In Vitro Reconstitution of a Functional Duck Hepatitis B Virus Reverse Transcriptase: Posttranslational Activation by Hsp90

Abstract

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,...

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

“…Under native conditions MiniRT2 and the TP and RT domains copurified with GroEL and DnaK, two bacterial chaperone proteins known to bind the DHBV and HBV RT, as well as degradation products and other contaminants (Fig. 2, lanes N) (10,12). In order to exclude the possibilities that the bacterial chaperone proteins and other contaminants might have influenced the results obtained, we also purified the 6ϫHis-tagged constructs from inclusion bodies under denaturing conditions and refolded them.…”

“…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).…”

“…During this so-called protein priming reaction, a short (3 to 4 nucleotides long) DNA oligomer is synthesized that is covalently attached to RT through the primer Y residue, using as a template an internal bugle of the ε RNA. Both the TP and the RT domains are essential for RT-ε interaction and protein priming but the RNase H domain is dispensable (10,23,33,51). Also, isolated TP and RT domains, when combined together, are able to reconstitute a functional RT protein that is able to carry out protein priming in a trans-complementation reaction (1,23,25,27).…”

“…(RRL) system (Promega), in the absence of any radiolabeling according to the manufacturer's instructions (9,10,50). GST-MiniRT2, -RT, and -TP were expressed in BL21DE3-CodonPlus-RIL cells and purified using the glutathione resin (10,12).…”

“…GST-MiniRT2, -RT, and -TP were expressed in BL21DE3-CodonPlus-RIL cells and purified using the glutathione resin (10,12). His-MiniRT2, -RT, -TP, and -MBP-TP were expressed in M15-(pREP4) cells and purified using Ni ϩ affinity resins under native conditions (10). For His-MBP-TP, the eluate from the Ni ϩ affinity resins was further purified using amylose affinity resins (New England Biolabs).…”