Reconstitution of a functional duck hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in Escherichia coli

Abstract

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (epsilon) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by epsilon and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of epsilon-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.

FIG. 1

(A) Domain organization of hepadnaviral P proteins. Numbers indicate amino acid positions of DHBV P. (B) Model of the DHBV replication initiation complex. P protein with its TP and RT-RH domains connected via the spacer (black angled bar) is bound to the RNA stem-loop Dɛ. A bulged region in Dɛ serves as a template for the synthesis of a short DNA primer that is covalently linked to a Tyr residue in the TP domain. Binding to Dɛ requires P protein to be present in a multicomponent complex composed of Hsp90, p23 (light gray objects), and, most likely, additional, as yet unknown, factors (designated X, Y, and Z).

FIG. 2

Functional trans-complementation of DHBV P protein domains coexpressed in RRL. (A) Schematic drawing of plasmid constructs and RNA transcripts used for in vitro translation. ORFs are given as boxes, and T7 promoter-driven transcripts are shown as wavy lines. Plasmid pT7AMVpol (Pɛ+) contains the complete 786-aa P protein ORF; the approximate borders of the four P protein domains are indicated by amino acid positions. Constructs Pɛ+, pT7-TP1-220ɛ+ (TPɛ+), and pT7-RT209-786ɛ+ (RTɛ+) carry a cis Dɛ element downstream of the coding region, whereas pT7-TP1-220 (TP) and pT7-RT209-786 (RT) are Dɛ deficient. (B) [³⁵S]Met-labeled in vitro translation products of the constructs shown in panel A. Proteins were analyzed by SDS-PAGE and autoradiography. Molecular size markers are given in kilodaltons. (C) In vitro cotranslation and protein priming assay in the presence of [α-³²P]dATP. Priming assays were performed as described in Materials and Methods. In the sample shown in lane 10 a 76-nt Dɛ RNA in vitro transcript was supplied in trans at a 1 μM concentration. An equal volume of each sample (except for Pɛ+ [20%]) was analyzed by SDS-PAGE and autoradiography. The band of about 32 kDa in the Pɛ+ sample corresponds most likely to a degradation product of the ³²P-primed full-length P protein and did not occur as prominently in similar experiments.

FIG. 3

In vitro-translated DHBV TP and RT-RH domains interact posttranslationally in a Dɛ-dependent manner. The indicated constructs were separately expressed in RRL, and translation was stopped by adding cycloheximide to a final concentration of 20 μg/ml. The TP- and RT-containing lysates were mixed and incubated together with Dɛ RNA (only lane 3; final concentration, 1 μM) for 1 h at 30° C. The samples were subjected to priming assays in the presence of [α-³²P]dATP and analyzed by SDS-PAGE on the same gel as those in Fig. 2C; the exposure shown here is four times longer. See the legend to Fig. 2A for details about constructs.

FIG. 4

Bacterial expression and purification of the DHBV TP domain. (A) Expression of TP1-220 in E. coli BL21-CodonPlus-RIL cells. E. coli cultures transformed with plasmid pET-TP1-220 were grown in the presence (+) or absence (−) of IPTG, and the pelleted cells were lysed by boiling in SDS sample buffer. Total lysates were analyzed by SDS-PAGE and Coomassie blue staining. (B) Purification of TP1-220. The soluble fraction of TP1-220 was subjected to IMAC and subsequent dialysis against a physiological buffer. The insoluble faction was solubilized in 8 M urea and purified by IMAC under denaturing (denat.) conditions. The protein was refolded by two-step dialysis against buffers containing 3 and 1 M urea, respectively. Aliquots of both purified fractions were resolved by SDS-PAGE and stained with Coomassie blue. Note the presence in the natively purified fraction (lane 1) of additional proteins of about 70 , 60, and <14 kDa. The former two correspond to E. coli DnaK and GroEL, as indicated by Western blotting with specific antibodies (not shown).

FIG. 5

In vitro reconstitution of functional priming complexes from in vitro-translated RT-RH and E. coli-expressed TP. RT209-786 (RTɛ+) was expressed in RRL from construct pT7-RT209-786ɛ+ in the presence of the indicated amounts of E. coli-derived TP1-220 (TP) purified under native conditions (nat.) (lanes 4 to 6), or purified under denaturing conditions followed by renaturation (renat.) (lanes 7 to 9), or without TP1-220 (lane 3). As negative controls, the TP fractions were incubated in RRL in the absence of RT209-786 (lanes 1 and 2). For comparison an in vitro cotranslation of TP1-220 and RT209-786 was performed (lane 10). After in vitro translation the samples were assayed for priming with [α-³²P]dATP. ³²P-labeled TP was detected by SDS-PAGE and autoradiography.

FIG. 6

Bacterial expression and purification of the RT-RH domain. (A) Schematic drawing of C-terminally truncated RT-RH constructs analyzed for protein expression in E. coli. Each construct starts with 50 vector-derived aa (open boxes at left) followed by DHBV P sequence from aa 349 to the indicated C-terminal position. Some constructs contain short vector-encoded C-terminal tags (open boxes at right). Numbers on the left represent abbreviations of the construct names; e.g., “661” refers to plasmid pET-RT349-661. Construct pET-RT349-786 c.o. is codon optimized for E. coli in the C-terminal region (black box). Construct pET-RT349-786Pol⁻ contains a D-to-H amino acid substitution in the YMDD motif of the polymerase active site (asterisk). Borders of the RT and RH domains are indicated by thick vertical broken lines. The conserved region of the RH domain from aa 660 to 759 (9) is labeled by thin broken lines. (B) Expression of RT-RH constructs in E. coli strain BL21-CodonPlus-RIL. Total cell lysates of induced bacterial cultures were analyzed by SDS-PAGE and Coomassie blue staining; molecular mass markers are given in kilodaltons. (C) Purification of bacterially expressed RT349-761. The soluble fraction of RT349-761 was purified by IMAC and subsequent dialysis against a physiological buffer. The purified protein fraction was analyzed by SDS-PAGE and Coomassie blue staining. The nominally 54-kDa RT349-761 protein is marked by an arrow. Additional bands of 60 kDa (strong) and 70 kDa (weak) were identified by immunoblotting as E. coli GroEL and DnaK.

FIG. 7

In vitro reconstitution of functional priming complexes from E. coli-derived TP and RT-RH domains in RRL. TP, RT, RRL, and Dɛ RNA were mixed, incubated for 1 h at 30°C to allow RNP formation, and subsequently assayed for protein priming activity in the presence of [α-³²P]dATP (for details, see Materials and Methods). The reaction products were resolved by SDS-PAGE, and ³²P-labeled TP protein was detected by autoradiography. Abbreviations: TP, bacterially expressed TP1-220 (25 ng/μl); RT, bacterially expressed RT349-761 (10 ng/μl); Dɛ, in vitro-transcribed Dɛ RNA (1 μM); RRL, rabbit reticulocyte lysate; +CHX, sample supplemented with cycloheximide (20 μg/ml) at the beginning of the incubation period (lane 6); -binding, the indicated components were mixed and subjected to priming conditions without prior incubation at 30°C (lane 7). For comparison, a priming assay with in vitro-cotranslated TP1-220 and RT349-761 proteins from Dɛ-deficient constructs, supplemented with 1 μM Dɛ RNA in trans, was performed in parallel (lane 8).

FIG. 8

Alignment of RNase H domains of DHBV, HBV, and HIV-1 RTs. The alignment is based on a previously published version (9). Amino acid residues conserved between DHBV and HBV are boxed. Residues conserved between hepadnaviral and retroviral RH domains are shaded. Invariant Asp and Glu residues implicated in catalysis are marked with asterisks. Numbers above and below the sequence correspond to amino acid positions of DHBV P and HIV-1 RT, respectively. The horizontal bars and boxes below the sequence indicate β-sheets and α-helices of the RH domain in HIV-1 RT as determined by X-ray crystallography (11). Helix αE is unlikely to exist in hepadnaviral RHs due to multiple Pro residues in this region. Note that DHBV residue 759 represents the C-terminal border of the homologous region.