nm in C and 100 nm in D.

Fig. 2

Ultrastructure of BHK-21 cells infected with rubella virus and processed by freeze-substitution. Low-magnification fields of (A) control uninfected BHK-21 cells and (B) rubella-infected cells (24 h.p.i.). Mitochondria are marked with white asterisks (A) or white arrowheads (B). Cytopathic vacuoles (black arrows in B) accumulate in the perinuclear region of infected cells. (C) Higher magnification field of the perinuclear region showing a budding profile (arrow) and a dense viral particle (white arrowhead) in smooth membranes (black arrowheads) close to a characteristic cytopathic vacuole (CV). N, nucleus; c, centrioles; RER, rough endoplasmic reticulum. Bars, 1 μm in A and B; 100 nm in C.

Fig. 3

Rubella virus polymorphism in the Golgi complex. (A and B) Golgi membranes in infected Vero cells at 16 h.p.i. contain maturation arcs (arrows) and dense spherical particles (arrowheads). (C–E) Higher magnification fields showing the structure of dense rubella particles. Connections with budding membranes are marked with arrows. Focal contacts between the particle content and the envelope are marked in (E). (F) Viral particles (white arrows) with an annular-like morphology (dense periphery and less dense center) are seen as a minor class inside Golgi stacks. (G) Mature rubella virion inside the Golgi stack. Note the dense envelope and internal core well separated from the envelope, features also found in extracellular virions (H). (I–K) Rubella virus-infected BHK-21 cells. (I) Perinuclear budding profile (black arrowhead) and dense viral particle (white arrowhead) in smooth membranes (black arrowheads). (J) Mature viral particles (arrows) containing assembled cores inside a Golgi stack. (K) Extracellular virion attached to the cell surface containing a central dense core and connections between core and envelope (arrows). A conventionally processed sample is shown in A, while B to K correspond to freeze-substituted cells. Bars, 100 nm in A and B; 60 nm in C–K.

Fig. 4

Structural details in rubella extracellular virions. Minor differences in the apparent density of the internal core are shown: (A and B) Virions with a denser core and (C and D) extracellular particles with less dense cores. (E–I) Shown are some internal details in extracellular virions. In (E) a layer (probably the core wall) is resolved (arrow), while in (F) and (G) envelope–core connections are clear (arrows). In distorted particles these contacts become more evident (arrows in H and I). All fields correspond to freeze-substituted samples. Bars, 60 nm.