In Vitro Reconstitution of Functional Hepadnavirus Reverse Transcriptase with Cellular Chaperone Proteins

Abstract

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...

p23, a simple protein with complex activities

“…This is consistent with genetic studies in yeast demonstrating that p23 is important but not essential for the activity of Hsp90 clients (Bohen 1998;Fang et al 1998;Knoblauch and Garabedian 1999). More recently, studies have demonstrated chaperoning of hepadnavirus reverse transcriptase with this system (Hu et al 2002). In what appears to be a situation similar to that of steroid receptors, p23 was not absolutely essential for reconstitution of in vitro transcriptase activity (protein priming), but p23 enhanced the kinetics of in vitro reconstitution.…”

“…It was first discovered as part of the complex of Hsp90 with the progesterone receptor . However, it has been found in complexes with a variety of Hsp90 clients, including the Fes tyrosine kinase (Nair et al 1996), the heme-regulated kinase HRI (Xu et al 1997), the transcription factor Hsf1 (Nair et al 1996), the Ah receptor (Nair et al 1996), and polymerases such as telomerase (Holt et al 1999) and hepadnavirus reverse transcriptase (Hu et al 2002). Whether p23 itself interacts with these client proteins or only with Hsp90 is not clear.…”

Hepatitis B virus replication

“…This system faithfully mimics several of the authentic replication steps, however its complexity precluded elucidation of many mechanistic details. The recent establishment of in vitro systems, culminating in the complete in vitro reconstitution of active DHBV replication initiation complexes from purified components [52][53][54] , overcame these restrictions (see below). It should be noted, however, that such minimal systems have their own limitations; for instance, the crucial role of the proper capsid environment for RC-DNA formation has not yet been modeled in vitro, and HBV P protein has thus far proven refractory to in vitro reconstitution of DNA synthesis activity.…”

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

“…The fulllength RT requires the assistance of the host cell chaperone proteins in order to establish and maintain a conformation that is competent to recognize the ε RNA and to initiate protein priming (9,10,13,15,17,18,41,42). However, a truncated DHBV RT protein, MiniRT2, with deletion of the entire RNase H domain, the N-terminal third of the TP domain, and most of the spacer, retains ε RNA binding and protein priming activity but no longer requires the host chaperones (55).…”