Abstract Hepatitis B virus (HBV) is a major cause of chronic viral hepatitis that increases dramatically the risk of liver cancer and other end-stage liver diseases such as cirrhosis. HBV is a small DNA virus and replicates its DNA genome via reverse transcription of a RNA intermediate called the pregenomic RNA (pgRNA). Viral replication depends critically on the assembly of a nucleocapsid (NC) that is composed of the viral core or capsids protein (HBc) and encapsidates a copy each of pgRNA and the reverse transcriptase (RT), which converts the RNA pregenome to the DNA genome within the NC. Viral capsids also encapsidate the host cyclin-dependent kinase 2 (CDK2) via unknown mechanisms. Challenging the current dogma that capsid assembly depends solely on the N-terminal domain (NTD) of HBc, we have recently discovered that under physiological conditions, capsid assembly critically depends on the C-terminal domain (CTD) of HBc. Furthermore, we have developed a mammalian cell-free system that recapitulates CTD-dependent capsid assembly and further regulates capsid assembly through host-mediated CTD phosphorylation and dephosphorylation. The cell-free capsid assembly system also recapitulates the specific encapsidation of CDK2. Building on these developments, we propose to dissect the role of CTD, and its state of phosphorylation as regulated by cellular protein kinases and phosphatases, in capsid assembly (Specific Aim 1). We also plan to elucidate the HBc and CDK2 requirements for CDK2 encapsidation (Specific Aim 2). Furthermore, we have recently developed methods to isolate the viral pgRNA in complex with RT, which is the substrate recognized by HBc during NC assembly to achieve specific encapsidation of pgRNA and RT. We now propose to combine the isolation of the pgRNA-RT complex together with the cell-free capsid assembly system to develop cell-free systems for the specific packaging of pgRNA (and RT) into NCs (Specific Aim 3).