Characterization of the flavivirus capsid protein and the development of replicon-based systems for studying genome replication and virus assembly

Flavivirus, a genus of enveloped, positive strand RNA viruses, contains over 70 members, including yellow fever virus (YFV), dengue virus (DEN), and West Nile virus. The flavivirus genome encodes three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The flavivirus particle is assembled from the three structural proteins, a host-derived lipid bilayer, and a single copy viral RNA genome. The C proteins from YFV (YFC) and DEN (DEN2C) have been expressed in E. coli and purified to near homogeneity. Analysis of purified C protein by a variety of techniques established that the C protein was a dimeric, alpha-helical protein, with an unstructured N-terminus. The ability to produce significant quantities of purified C protein enabled solution structure determination of DEN2C by NMR techniques through a collaboration with Dr. Carol Post and Dr. Lixin Ma. The most prominent features of the DEN2C structure include an extensive dimerization interface and a solvent exposed apolar cleft. The flavivirus nonstructural proteins are responsible for genome replication and have been implicated in virus assembly. To facilitate study of these processes, replicons of YFV were constructed by deletion of the structural protein coding region. Using these replicons a trans-complementation system was developed to identify the cis- and trans-acting elements of flavivirus genome replication. While trans-complementation of NS1 occurred readily, the level of NS5 trans-complementation was significantly less and suggested the necessity for NS5 in cis for genome replication. A replicon-based system was also developed to study the process of virus assembly. Replicons of YFV could be packaged into virus particles by supplying the YFV structural proteins in trans. Additionally, it was found that while the C protein was essential for virus assembly, the N-terminus of the C protein was dispensable in this regard