The establishment of a cell culture system for producing infectious hepatitis C virus (HCV) prompted genetic and functional studies of viral proteins and their roles in the assembly process. Since then, all ten viral proteins have been implicated in HCV assembly. Nonetheless, the exact location of the assembly site within an infected host cell remains unknown. Moreover, an understanding of the chronology of events and individual protein contributions at different stages of the assembly process has been difficult to obtain.
The two studies comprising this dissertation apply high-resolution genetics and high-resolution imaging to the study of HCV in cell culture. Study 1 employs a high-resolution genetics approach to reveal a functional map of the entire HCV genome. Next-generation sequencing of an insertion mutant library following passage in cell culture revealed genetic footprints that reflected known biological functions of the underlying protein regions. We show how these genetic footprints can serve as a resource to identify flexible regions that tolerate insertion of tags useful for a variety of protein detection methods. Moreover, using the genetic footprints, we identify a region in the NS4B protein that plays a role in post-RNA-replication steps. Study 2 examines HCV assembly through imaging, applying electron microscopy, electron tomography, superresolution light microscopy, multi-color fluorescence microscopy and live-cell imaging to the study of virus assembly. Our results indicate a juxtaposition of LDs, virus-like particles, membrane vesicles, clusters of HCV core protein and areas containing core-E2-NS5A proteins. The high-resolution snapshots underscore the functional compartmentalization of the LD environment, which provides viral proteins with membranous platforms to carry out a complex process such as virion assembly. We also show how our imaging platform can aid in the phenotypic characterization of an assembly-deficient mutant NS5A virus.
The two studies presented in this dissertation further our understanding of the contributions of non-structural proteins such as NS4B and NS5A to the HCV assembly process. We suggest that the combination of the high-resolution genetic platform of study 1 and the high-resolution imaging platform of study 2 facilitates the identification and phenotypic characterization of viral protein regions involved in HCV assembly. A streamlined approach that integrates these two methods has the potential to identify additional targets for therapeutic intervention at post-genome-replication steps.