Structure of the recombinant alphavirus Western equine encephalitis virus revealed by cryoelectron microscopy.

Western equine encephalitis virus (WEEV; Togaviridae, Alphavirus) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV's structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.

In vitro-assembled alphavirus core-like particles maintain a structure similar to that of nucleocapsid cores in mature virus.

In vitro-assembled core-like particles produced from alphavirus capsid protein and nucleic acid were studied by cryoelectron microscopy. These particles were found to have a diameter of 420 A with 240 copies of the capsid protein arranged in a T=4 icosahedral surface lattice, similar to the nucleocapsid core in mature virions. However, when the particles were subjected to gentle purification procedures, they were damaged, preventing generation of reliable structural information. Similarly, purified nucleocapsid cores isolated from virus-infected cells or from mature virus particles were also of poor quality. This suggested that in the absence of membrane and glycoproteins, nucleocapsid core particles are fragile, lacking accurate icosahedral symmetry.

An interspecific mosquito model for the mesenteronal infection barrier to western equine encephalomyelitis virus (Culex tarsalis and Culex pipiens).

Culex tarsalis, a putative mosquito vector of western equine encephalomyelitis (WEE) virus, is susceptible to peroral infection by WEE virus. The nonvector mosquito, Culex pipiens, has a very high peroral threshold of infection and is considered to be refractory. By parenteral inoculation, both mosquito species are equally susceptible. Thus, Cx. tarsalis and Cx. pipiens represent an excellent model system to examine the mechanisms for the mesenteronal infection barrier to WEE virus. Diethylaminoethyl (DEAE)-dextran (1.6 mg/ml), when added to a pledget bloodmeal that contained high concentrations of WEE virus, enhanced peroral infection rates of Cx. pipiens. In Cx. tarsalis, a reduction in infection rates was observed when low WEE concentrations of virus were ingested. DEAE-dextran had no apparent effects on the dissemination of WEE after infection of either mosquito species. It is suggested that the peroral enhancement of WEE viral infection observed with Cx. pipiens may be related to random, nonspecific mechanisms of infection, since it requires high titers of ingested WEE virus. Interference with specific binding of WEE virus to cellular receptor sites is suggested to explain the reduction in WEE viral infection rates in Cx. tarsalis. Altering the pH of the ingested infectious bloodmeal did not affect the WEE viral infection rate of Cx. pipiens, within the range of 6.0-8.5. Cx. tarsalis was optimally infected when the infectious bloodmeal was pH 8.0, and the infection rate was significantly reduced when the infectious blood was at either extreme of the pH range tested. This is again interpreted to indicate that different mechanisms control the peroral infection of Cx. tarsalis and Cx. pipiens with WEE virus. The mesenteronal infection barrier to WEE virus in Cx. pipiens is associated with an inability of the virus to adsorb and/or penetrate mesenteronal epithelial when administered perorally. The barrier is not related to an inability of the Cx. pipiens mesenteronal epithelial cells to support viral multiplication since these cells become infected when the virus is administered parenterally.

Permeability of the midgut basal lamina in the mosquito, Culex tarsalis Coquillett (Insecta, Diptera).

The permeability of the basal lamina of the midgut of the mosquito, Culex tarsalis, was determined for engorged and unfed mosquito midguts both pre- and postfixed. A range of materials, with diameters from less than 2nm (lanthanum) through 90 nm, (polystyrene spheres), was examined. The permeability of the basal lamina was constant under all experimental conditions; 5--8 nm particles (colloidal thorium) were the largest to consistently permeate. The discussion is centered on the question of how a virus particle, often 10 times the diameter of the established permeability limits of the basal lamina, can traverse this structure. Possible explanations are: 1. The basal lamina is a dynamic, plastic structure that easily distorts under physical and/or biochemical stresses, 2. The virion may possess enzymatic activity that locally alters the structure of the basal lamina, or 3. The extracellular surfaces of the midgut epithelium or the basal lamina itself may possess enzymatic properties that alter the size or possibly the structure of the virion as it passes through.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus.

Morphological and physical properties of a multiploid-forming mutant of Western equine encephalitis virus were studied. Electron micrographs of the infected cells showed that most of mutant virions bud from the plasma or vacuolar membrane as a multiploid particle containing a various number of nucleocapsids enclosed with a defined common envelope. The mutant virions contained three polypeptides which migrated to the position identical with those of wild type on discontinuous acrylamide gels. Cells infected with the mutant virus synthesized the same intracellular viral RNA species as was made after infection of wild type. Cytoplasmic nucleocapsids of the mutant sedimented at 140S and contained 42S virion RNA as those of wild type; they were indistinguishable from those of wild type in an electron microscope examination. On the other hand, mutant nucleocapsids isolated from extracellular virions sedimented as heterogeneous particles larger thant 140S and were shown to be pleomorphic and aggregate in electron micrographs. The budding process of this mutant seemed to be modified, so that it might form the multiploid with the alteration of its nucleocapsids.