Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

Abstract

The heat shock protein Hsp9O is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp9O interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp9O pathway in the formation of an RNP.Hepatitis B viruses (hepadnaviruses) are a group of small DNA viruses that replicate through a reverse transcription pathway. Reverse transcription in these viruses is initiated by a protein-priming mechanism as depicted in Fig. 1. The virally encoded reverse transcriptase binds to a short RNA sequence located at the 5' end of the viral pregenomic RNA (the template for reverse transcription), termed £, and initiates DNA synthesis de novo by using a tyrosine residue within the polymerase polypeptide as the primer (the protein-priming reaction) (2, 3). In addition, binding of the reverse transcriptase to e triggers nucleocapsid assembly, thus ensuring packaging of the RNA pregenome and the polymerase into nucleocapsids where reverse transcription takes place (4, 5). Therefore, the formation of a specific ribonucleoprotein (RNP) complex between the reverse transcriptase and s is critical for viral assembly and DNA synthesis (1, 4, 6-10).Since the RNP complex between the reverse transcriptase and £ RNA is transient (1, 7), its formation most likely depends on a particular conformation of the polymerase that is competent for binding to s. An important question is how the polymerase is stabilized in such a conformation that allows for formation of the RNP complex. To address this problem, we have exploited a recently developed cell-free system that permits expression of an enzymatically active reverse transcriptase of an avian hepadnavirus, the duck hepatitis B virus (DHBV), in a rabbit reticulocyte lysate (RL) (2, 3, 7). With the help of this system, we have identified a molecular chaperone, the heat shock protein Hsp9O, as an essential host factor required for the polymerase activity in vitro and in vivo. Hsp90 binds to the polymerase and facilitates the interaction between the polymerase and the £ RNA and thus plays an essential role in initiation of viral DNA synthesis and RNA packaging. MATERIALS AND METHODSPlasmids. Plasmids pHP and pHTP for expression of the DHBV polymerase in vitro have been described (3). pT3.1118 (11), used for in vitro expression of the glucocorticoid receptor (GR), was provided by K. Yamamoto (University of California, San Francisco). pCMV-DHBV directs expression of the DHBV pregenome from the cytomegalovirus immediate early promoter (12, 13). pTet-DHBV was derived from pCMVThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

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

“…One prerequisite for a cellular protein to be subjected to such a modification would likely be a close interaction with RT so that it can gain access to the RT active site. The RT protein is known to interact with a number of cellular proteins, including molecular chaperone proteins (13,15), the helicase DDX3 (52), and the translation factor eIF4E (22). The RT protein is also thought to be cytotoxic when overexpressed and has been reported to affect cellular functions such as interferon signaling and gene expression (2,8,14,53).…”

“…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).…”

Role of heat-shock protein 90 in hepatitis E virus capsid trafficking

“…Use of GA. GA has been reported as an inhibitor of HSP90 and is used widely in HSP90 research (Hu & Seeger, 1996). It is known that GA binds to a conserved binding pocket in the N-terminal domain of HSP90, inhibits ATP binding, and consequently blocks ATPdependent HSP90 chaperone activity (Hu & Seeger, 1996).…”

“…It is known that GA binds to a conserved binding pocket in the N-terminal domain of HSP90, inhibits ATP binding, and consequently blocks ATPdependent HSP90 chaperone activity (Hu & Seeger, 1996). In order to explore further the role of HSP90 on p239 translocation, HepG2 cells were incubated with p239 for 30 min at 4 uC, then with 10 mM GA (dissolved within DMSO; both from Sigma Aldrich) or with DMSO as a vehicle control for additional 30 min at 4 uC, or cells were pre-treated with 10 mM GA for 30 min, then incubated with p239 for additional 30 min at 4 uC.…”

Cis -preferential recruitment of duck hepatitis B virus core protein to the RNA/polymerase preassembly complex

“…4,5 Pregenomic RNA also serves as mRNA for both the viral polymerase and core protein. Assembly of replication-competent core particles is a highly orchestrated process that involves at least 3 steps: (1) formation of the viral preassembly complex, consisting of pregenomic RNA, polymerase, and cellular proteins [6][7][8][9][10][11][12][13] ; (2) docking of core protein to the preassembly complex; and (3) multimerization of core protein dimers to form the capsid shell.…”