Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.Hepatitis B virus (HBV) infection is a major public health problem, with over 300 million chronically infected people worldwide (28). Chronic HBV infection carries a great risk of developing severe liver diseases such as cirrhosis and hepatocellular carcinoma (5, 10). HBV belongs to the family Hepadnaviridae, a group of small, hepatotropic DNA viruses that infect mammalian and avian hosts. All hepadnaviruses replicate their DNA genome through an RNA intermediate (the pregenomic RNA [pgRNA]) by reverse transcription carried out by a virally encoded reverse transcriptase (RT) (13,40,43).The hepadnavirus RT has several unique functional and structural properties compared to all other known RTs (for a review, see reference 20). It can specifically recognize its RNA template via a short RNA signal ε, located at the 5Ј end of the pgRNA, and can form a stable ribonucleoprotein (RNP) complex with ε (34, 48). The formation of this RT-ε complex triggers the assembly of viral core particles, leading to the selective encapsidation of both the RT and the pgRNA into replication-competent nucleocapsids (4, 34). Thus, ε functions as the signal for pgRNA packaging, a process also dependent on the RT (1,18,19,25,33). In addition, the formation of this RT-ε complex initiates viral reverse transcription through a unique protein priming mechanism. Thus, the RT is able to initiate viral DNA synthesis de novo, using the RT itself as a protein primer and ε as a specific RNA template (12,...

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions. Reverse transcription in hepadnaviruses (hepatitis B viruses[HBVs]) is carried out by a virally encoded reverse transcriptase (RT), which is unique among all known RTs in several aspects (39, 43). The RT initiates DNA synthesis de novo using a specific tyrosine residue located within its unique N-terminal domain as a protein primer (27,46,48,51; for a recent review, see reference 19). This protein-priming reaction requires interaction between the RT and a specific RNA signal, termed ε, located on the viral pregenomic RNA (pgRNA) (the template for reverse transcription) (33, 47). The ε RNA forms a conserved stem-loop structure with an internal bulge, which is used as the specific template for protein priming (and thus, the origin of reverse transcription) (29,45,47; for a recent review, see reference 21). In addition, ε serves as the RNA packaging signal (16, 24) and, through its interaction with the RT, directs the selective encapsidation of both the pgRNA and the RT into viral nucleocapsids (1, 33). Therefore, the specific interaction between the RT and ε triggers two essential early steps in hepadnavirus assembly and replication, i.e., the proteinprimed initiation of reverse transcription and the assembly of replication-competent nucleocapsids.Using the duck hepatitis B virus (DHBV) RT as a model system, we have recently found that the RT requires the assistance of host cell factors in order to carry out specific ε binding and protein-priming functions (20,22). One such cellular factor is the 90-kDa heat shock protein (Hsp90). Hsp90 associates with the DHBV RT and is required for RT-ε interaction and protein priming in vitro and for pgRNA packaging and DNA synthesis in vivo. Furthermore, the chaperone is specifically incorporated into viral nucleocapsids via associatio...