The Arenaviredae are a family of viruses whose members are generally associated with rodent transmitted disease in humans which currently comprises 24 viral species. Arenavirus infections are relatively common in humans in some areas of the world and can cause' severe illnesses including several haemorrhagic fevers. The virus particles vary in diameter from 60 to more than 300 nm. They are spherical and have a reported average diameter of 92 nanometres. All are enveloped in a lipid bilayer and have a bisegmented ambisense RNA genome, but relatively little is known about how virions are assembled and how virion structure relates to transmissibility. To investigate the role of each viral structural protein in forming and maintaining the structure of the virion, we have imaged particles of arenaviruses LCMV, PICV and TCRV, and compared their shape and structural characteristics to similar sized phospholipid vesicles. A very strong association between particle size and shape was found for all arenavirus particles: small virions were significantly rounder than vesicles of similar size, while large particles tended to be more elliptical in appearance. The natural variation in surface glycoprotein decoration and ribonucleoprotein incorporation was then measured. From this data it was concluded that there is no strong evidence relating particles size to decoration for arenaviruses as a group, but we did detect Significant correlations between internal density and virion shape. Overall, we are able to conclude that small virions are round and relatively rigid compared to vesicles of the same size, while large virions are not. By comparing relative density of the membrane- proximal region it was discovered that arenavirus shape is controlled by complexes containing GPC, Z and NP at the surface of the virion, and that an unbroken inner shell of NP is essential for maintaining a rigid spherical shape. Furthermore, it was revealed that the inner leaflet of intact arenaviruses has a lower density than the inner leaflet of vesicles consistent with the interpretation that viral proteins are displaying lipid molecules from the inner leaflet of the viral membrane. These data provide a new way of assessing the function of viral protein interactions on virion structure and may be of use in designing antiviral drugs that act at the level of virion structure.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Morbilliviruses can cause severe disease in a wide range of terrestrial and marine mammals with measles still responsible for large numbers of fatalities in developing countries despite the availability of an efficacious vaccine. Identification of morbillivirus receptors, the major determinant of host range and tissue tropism, is of paramount importance in understanding the disease induced by these viruses. Identification and characterisation of receptors also facilitates the development of novel antiviral drugs which block viral attachment and entry. SLAM, also known as CD 150, is the entry receptor for wild-type strains of measles virus (MY). Whereas laboratory-adapted MY can use both SLAM and CD46. The susceptibility of SLAM negative oligodendroglioma, astrocytoma and microglioma derived cell-lines to infection with recombinant (r) CD46 "receptor blind" rM suggests the expression of an unknown virus entry receptor. While it is currently unknown whether wild-type MY uses the same receptor molecule to infect glial and epithelial cells, the absence of infected glial cells following infection with an epithelial "receptor blind" rMY suggests that MY may use the same cellular receptor to infect both cell types. This unknown receptor could be identified through the construction of a cDNA library from these cell lines and subsequent analysis using the novel bimolecular fluorescent complementation (BiFC) fusion assay developed in this study. This assay, which was developed to identify unknown viral fusion receptors, has been validated using expression plasmids encoding MY glycoproteins and known cellular receptors. A BiFC based MY receptor screen using a human spleen cDNA library produced fluorescent fusion events. These were isolated and PCR amplification was used to attempt to identify the unknown receptor Fusion assays using expression plasmids encoding canine distemper virus (CDY) heamagglutinin (H) and fusion (F) glycoproteins and BiFC fusion assay demonstrated that the H glycoprotein is the key determinant of cell tropism.

Simple (non-enveloped) small, positive-sense single-stranded RNA viruses infect hosts from all kingdoms of life. However, their assembly and uncoating processes remain poorly understood. For turnip crinkle virus (TCV), 3D reconstructions by cryoelectron microscopy (cryo-EM) are shown for the native and the expanded form. The expanded form is a putative disassembly intermediate and exhibits pores that are large enough to allow exit of single-stranded RNA. Biochemical experiments revealed the expanded form is protease-sensitive, although the RNA genome remains protected from ribonuclease. Virus particles complexed with ribosomes are shown by negative stain EM. Proteolysis causes release of some coat protein from the capsid, while the capsid remains largely intact. Proteolysed particles have lost their icosahedral symmetry and show a protuberance in negative stain EM. Taken together, these results suggest expansion and subsequent proteolysis are essential steps in the uncoating process of TCV, and that the capsid plays multiple roles consistent with ribosome-mediated genome uncoating to avoid host anti-viral activity. Similarly, 3D cryo-EM reconstructions are presented for native equine rhinitis A virus (ERAV) an expanded particle containing no RNA. The native virus fits well with the ERAV crystal structure. The empty particle is a putative disassembly intermediate representing a stage after the release of the RNA genome. A mechanism is suggested that is consistent with the RNA release from the endosome without exposure to the endosomal contents. A crystal structure is presented of satellite tobacco necrosis virus (STNV) virus-like particles containing a small RNA fragment. The coat protein structure is identical to that of native STNV. Although density internal to the coat protein shell has been observed in the experiment that corresponds to earlier experiments, no unambiguous RNA structure can be built into the density. Together, the results presented here shed some light on the life cycle of three of these viruses.