The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, ) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific binding. We now report that Hsp90 is also required for HBV RT-interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RTinteraction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-interaction in HBV, as in DHBV. Specific binding required sequences from both the amino-terminal terminal protein and the carboxyterminal RT domain. Only the cognate HBV , but not the DHBV , could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-interaction and chaperone activation.Hepatitis B virus (HBV) infection is a major global public health problem with over 300 million chronically infected patients worldwide (34). Patients with chronic HBV infection carry a great risk of developing severe liver diseases, including cirrhosis and liver cancer, which result in a million mortalities annually (4, 10). HBV is a member of the Hepadnaviridae family, a group of small hepatotropic DNA viruses that also includes related animal viruses, such as the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus. All hepadnaviruses carry a small (ca. 3.2 kb), relaxed circular, partially double-stranded DNA genome and replicate this DNA genome through an RNA intermediate, the pregenomic RNA (pgRNA), by reverse transcription (52). The reverse transcription pathway employed by hepadnaviruses is similar to, yet distinct from, that used by retroviruses (for reviews, see references 49 and 50).All hepadnaviruses encode a novel, multifunctional reverse transcriptase (RT). Like its retroviral counterparts, the hepadnavirus RT catalyzes RNA-and DNA-dependent DNA polymerization and has an intrinsic RNase H activity (12,43,56). Reflecting this functional conservation, the central catalytic RT domain and carboxy (C)-terminal RNase H domain of the hepadnavirus RT are homologous to the corresponding domains of retroviral RTs. However, all hepadnavirus RTs share an amino (N)-terminal domain, called the terminal protein (TP) (2,12,43). The TP domain is absent from retroviral RTs. Sequence database searches indicate tha...