Flaviviruses are major human pathogens. They include West Nile, yellow fever and dengue viruses. These viruses cause millions of deaths each year in temperate and tropical regions of the Earth. West Nile virus has spread throughout the US in the last decade, causing more than 100 deaths annually. Similarly the incidence of dengue increased 30-fold worldwide between 1960 and 2010, probably due to lack of suitable insecticide to stop the spread of the mosquito vector. We intend to continue our structural studies of the viral life cycle in order to map the structural changes that occur when the virus assembles into immature particles, when the immature particles change to infectious virus and, finally, when these become fusogenic in order to invade a new host for synthesis of new viral components. The immature particles contain 60 trimers of prM:E heterodimer, where prM is the pre-membrane protein and E is the envelope glycoprotein. The mature virus contains 90 dimers of M:E heterodimers and the post-fusion particles contain at least some trimers of M:E. Because of the different environment of the three subunits within the icosahedral framework of the immature and mature particles, the three types of subunits within each of the 60 icosahedral asymmetric units can be labeled differently. Thus, the re-assortment of the subunits during maturation (30 trimers to 90 dimers) or fusion (90 dimers to at least some trimers) can be followed by virtue of the labels. The purpose will be to study the various pathways of the subunit motions by inspecting the labeled subunits at the beginning and end of their motions using cryo-electron microscopy, and also to study the pleomorphic intermediates with cryo-electron tomography. Furthermore, the labels have been shown to block (at least in one instance) the full transformation and catch the particles at various intermediate steps. We plan to use three types of labels: Fab fragments of antibodies, heavy metal clusters;and the removal of the more exposed glycans on the E protein surface. Preliminary results of asymmetric labeling (different labels to the three different subunits in each icosahedral asymmetric unit) have been successful. We have learned to produce purified dengue virus and West Nile virus in milligram quantities of sufficient quality for structural studies. West Nile virus is especially suitable because of its greater stability, but requires biosafety level 3 facilities and precautions. We also plan to continue structural studies of the initial recognition and subsequent infection of a suitable host by the virus. We will examine some of the many cell surface molecules that have been tentatively shown to be flavivirus receptors by binding recombinantly produced fragments of potential receptor molecule to the virus. This should initiate fusion with the host cell endoplasmi membrane during endocytosis and, eventually, trigger release of the viral genome into the host cell's cytoplasm.
Flaviviruses, which include West Nile, yellow fever and dengue viruses, are significant human pathogens that give rise to major concerns for human health the World over. We propose in-depth studies of the structural changes that occur in the assembly pathway and infection process during the life cycle of flaviviruses, in particular West Nile and dengue viruses which each year cause illness or death to millions worldwide. Such information is essential for the development of antiviral drugs and vaccines and for determining the best response in the event of any possible epidemic.
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