PROJECT SUMMARY Hepatitis B virus (HBV) is a DNA virus belonging to the hepadnavirus family and is responsible for chronically infecting over 250 million individuals worldwide, resulting in nearly a million annual deaths due to severe liver diseases such as cirrhosis and hepatocellular carcinoma. Unfortunately, anti-viral treatments fail to cure HBV infection due to the tenacity of covalently closed circular DNA (cccDNA), which is the template for viral transcription and subsequent translation of all viral products necessary to establish and sustain HBV replication. Much is learnt about HBV replication events post cccDNA production, however little is known about the intracellular trafficking steps that are required for the initial cccDNA production to establish productive infection. My PhD dissertation research aims to define the role of the viral capsid in endocytic trafficking during HBV infection (Aim 1). We have identified a series of HBV capsid mutants which have a unique phenotype in that during infection, they do not lead to productive infection (i.e., no cccDNA formation), however cccDNA can form during transfection via intracellular amplification, which bypasses the viral entry steps during infection, indicating that there is a block in the viral entry steps during infection with the mutant viruses that are not required during transfection. We plan to monitor post-entry trafficking events of WT and mutant HBV (Aim 1.1) and will further define the role of the viral capsid in directing endosomal trafficking through use of capsid inhibitors that induce a similar effect to our capsid mutants (Aim 1.2). These capsid inhibitors are in active clinical development; therefore, it is crucial that we understand what entry steps are modulated by them. Addressing questions related to HBV entry has been made possible by the identification of the viral entry receptor, hNTCP, however its expression cannot confer susceptibility to HBV infection in HEK293 cells, which can support cccDNA formation during transfection but cannot support cccDNA formation during infection, phenocopying the effects of the HBV capsid mutations and inhibitors. The second portion of this project will focus on defining the role of host cells in HBV trafficking during infection (Aim 2). We will address the endocytic trafficking of HBV in HEK293-hNTCP cells to identify where the block(s) in infection occurs, rendering these cells non-susceptible (Aim 2.1). To help identify the host determinants, beyond the entry receptor hNTCP, that may play a role in HBV entry, we have designed a split GFP-based HBV reporter system that will be used to isolate a subpopulation of HepG2-hNTCP cells that are highly susceptible to HBV infection (Aim 2.2) and perform RNA sequencing analysis to identify differentially expressed genes between susceptible vs. non-susceptible cells. These proposed studies will increase our understanding of HBV entry and identify the essential endocytic trafficking steps tha...