Project Summary Hepatitis B virus (HBV) infections affect the lives of more than 296 million people who are chronically infected with HBV and are at risk of developing liver cirrhosis and hepatocellular carcinoma. Every year approximately 820,000 people die due to HBV-related complications and currently there is no effective cure. At the same time human immunodeficiency virus (HIV) infects about 38 million people worldwide. Among those, 7.4% globally and 15-28% in highly endemic areas are also infected with HBV. HIV/HBV co-infected individuals are at higher risk to develop severe liver disease and liver cancer than HBV mono-infected people with mechanisms that are not well understood. Chronicity of HBV infections is linked to the stability and maintenance of the covalently closed circular DNA (cccDNA), which is the nuclear form of the HBV genome. The goal for a functional HBV cure relies on strategies that will either eliminate or inactivate the cccDNA. Therapies that are based on nucleoside analogs do not target the cccDNA while the mechanisms by which IFNα blocks HBV replication are not entirely understood in respect to the direct or indirect effects on the cccDNA. Several investigational direct acting antivirals and host targets that are implicated in HBV lifecycle are currently under investigation with the hope of achieving a cure. CRISPR screens have emerged as robust tools to discover host restriction and host dependency factors across multiple viral families and druggable genes provide information for the development of novel host-targeting therapeutics. HBV is exclusively a hepatotropic virus that infects only human hepatocytes and for studies that aim to characterize host responses primary human hepatocytes are the only cells for such studies. In parallel, the main target primary cell types for HIV are CD4+ T cells. Co-culture systems that incorporate these primary cell types are optimal for understanding the crosstalk between HIV and HBV infected cells and characterize host factor changes at the gene and protein level. Our robust and innovative co-culture systems provide unique opportunities to study HIV/HBV co-infections. These, together with a series of rigorous and analytical methods spanning from CRISPR screens in primary cells to proteomics, phosphoproteomics and secretomics approaches will be used here for the discovery and validation of host factors and antivirals against HIV/HBV co-infections with the potential to generate therapeutics for a functional HBV cure.