INTRODUCTION

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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 , located at the 5' end of the pgRNA, and can form a stable ribonucleoprotein (RNP) complex with  (34, 48). The formation of this RT- 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,  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- 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  as a specific RNA template (12, 26, 32, 46-49, 52). As a result of this protein priming mechanism, the RT becomes covalently attached to the viral minus-strand DNA (3, 14, 31). To carry out these functions, the hepadnavirus RT contains a unique N-terminal domain (the terminal protein [TP]), in addition to the central RT and C-terminal RNase H domains, which are homologous to those of other RTs. Both the TP and RT domains are required for RNP formation and protein priming (34, 39, 48).

It has proven difficult to study the hepadnavirus RT functions through biochemical and structural analyses, mainly due to problems in obtaining sufficient amounts of purified RT proteins. The RT is present only at very low levels in virions and in infected host cells; efforts to extract an active RT from either viral particles or infected cells for in vitro analysis have been unsuccessful (2, 37). Similarly, attempts to express and purify an active, recombinant RT using a variety of expression systems have had only limited success (for a summary, see reference 20). The most useful system to date has been a cell-free translation system developed using the duck hepatitis B virus (DHBV) RT. The DHBV RT, expressed in a rabbit reticulocyte lysate in vitro translation system, is active in both RNP formation and protein priming (46-48, 52). With the help of this cell-free system, we have recently shown that specific cellular factors, including components of the molecular chaperone complex, heat shock protein 90 (Hsp90), are required for the RT to carry out these functions (21, 23). However, only very small amounts of RT proteins can be produced in this cell-free translation system. Recombinant baculoviruses have also been used to express the HBV RT in insect cells at relatively high levels (26, 27, 45). However, it has not yet been possible to reconstitute RT- interaction and -dependent protein priming activity in vitro using the insect cell-expressed RT. These limitations have hampered a systematic search for additional factors required for RT functions, as well as biochemical and structural analyses of the mechanisms of RT activation by host factors.

Hsp90 is required for the activities of a specific subset of cellular proteins (so-called target or substrate proteins) which are diverse both in structure and function (9, 24, 35, 36). In facilitating the functions of its various target proteins, Hsp90 forms multicomponent chaperone complexes with different cofactors (cochaperones), which seem to vary depending on the particular target protein. The molecular basis of specific target recognition by Hsp90 is still not well understood. We have shown previously that both Hsp90 and one of its cofactors, p23, can bind specifically to the RT (21, 23) even though the RT does not share any apparent sequence similarities to other known Hsp90 target proteins. In addition, we proposed earlier that the role of Hsp90 in RT functions is to facilitate the conformational (and functional) maturation of the RT to a state that is competent for  binding and protein priming (21, 23). This notion is consistent with the known functions of Hsp90 as a molecular chaperone; it is not clear, however, what such an RT conformation may be or how the chaperone may help to establish that conformation. It is also unknown when Hsp90 exerts its effect on the RT during RT synthesis and maturation. The fact that the RT preferentially interacts with and packages the pgRNA from which it is translated (cis preference) (1, 18) suggests that the RT may become competent for  binding even while it is still being translated. This, in turn, implies that Hsp90 may activate the RT cotranslationally. On the other hand, Hsp90 is thought to act at a relatively late stage during the functional maturation of the steroid hormone receptors, the best-studied Hsp90 target proteins (9, 36). Furthermore, the chaperone components that we have identified so far are not sufficient for RT activation (21, 23), suggesting that an additional chaperone cofactor(s) required for RNP formation and protein priming remains to be identified.

To begin to address these questions, we have defined the RT sequences that are required for chaperone association,  RNA binding, and protein priming. With this information, we were then able to express and purify two minimal RT proteins using bacterial as well as eukaryotic expression systems. Using the purified RT proteins, we were able to develop a cell-free, Hsp90-dependent RT activation system and to reconstitute both the RT- interaction and protein priming in vitro. We present here an initial analysis of the mechanisms of RT-Hsp90 interaction and RT activation using the newly established reconstitution system. Our results suggest a model for Hsp90 function whereby the chaperone acts as an active bridge to bring together the two domains of the RT necessary for  binding, thus helping to establish and maintain a poised but unstable RT conformation competent for RNP formation and protein priming.

	MATERIALS AND METHODS

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Plasmids. The DHBV RT, tagged with a synthetic hemagglutinin (HA) epitope inserted into the nonessential spacer region between the TP and RT domains, was expressed in vitro in the reticulocyte lysate translation system from plasmid pHTP (52). C-terminally truncated RT mutants were translated from pHTP that was linearized at various restriction sites within the RT coding sequences. The internal deletion mutant pHTP-dBX was derived from pHTP by removing codons 74 (at the unique BglII site) to 352 (XhoI site) of the RT coding sequence. pcDNA-miniRT1 encodes an RT polypeptide that has deletions from the N terminus (amino acids 1 to 74), the C terminus (amino acids 734 to 786), and the spacer region (amino acids 245 to 352). Similarly, pcDNA-miniRT2 encodes a short RT polypeptide that has the same N-terminal and spacer deletions as that encoded by pcDNA-miniRT1 but a more extensive C-terminal truncation (from amino acid 575 to 786). Both constructs contained c-Myc epitope tags fused to the C termini of the RT proteins, in addition to the internal HA tag derived from pHTP. Both of these were constructed using the pcDNA3 (Invitrogen) vector, and they were used to express the mini-RT proteins in vitro and in hepatoma cells.

Antibodies and reagents. The monoclonal antibody (MAB) against Hsp90 (clone 3G3) was purchased from Affinity Bioreagents. The MAB against p23 (clone JJ3) was generously provided by David Toft (Mayo Clinic). The MABs against DnaK and GroEL were purchased from Stressgen. The goat anti-mouse immunoglobulin G (IgG), control mouse IgG, and anti-c-Myc epitope MAB (clone 9E10) were purchased from Sigma. The anti-HA MAB (clone 12CA5) was purchased from Berkeley Antibody. The antibiotic geldanamycin was obtained from the Drug Synthesis and Chemistry Branch, National Cancer Institute.

In vitro transcription and translation. RNAs used for in vitro translation were transcribed from linearized plasmids using an in vitro transcription kit (MEGAscript; Ambion) and purified as described before (21, 47). Purified RNAs were then translated using the rabbit reticulocyte lysate in vitro translation system (Promega). Protein expression in the coupled transcription and translation reaction using the TnT rabbit reticulocyte lysate system (Promega) was carried out according to the manufacturer's instructions.

Immunoprecipitation. A mixture of two MAbs, anti-HA and anti-c-Myc, was bound to the protein A/G resin (Pierce) and used to immunoprecipitate the tagged mini-RT proteins from the in vitro translation reactions, as described before (21). The binding and wash buffer used was the radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.05% NP-40, 10% glycerol, 2 mM dithiothreitol (DTT), and a cocktail of protease inhibitors [Protease Inhibitors Complete; Boehringer Mannheim]). Hsp90 or p23 with its associated RT proteins was immunoprecipitated from the in vitro translation reactions using Hsp90-specific MAB 3G3 or p23 MAB JJ3 under detergent-free conditions (homogenization buffer, 50 mM KPO₃ [pH 7.4]-10 mM thioglycerol-10 mM sodium molybdate), as described previously (21).

Sucrose gradient centrifugation. For sedimentation under standard conditions, the in vitro translation reaction mixtures were diluted 20-fold in TNMN buffer (50 mM Tris [pH 7.5], 100 mM NaCl, 5 mM MgCl₂, 0.02% NP-40) and layered over a linear 20 to 40% sucrose gradient made in TNMN. The gradient was then centrifuged at 40,000 rpm at 4°C for 4 h in a Beckman SW41 rotor. To dissociate the RT complexes, the translation reaction mixtures were first diluted 20-fold into a high-salt and -detergent buffer (same as TNMN except with 1 M NaCl and 1% NP-40). The samples were then fractionated as described above except that the sucrose gradient was made in the high-salt and -detergent buffer. Individual fractions were then collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The ³⁵S-labeled RT was detected by autoradiography.

Bacterial expression and protein purification. Plasmids encoding GST-MiniRT1, GST-MiniRT2, His-MiniRT1, and His-MiniRT2 were transformed into protease-deficient bacterial strain BL21. The cells were grown to an optical density at 600 nm of 0.5 to 1, and mini-RT expression was then induced by adding isopropyl--D-thiogalactopyranoside to a final concentration of 0.5 to 1 mM. The cultures were continued for another hour to overnight at room temperature before harvest. The cells were then pelleted and lysed by sonication in lysis buffer (50 mM Tris [pH 8.0], 500 mM NaCl, 0.5% Triton X-100, 10% glycerol, 10 mM DTT, and a cocktail of protease inhibitors [Protease Inhibitors Complete; Boehringer Mannheim]). The mini-RT proteins were then purified from the soluble fraction (clarified lysate) by affinity purification. Glutathione-agarose beads (Sigma) were used for purifying the GST fusion proteins and Ni⁺-nitrilotriacetic acid resins (Qiagen) were used for purifying the six-histidine-tagged proteins, according to the manufacturer's instructions. To further separate the full-length fusion proteins from degradation products, the eluates from the glutathione or Ni⁺ beads were then subjected to a second immunoaffinity purification step by immunoprecipitation with the anti-c-Myc antibody. As the GST (or the six-histidine tag) and the c-Myc epitope were fused to the N and C termini of the mini-RT proteins, respectively, this sequential two-step purification could effectively separate the full-length fusion proteins from degradation products truncated from either end.

Reconstitution of protein priming and  binding with purified mini-RT. The purified mini-RT proteins used for in vitro reconstitution included the following: (i) immunoprecipitate from the in vitro translation reactions (still bound to the immunoaffinity resin), (ii) eluate from the glutathione or Ni⁺ affinity resins, and (iii) immunoprecipitate from the eluate described in item ii. In vitro protein priming and  binding reactions were performed as previously described (21, 47), with the following modifications. The reaction buffer used for both the protein priming and RNA binding reactions was TMNK (20 mM Tris [pH 7.5], 2 mM MgCl₂, 15 mM NaCl and 20 mM KCl, 2 mM DTT, 10 mM sodium molybdate). Following the RNA binding reaction, the ³²P-labeled  RNA was detected directly by resolving the -RT complex using SDS-PAGE (15% acrylamide) and autoradiography, omitting the RNA purification steps of phenol extraction and ethanol precipitation. We found it unnecessary to separate the RNA from the immunoprecipitates since the ³²P-labeled  ran towards the bottom of the gel and was well resolved by SDS-PAGE. Various supplements were added to the protein priming or RNA binding reactions; these included rabbit reticulocyte lysate and an ATP regenerating system consisting of 5 mM ATP, 10 mM creatine phosphate, and 50 µg of creatine phosphokinase/ml (21).

	RESULTS

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Mapping of RT sequences required for chaperone association,  binding, and protein priming. As a first step towards understanding the molecular basis for the RT-chaperone interaction, we sought to delineate the regions of the RT that can mediate chaperone binding. To this end, we translated the full-length RT and various RT deletion mutants in reticulocyte lysate and determined their association with the two components of the chaperone complex that we have previously shown to bind the RT, i.e., Hsp90 and p23 (21, 23). As shown in Fig. 1A and B, two segments of the DHBV RT could each independently mediate binding to the chaperone proteins, as determined by immunoprecipitation using Hsp90- and p23-specific MAbs. Both segments were approximately 200 residues long and were localized to the N-terminal TP and the central RT domains, respectively. We also assayed for the abilities of the various deletion mutants to bind  RNA and carry out protein priming. Consistent with previous reports (34, 39, 48),  binding required part of the TP and a short segment from the RT domain whereas protein priming required the TP and a longer sequence from the RT and RNase H domains.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   RT sequences required for chaperone association,  binding, and protein priming. (A) Schematic diagram (top) of the domain structure of the DHBV RT. The primer tyrosine (residue 96) in the TP domain and the two aspartic acids (residues 513 and 514) at the RT active site are indicated, as are the unique restriction sites used to construct the various deletion mutants: Bl, BglII; Bp, BspHI; Bt, BstEII; M, MscI; T, Tth111I; X, XhoI. Below are the activities of the wild-type (WT) RT and the various truncation and deletion RT mutants in chaperone binding,  binding, and protein priming, as determined by coimmunoprecipitation and in vitro protein priming assays (see Materials and Methods). The two independent chaperone-binding regions of the RT are shown schematically. ND, not determined. (B) Examples of coimmunoprecipitation of full-length RT and deletion variants of the RT with p23. Tth111I harbored the N-terminal chaperone-binding region in the TP domain, and B-X/BstE contained the central chaperone-binding region in the RT domain (A). The RT proteins, along with the luciferase protein (Lucif; used as a negative control for immunoprecipitation), were translated in reticulocyte lysate, and the translation reaction mixtures were subjected to immunoprecipitation with a MAB (clone JJ3) specific for p23 (-p23; lanes 2, 5, 8, and 11) or a nonimmune control antibody (IgG; lanes 3, 6, 9, and 12). The immunoprecipitates, along with the translation reaction products (input; lanes 1, 4, 7, and 10), were resolved by SDS-PAGE and the 35S-labeled RT proteins were detected by autoradiography. (C) WT RT and MiniRT1 were translated in reticulocyte lysate, and their protein priming activities were assessed by the in vitro priming assay, with or without treatment with Hsp90 inhibitor geldanamycin (GA). Lanes 1 and 2, 35S-labeled translation product (lane 1, WT RT; lane 2, MiniRT1); lanes 3 to 8, 32P-labeled WT RT (lanes 3 and 4) and mini-RT (lanes 5 to 8) as a result of the protein priming reaction in the presence of [-32P]dATP. Following translation but before the priming reaction, aliquots of the translation reaction mixture, as indicated, were treated with GA (100 µg/ml).  was added during either the translation [i.e., before GA treatment; (T)] or priming [i.e., after GA treatment; (P)] step.

View larger version (51K): [in this window] [in a new window]   FIG. 2.   Purification of the RT following in vitro translation and reconstitution of  binding and protein priming. (A) Dissociation of the RT complexes. 35S-labeled RT (upper and middle sections) and luciferase (Lucif) (lower section) were synthesized in reticulocyte lysate and centrifuged over a linear 20 to 40% sucrose gradient at 40,000 rpm for 4 h in an SW41 rotor. For the RT sample shown in the middle section, the translation reaction mixture was first treated with 1 M NaCl and 1% NP-40 before centrifugation over the sucrose gradient made in the same high-salt and -detergent buffer. Individual fractions were then collected and resolved by SDS-PAGE. The direction of centrifugation is indicated. (B) Dissociation of RT-p23 binding. MiniRT1, tagged with the c-Myc and HA epitopes, was expressed in TnT reticulocyte lysate (lane 1) and immunoprecipitated (IP) with either a control IgG (lanes 2 and 5), an anti-p23 MAb (lanes 3 and 6), or a mixture of anti-c-Myc and anti-HA (lanes 4 and 7) antibodies. The binding and washing buffers contained either 50 mM phosphate buffer-10 mM sodium molybdate (lanes 2 to 4) or 0.5 M NaCl-0.2% NP-40 (lanes 5 to 7). The immunoprecipitated, 35S-labeled mini-RT was then resolved by SDS-PAGE and detected by autoradiography. (C) Immunoaffinity purification of mini-RT proteins and reconstitution of  binding. MiniRT2 and the corresponding -binding-defective mutant, MiniRT2/CA29, were expressed in TnT reticulocyte lysate supplemented with [35S]methionine and purified by immunoprecipitation under high-salt and -detergent conditions (as described for panel B). 32P-labeled  RNA was then incubated with the purified RT proteins with (lanes 4 and 6) or without (lanes 3 and 5) the addition of reticulocyte lysate. Unbound RNA was washed away, and the bound  was detected following SDS-PAGE (lanes 3 to 6). As controls, labeled  RNA was added to the translation reaction mixtures and thus allowed to bind to RT during translation; the RT- complex was then immunoprecipitated and detected by SDS-PAGE and autoradiography (lanes 1 and 2). (D) Reconstitution of protein priming. In vitro-translated MiniRT1 was isolated by immunoprecipitation under high-salt and -detergent conditions. The  RNA was then added to the purified RT to initiate protein priming. The 32P-labeled priming reaction products were detected by autoradiography following SDS-PAGE. The priming reactions were carried out with the following supplement: nonsupplemented control (lane 1), unfractionated reticulocyte lysate (RL; lanes 2 and 5), reticulocyte lysate desalted by passing through a Sephadex G-25 column (RL/G25; lane 3), desalted reticulocyte lysate supplemented with an ATP regenerating system (see Methods and Materials) (RL/G25/ATP RS; lane 4), reticulocyte lysate plus geldanamycin (100 µg/ml) (RL/GA; lane 6). Solid circle (lane 4) reaction product observed sometimes under the indicated conditions, the nature of which is currently unknown.

Continuous requirement of Hsp90 for the maintenance of RT functions. We reported previously that Hsp90 is required to establish an RT state that is active in  binding and protein priming (21, 23). However, it was not known whether Hsp90 association and function would also be required to maintain such an RT state once it has been established. To address this issue, we attempted to separate the RT from associated chaperone proteins and determine the ability of the purified RT to carry out  binding and protein priming.

Reconstitution of a functional RT following bacterial expression and purification: posttranslational RT activation by Hsp90. Our success in reconstituting RT activities using the mini-RT proteins purified from the in vitro translation system prompted us to test whether it would be possible to purify the RT from bacteria and reconstitute its activities using similar approaches. To this end, we fused the N termini of the two mini-RT proteins to GST or a stretch of six histidines. The GST-mini-RT and six-His-mini-RT fusion proteins were then purified by using the glutathione and Ni⁺ affinity resins, respectively. Similar results were obtained with the mini-RT proteins purified using either method. However, the GST fusion proteins proved to be more easily purified, and the results reported below were obtained using the purified GST-mini-RT proteins.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Bacterial expression and purification of mini-RT. (A) Two GST mini-RT fusion proteins, GST-MiniRT1 and GST-MiniRT2, were expressed in BL21 cells as described in Materials and Methods. Uninduced (lanes 1 and 3) and induced (lanes 2 and 4) bacterial lysates were prepared. The fusion proteins were first purified using glutathione-agarose beads (lanes 5 and 6) and then further purified by immunoprecipitation (IP) with the anti-c-Myc antibody (lanes 7 and 8). The lysate and purified protein samples were resolved by SDS-PAGE and stained with Coomassie blue. Stars, full-length mini-RT fusion proteins; arrowheads, two major copurifying bacterial proteins (DnaK and GroEL; see Fig. 5). The Ig heavy (IgH) and light chains (IgL) and GST are also indicated. (B) GST-MiniRT1 (lane 2) and GST-MiniRT2 (lane 1) purified on glutathione-agarose beads were resolved by SDS-PAGE and detected by Western blot analysis using the anti-c-Myc antibody. Stars, full-length mini-RT fusion proteins.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Reconstitution of RNA binding and protein priming with mini-RT purified from bacteria. (A) GST-MiniRT1, purified either by glutathione-agarose beads (lanes 2 and 3) or by glutathione beads plus the second step of immunoaffinity purification (lanes 4 and 5), was used for the in vitro protein priming reaction in the presence of  and [-32P]dATP, with (lanes 3 and 5) or without (lanes 2 and 4) supplementation with reticulocyte lysate (RL). As a positive control, MiniRT1 (without the GST fusion) was translated in reticulocyte lysate and assayed in the priming reaction (lane 1). The 32P-labeled RT was then detected by SDS-PAGE and autoradiography. (B) Purified GST-MiniRT1 was used in the protein priming reaction as described for panel A, with the indicated supplements. Abbreviations are as defined in the Fig. 2D legend. RL/no , priming reaction mixture supplemented with reticulocyte lysate but without  RNA. (C) Purified GST-MiniRT2 was used in the  RNA binding assay as described for Fig. 2C, except that GST-MiniRT2 was bound to glutathione-agarose beads. 32P-labeled  RNA precipitated by the RT-bound beads was then detected by SDS-PAGE and autoradiography. Reticulocyte lysate was added as indicated. Lane 3, RNA input.

Association between the RT and the bacterial chaperones GroEL and DnaK. During the process of purifying the mini-RT proteins expressed in bacteria, we noticed that two prominent bacterial proteins copurified with the RT when the GST affinity and immunoaffinity beads were used (Fig. 3A, lanes 5 to 8, and Fig. 5). Their apparent sizes, approximately 70 and 57 kDa, and the fact that the RT associates with eukaryotic chaperones prompted us to determine whether these might be the two abundant bacterial chaperone proteins, the 70-kDa DnaK and the 57-kDa GroEL (11, 17). Immunoblot analysis using specific antibodies confirmed that these two proteins were indeed DnaK and GroEL (Fig. 5).

View larger version (44K): [in this window] [in a new window]   FIG. 5.   Association of DnaK and GroEL with RT. GST-MiniRT1 and -MiniRT2 were expressed in BL21 cells and purified by using glutathione beads. The bacterial lysate (lysate) and different fractions from the purification protocol were then analyzed by Coomassie staining (top) or Western blotting using anti-DnaK (middle) or anti-GroEL (bottom). ATP wash, proteins washed off the beads following a 30-min incubation with ATP (5 mM) at room temperature; eluate, proteins eluted from the beads by glutathione; HS (high salt) wash, proteins washed off the beads by 3 M NaCl (used to regenerate the affinity resin). Stars, mini-RT fusion proteins; arrows, DnaK and GroEL.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Recent studies demonstrated that the hepadnavirus RT requires the assistance of specific host cofactors, including the Hsp90 chaperone and its cofactors, in order to interact with  and to carry out protein priming (21, 23). The experiments reported here were carried out in order to gain a better understanding of the molecular basis for the specific interaction between the chaperone and the RT and the mechanisms of RT activation by the chaperone. Our results showed that (i) two RT segments located in the TP and the RT domains can each mediate Hsp90 and p23 binding independently; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in an -binding-competent state; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. These findings were made possible largely due to the development of a biochemical reconstitution system for RT- interaction and protein priming, which we anticipate will facilitate future studies on these critical steps in hepadnavirus assembly and replication.

One of the important, unresolved issues in the Hsp90-RT interaction and in Hsp90 function in general is the molecular basis for the specific recognition by Hsp90 of the RT and other target proteins, including the steroid receptors and certain protein kinases (9, 24, 35). These various target proteins differ greatly in both structure and function. The two independent chaperone-binding regions of the RT that we have now defined do not have any apparent sequence similarity with other Hsp90 target proteins or, for that matter, with each other. One possibility arising from studies on the kinase targets is that distinct Hsp90 chaperone complexes exist in the cell, each with a specific "targeting" cofactor, which directs each distinct Hsp90 complex to its cognate substrate. For example, Hsp90 cofactor p50/CDC37 can specifically bind to certain protein kinases, e.g., CDK4 and Raf, and is thought to target a Hsp90-p50 complex to its protein kinase substrates (15, 42). If this is the case for Hsp90 targeting to the RT, two distinct targeting cofactors would presumably be required to target Hsp90 to the two distinct domains of the RT. Alternatively, the Hsp90 complex may recognize some as yet unknown structural properties shared by the various targets, including the two distinct domains of the RT, such as an intrinsic structural instability or some common folding characteristics. The resolution of this important issue will likely require the identification of all the essential components of each chaperone complex and high-resolution structural studies.

Although each of the two chaperone-binding regions of the RT can bind independently to the chaperone, both of these together are required for  binding. Indeed, a minimal RT protein (MiniRT2) consisting only of these two segments (less than 400 amino acids long) can bind  RNA as efficiently as the full-length RT in a chaperone-dependent manner. These findings lead us to suggest a plausible model for how Hsp90 may facilitate the RT- interaction (Fig. 6). We propose that the Hsp90 chaperone complex, by binding both to the TP and to the RT domains of the viral polymerase, acts to mediate interdomain interactions between these two regions. In doing so, the chaperone functions as a dynamic bridge to help bring the amino acid residues critical for  recognition in both of these domains (34, 39, 48) into precise spatial arrangement for specific  binding and protein priming.

View larger version (9K): [in this window] [in a new window]   FIG. 6.   Model of RT folding and activation by cellular chaperones. The nascent RT polypeptide (I) exiting the translating ribosomes is first recognized by Hsp70 (or DnaK when expressed in bacteria). With Hsp70 assistance, the TP and RT domains can then partially fold (initial folding; II), independent of each other. However, the RT remains incompetent for  binding. The Hsp90 complex then recognizes the partially folded TP and RT domains and brings them together to establish an RT conformation able to bind  (activation; III). This RT conformation is intrinsically unstable and requires the continued assistance of Hsp90 to maintain it. Finally,  binding stabilizes this RT conformation while Hsp90 remains bound to the RT (IV).

The definition of the minimal functional domains of the RT and the ability to strip the RT of its associated cellular proteins allowed us to purify mini-RT proteins using both in vitro translation and bacterial expression systems. Our results showed that the purified RT proteins, dissociated from the chaperone complex, lost their ability to recognize  and to carry out protein priming and required reconstitution with the chaperone to regain these activities. We also attempted to express the same mini-RT proteins in hepatoma cells that can support efficient viral replication. Again, we found that the mini-RT proteins, even when expressed in authentic host cells, still could not maintain their activities when stripped of the necessary host factors and required in vitro reconstitution to recover the  binding and protein priming activities (J. Hu, unpublished results). These results thus indicate that Hsp90 is required not only to establish but also to maintain the RT in a state competent for  binding (Fig. 6). Also, since the RT dissociated from the chaperone showed no evidence of aggregation, Hsp90 apparently was not required to keep the RT in a soluble (and hence, at least partially folded) state. Furthermore, since the mini-RT proteins expressed in bacteria, in the absence of a functional Hsp90 chaperone system, could also be activated by Hsp90 following purification, the chaperone does not appear to have an essential cotranslational role in RT activation.

Taken together, these results point to a distinct, posttranslational role for Hsp90 in establishing and maintaining a specific functional conformation of the RT, rather than acting simply as a "generic chaperone" to keep the RT from aggregating. This notion agrees well with the interdomain bridging model proposed above (Fig. 6). Thus, the TP and RT domains may be able to fold to certain degrees independently of Hsp90, perhaps with the assistance of other host chaperones (see below). Subsequently, the Hsp90 complex may recognize the partially folded TP and RT domains and bring them together to establish a functional, -binding-competent conformation. This posttranslational, late-folding role for Hsp90 in RT function is consistent with the current view that Hsp90 mainly recognizes some late, metastable folding intermediates in the folding pathways of its target proteins (9, 35). Such folding intermediates, for Hsp90 target proteins such as the RT, are apparently labile and require the continuous assistance of the chaperone to maintain them in so-called "poised" states (7) ready for interaction with specific partners or ligands (e.g., the  RNA). This proposed role for Hsp90 during a later stage in the RT folding and activation pathway in vitro does not contradict the cis preference phenomenon in the RT- interaction observed in vivo (1, 18). Thus, Hsp90 may interact with the RT and activate RT- binding in the cell immediately following translation (or even cotranslationally), as soon as the TP and RT domains are synthesized and partially folded. This would allow pgRNA packaging to proceed immediately following or during RT translation.

The task of keeping the RT in a soluble, albeit nonfunctional, state may instead fall on other chaperone proteins. We found that bacterial chaperones DnaK and GroEL tightly associated with the mini-RT proteins expressed in bacteria. DnaK, the homologue of the eukaryotic chaperone Hsp70, is known to bind short segments of hydrophobic peptides and is thought to act cotranslationally, keeping the elongating polypeptide chains from aggregating (11, 17). GroEL, on the other hand, is thought to act posttranslationally to provide a sequestered, folding-productive environment for its substrates by enclosing them in its central cavity (11, 17). Although the role of these chaperone proteins in RT folding and function remains to be investigated, it is possible that they may help to keep the RT in a soluble, activation-competent state, from which it can then be folded into its functional conformation with the assistance of the Hsp90 complex. Consistent with this notion is our observation that stripping the RT of its associated bacterial chaperones seemed to decrease the extent of RT activation through reconstitution and that the stripped RT tended to aggregate (J. Hu, unpublished results). These results, together with our previous evidence for a role of the eukaryotic Hsp70 in the RT functions (23), suggest that the bacterial DnaK may be able to substitute for the eukaryotic Hsp70 in the initial stage of RT folding (Fig. 6). In the absence of a functional Hsp90 chaperone in bacteria, some of the partially folded RT proteins may then be recognized by GroEL, which, however, is unable to establish the final RT conformation competent for  binding. As a result, both DnaK and GroEL remain "trapped" by the incompletely folded, nonnative RT proteins.

Our results represent the first successful attempt to purify a recombinant hepadnavirus RT and reconstitute its  binding and protein priming activities in vitro. This reconstitution system now provides the opportunity to analyze the requirement for RT functions in a systematic manner. The role of additional components of the Hsp90 complex known to be important in other substrate systems (9, 24, 36) may now be tested using purified, individual components. The system will also permit a systematic search for other host factors required for RT- interaction and protein priming that may or may not be related to the Hsp90 chaperone complex, such as the putative  binding factor (34; J. Hu, unpublished results). The ability to purify a recombinant RT expressed in bacteria, in particular, raises the prospect that sufficient amounts of purified RT may be obtained so that high-resolution structural studies may be applied to this most unique RT.