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 belongs to the Hepadnaviridae, a family of para-retroviruses that have a small DNA genome and replicate through an RNA intermediate (the pregenomic RNA, or pgRNA), by a unique reverse transcription pathway. The initiation of both viral DNA synthesis and nucleocapsid assembly is triggered by the assembly of a specific ribonucleoprotein (RNP) complex between the viral reverse transcriptase (RT) and a specific RNA signal located at the 5' end of pgRNA termed epsilon. RNP formation is, in turn, facilitated by specific host cell factors, some of which have been identified in vitro, and furthermore, can be inhibited by unknown cellular factors under specific conditions. Following DNA synthesis, the mature, DNA-containing nucleocapsids are selectively enveloped and secreted as extracellular virions. The long-term goal of this application is to elucidate the viral and host factors that positively or negatively modulate viral replication and assembly, with a focus on the early step of initiation of reverse transcription and nucleocapsid assembly and the later step of nucleocapsid envelopment and virion secretion. Four specific aims are proposed. First, the role of the chaperone heat shock protein 90 (Hsp90), shown to be important for RT-epsilon interaction and protein priming in vitro using the duck HBV (DHBV) model system, to viral replication in vivo will be tested in host cells, through the use of specific pharmacological inhibitors, dominant negative mutants, and the RNA interference approach. Second, the viral and host requirements for RT-epsilon interaction in HBV will be determined using a recently developed RNA gel-mobility shift assay in vitro and verified in vivo. Third, cellular factors that can inhibit RNP formation will be isolated using recently established in vitro assay conditions and their role in cytokine-induced anti-HBV defense will be explored. Fourth, the state of post-translational modifications of the DHBV capsid protein at different stages of reverse transcription will be assessed through detailed biochemical and biophysical analyses of purified nucleocapsids at different stages of maturation; host factors responsible for mediating these modifications will be identified; and the role of these modifications and the relevant host factors in different steps of viral DNA synthesis and assembly will be determined. The proposed studies should provide important insights into the mechanisms of virus-host interactions and also facilitate the development of novel and effective anti-HBVstrategies.