Filovirus Filament Proteins.

Filoviruses are highly filamentous enveloped animal viruses that can cause severe haemorrhagic fevers. The filovirus ribonucleoprotein forms a highly organized double-layered helical nucleocapsid (NC) containing five different virally encoded proteins. The inner layer consists of NP, the RNA binding protein, complexed with the monopartite linear genome. A distinctive outer layer links individual NP subunits with bridges composed of VP24-VP35 heterodimers, which achieves condensation of the NP-RNA into tight helical coils. There are no vertical connections between the outer helical layers, explaining the flexibility of the NC and its ability to bend into tight curves without breaking the genomic RNA. These properties allow the formation of enveloped virions with varying polymorphisms, including single, linear, continuous, linked, comma-shaped and torroidal forms. Virion length is modular so that just one, or two or more genome copies may be present in each virion, producing polyploid particles. The matrix protein VP40, which drives budding and envelopment, is found in a layer adjacent to the inner cytoplasmic side of viral envelope and is arranged in a 5 nm lattice structure, but its exact symmetry is unclear. There is a constant low density gap between VP40 and the nucleocapsid, so that the latter is held rigidly centred on the long axis of the viral filament. This gap likely contains a region of flexible contacts between VP40 and the NC. The unique morphology of filoviruses may be related to high titre replication, their ease of transmission, and abilities to invade a wide range of host cells and tissues.

The scanning electron microscope in microbiology and diagnosis of infectious disease.

Despite being an excellent tool for investigating ultrastructure, scanning electron microscopy (SEM) is less frequently used than transmission electron microscopy for microbes such as viruses or bacteria. Here we describe rapid methods that allow SEM imaging of fully hydrated, unfixed microbes without using conventional sample preparation methods. We demonstrate improved ultrastructural preservation, with greatly reduced dehydration and shrinkage, for specimens including bacteria and viruses such as Ebola virus using infiltration with ionic liquid on conducting filter substrates for SEM.

The organisation of Ebola virus reveals a capacity for extensive, modular polyploidy.

BACKGROUND: Filoviruses, including Ebola virus, are unusual in being filamentous animal viruses. Structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. The present study provides unique insights into the structure of this deadly pathogen. METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. We show that the helical Ebola virus inner nucleocapsid containing RNA and nucleoprotein is stabilized by an outer layer of VP24-VP35 bridges. Elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. The matrix protein VP40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular. CONCLUSIONS: The results of this study demonstrate a modular organization in Ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope.