Venezuelan equine encephalitis is a mosquito-borne viral illness occurring primarily in parts of South and Central America. The causative agent (VEEV) is an RNA virus of the genus Alphaviridae. It is endemic in rodent populations in many areas and causes occasional epidemics in horse and human populations. Although the potential for serious illness in humans in naturally occurring outbreaks is rather low, aerosolized virus can be lethal if inhaled by susceptible individuals. Consequently, it has recently gained attention because of its potential to be used as an agent of bio-terrorism. The lack of a human vaccine and effective treatments for the disease make this virus an important subject for further study. The goal of our laboratory is to apply our cell-free virus assembly systems to the study of VEEV and to use our new knowledge of VEEV assembly to develop drugs that block this step in the viral life cycle. The cell-free system reconstitutes virus assembly using cell extracts, creating an environment that mimics the inside of target cells and allows for the manipulation of numerous variables that may have an impact on assembly. Such systems have been utilized in the past to understand assembly of poliovirus, hepadnaviruses, retroviruses, and herpesviruses. Translation machinery from either wheat germ extract or rabbit reticulocyte lysate was used to translate the VEEV capsid protein from a messenger RNA. Newly synthesized proteins were labeled with radioactive methionine during translation. Velocity sedimentation of cell-free reaction products followed by SDS-PAGE and autoradiography revealed the formation of high molecular weight complexes that are likely to represent assembled capsids. Assembly appeared to be very efficient, with greater than 90% of capsid protein present in high molecular weight complexes. Additional experiments suggested that VEEV assembled either co-translationally or soon after translation was completed. Thousand-fold dilution of VEEV transcript did not significantly reduce the percentage of capsid protein that assembled, nor did solubilization of lipid membranes with detergents. Future experiments will use mutational analyses to identify regions of the capsid protein that are necessary for capsid assembly and packaging of the viral RNA genome. These data suggest for the first time that alphavirus capsids can be assembled in a cell-free system.
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