This project will extend active collaborations of the Ahlquist group with the Loeb and Lambert groups to define key aspects of virion assembly, genome replication, and virus-host interactions for two of the most important human tumor viruses, hepatitis B virus (HBV) and human papillomavirus (HPV). These experiments use integrated studies in human cells and the genetically tractable yeast Saccharomyces cerevisiae, which the Ahlquist group has pioneered as a model host to study virus replication and virus-host interactions. The project has four aims: For HBV, we will (I) Use confocal microscopy, electron microscopy and cell fractionation to define the intracellular localization, localization determinants, and interaction of the HBV structural proteins [core, reverse transcriptase, and envelope proteins (surface antigens)] when expressed singly and in selected combinations in human hepatoma cells and in yeast; (II)Use targeted and global yeast genetic screens to determine if HBV envelope protein-driven budding into endoplasmic reticulum-associated compartments in yeast depends on host cell functions, including vacuolar protein sorting and ubiquitination functions involved in crucial late steps of plasma membrane budding by retroviruses and other viruses; (III) Extend ongoing experiments to duplicate pregenomic RNA encapsidation and subsequent genome replication steps in yeast for HBV and its major laboratory model, DHBV (duck HBV), to allow study of host contributions to these replication steps in cells whose genome can be freely and systematically manipulated. For HPV, we will (IV) combine the Ahlquist lab's optimization in yeast of HPV protein expression and capsid assembly with the Lambert lab's finding that yeast replicate and stably maintain HPV genomic DNA, to determine the protein and DNA requirements for HPV genome encapsidation and infectious virion assembly in yeast. In addition to defining the assembly determinants, these studies will overcome present limitations of organotypic raft cultures to provide a more facile source of genetically manipulatable, infectious HPV virions with which to study virus entry and early infection steps in mammalian cells.
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