Project summary Worldwide, over 257 million people are chronically infected with hepatitis B virus (HBV), which can lead liver cirrhosis and/or hepatocellular carcinoma (HCC). The scarcity of small animal models has hampered curative therapy development and a mechanistic understanding of viral pathogenesis since HBV has a narrow host tropism limited to humans and chimpanzees, which is poorly understood. Our lab and others have previously shown that expression of human sodium taurocholate co-transporting polypeptide (hNTCP), the HBV receptor, facilitates viral uptake into murine cells and the intranuclear conversion of HBV relaxed-circular DNA (rcDNA) into covalently closed circular DNA (cccDNA) is fully supported in mouse cells and yields infectious virions. These data suggest that a late post-entry step, presumably capsid disassembly and/or nuclear import, constitutes a key barrier in the murine HBV life-cycle. Prior work showed that heterokaryons formed from mouse and human hepatoma cells expressing hNTCP are susceptible to HBV infection, suggesting that human-specific factors critical for the HBV life cycle are missing in mouse hepatocytes rather than (dominant) restriction factor being present in mouse cells. This prompted us to investigate the role of nuclear import proteins in restricting the murine HBV life-cycle. My proposal is founded on strong preliminary data from our lab demonstrating that the expression of several human karyopherin alphas and betas renders mouse Hepa1.6-hNTCP cells susceptible to HBV infection. Excitingly, our data further show that human, but not mouse, KPNA2 expression increases the susceptibility to HBV, establishing firmly that the observed increases are not simply due to the higher abundance of KPNA2 but to the expression of the human orthologues. I now hypothesize that incompatibilities between one or more karyopherins and the virus preclude efficient import of the HBV genome into mouse nuclei and thereby block the viral life-cycle in mice. To test this hypothesis, I will first extend our analysis to all karyopherins involved in nuclear import; I will systematically compare the ability of the respective mouse and human orthologues to facilitate infection in mouse cells and map subsequently the species-tropism defining regions in KPNA2 and putatively other karyopherins. Results from this aim will highlight the human-specific dependency factors needed to break the species barrier for HBV infection in murine cells. Secondly, I aim to determine how human karyopherins enhance HBV infection in murine cells mechanistically. I hypothesize that human but not mouse KPNA2, and other karyopherins, can facilitate the import of rcDNA HBV capsids into the nucleus through direct physical interactions. To test this systematically, I will visualize the subcellular localization of HBV capsids in the presence of human versus mouse KPNA2 by confocal microscopy. I will further test the relative ability of human and mouse KPNA2 to bi...