Formation of a functional hepatitis B virus replication initiation complex involves a major structural alteration in the RNA template

Abstract

The DNA genome of a hepatitis B virus is generated by reverse transcription of the RNA pregenome. Replication initiation does not involve a nucleic acid primer; instead, the hepadnavirus P protein binds to the structured RNA encapsidation signal epsilon, from which it copies a short DNA primer that becomes covalently linked to the enzyme. Using in vitro-translated duck hepatitis B virus (DHBV) P protein, we probed the secondary structure of the protein-bound DHBV epsilon RNA (Depsilon) and observed a marked conformational change compared to free Depsilon RNA. Several initiation-competent mutant RNAs with a different free-state structure were similarly altered, whereas a binding-competent but initiation-deficient variant was not, indicating the importance of the rearrangement for replication initiation and suggesting a mechanistic coupling to encapsidation.

FIG. 1

Functional roles of the ɛ-P interaction. The straight line represents a linear version of the circular DHBV genome, the diamond represents the polyadenylation signal, and the open rectangles represent the three major open reading frames. Numbers are nucleotide positions. The wavy lines represent the terminally redundant RNA pregenome, which also serves as mRNA for the capsid (C) and the P protein (small and large circles). Dɛ is shown as a symbolic hairpin, and the direct-repeat elements DR1, DR2, and DR1* are shown as boxes. Region 2 is an as yet not well-defined second element required for DHBV pregenome encapsidation (7). Binding of P protein to 5′ Dɛ triggers the addition of dimeric capsid protein subunits and hence nucleocapsid assembly; in addition, replication is initiated by P copying 3 to 4 nt of the Dɛ bulge into a DNA primer that is covalently linked to the P protein. The temporal order, if any, of these events has not yet been established. The entire complex is then translocated to DR1*, and the primer is extended to form minus-strand DNA.

FIG. 2

In vitro assembly and purification of the ɛ-P RNP complex. N-terminally His₆-tagged DHBV P protein was expressed by in vitro translation in rabbit reticulocyte lysate from an RNA template lacking Dɛ sequences (step 1). Upon addition of in vitro-transcribed Dɛ RNA the ɛ-P complex was formed (step 2) and separated from free Dɛ RNA by IMAC (step 3).

FIG. 3

Authentic enzymatic activity of the immobilized and purified ɛ-P complex. (A) Schematic drawing of the DNA primer covalently linked to Tyr 96 of the DHBV P protein. Nucleotides in the bulge region of Dɛ serve as the template. (B) The immobilized and purified ɛ-P complex exhibits sequence-specific priming activity. Priming reactions were performed in the presence of [α-³²P]dATP plus various combinations of unlabeled dNTPs as indicated in the figure, and samples were analyzed by sodium dodecyl sulfate-PAGE. Strong labeling of P protein was dependent on both dGTP and dTTP (left lane), in agreement with the synthesis of a primer with an authentic sequence.

FIG. 4

Secondary-structure analysis of free and P-protein-bound Dɛ RNA. (A) Nuclease probing. (Left) Free and P-protein-bound 5′-³²P-labeled wt Dɛ RNA was subjected to limited digestion with RNase A (A), RNase T₁ (T), and V₁ nuclease (V), and the products were analyzed by denaturing PAGE. The minus signs represent an undigested control; a reference nucleotide ladder obtained by partial alkaline hydrolysis of 5′-³²P-labeled wt Dɛ RNA is shown in lanes OH⁻. Right (“released”), the structural analysis of wt Dɛ RNA dissociated from the ɛ-P complex is shown. G represents a G-sequencing track obtained by digestion of wt Dɛ RNA with RNase T₁ under denaturing conditions. (B) Lead probing. Free and P-protein-bound 5′-³²P-labeled wt Dɛ RNA was treated with increasing amounts of lead acetate, and the products were analyzed by denaturing PAGE. The hypersensitive region of Dɛ in the ɛ-P complex is indicated by a solid bar, and regions showing reduced sensitivity are labeled with shaded bars. (C) Comparison of experimental nucleotide accessibilities in free and P-protein-bound Dɛ RNA with the secondary-structure model. Major sites accessible to single-strand-specific nucleases are indicated by arrows (A, RNase A; T1, RNase T₁); The major V₁ nuclease cleavage site is labeled by a large arrowhead (V1). Pb²⁺ cleavages in free RNA are denoted by small arrowheads. Lead cleavage sites enhanced in the ɛ-P complex have a black background, and regions protected from lead cleavage are shaded in gray. Numbers represent nucleotide positions. Nucleotides mutated in the Dɛ variants L5 (U2591A) and L56 (U2591C, G2592A) are encircled.

FIG. 5

Enzymatic secondary-structure analysis of Dɛ mutants L5 and L56 compared with wt Dɛ in their free state and bound to P protein. For further explanations and abbreviations, see the legend to Fig. 4. G-sequencing tracks on the left side of both panels (“free” and “bound”) were obtained from wt Dɛ RNA, and those on the right side were obtained from mutant L56 RNA. Note that a band representing G2592 (labeled with an asterisk) appears only with wt Dɛ, since L56 contains an A at that position.

FIG. 6

Lead probing of Dɛ mutants L5 and L56 in their free state (lanes f) and bound to P protein (lanes b). Free and P-protein-bound 5′-³²P-labeled wt Dɛ RNA was treated with lead acetate (30 mM final concentration), and the products were analyzed by denaturing PAGE. The hypersensitive region of L5 is indicated by a solid bar, and regions showing reduced sensitivity are labeled with shaded bars. Numbers represent nucleotide positions.

FIG. 7

Comparison of experimental nucleotide accessibilities in free L5 RNA and in P-protein-bound L5 and L56 RNA with their secondary-structure models. (Left) Model of free L5 RNA. For comparison, nucleotides corresponding to the 6-nt bulge and the 4-nt apical loop of wt Dɛ are boxed. Nucleotides different from wt Dɛ are encircled. (Middle and right) Models of L5 and L56 RNA in the P-protein-bound state. Only the right half of each structure is shown. The lead-hypersensitive region of L5 has a black background. Regions significantly protected from lead cleavage in the ɛ-P complex have a gray background.

FIG. 8

Enzymatic secondary-structure analysis of Hɛ and Dɛ mutant V3 in their free state (lanes f) and in the ɛ-P complex (lanes b). (A) Nuclease probing. Hypersensitive sites are marked with arrowheads. For further explanations and abbreviations, see the legend to Fig. 4A. (B) Location of hypersensitive cleavage sites in the ɛ-P complex with respect to the secondary-structure model of Hɛ. Nucleotides different between Hɛ and wt Dɛ are encircled. The sequence of V3 differs at six positions from that of Hɛ and is denoted inside the large apical loop. Hypersensitive RNase A sites of P-protein-bound Hɛ and V3 RNA are indicated by arrows labeled Hɛ and V3.

FIG. 9

Model for replication initiation complex formation in hepatitis B viruses. Free wt and mutant Dɛ RNAs are shown as schematic stem-loops representing their most likely secondary structures. Positions of nucleotides exchanged in the mutants L5 and L56 are symbolized by circles. P protein is represented by the U-shaped gray object. Binding of the protein to wt Dɛ and L5 RNA, probably via regions at the base of the bulge and in the vicinity of the apical loop, induces a distinct conformational change in the RNA and the reverse transcriptase. As a result, the primer template region, corresponding to the 3′-half of the bulge in free wt Dɛ RNA, becomes properly oriented with respect to the active site of the enzyme and the anchoring Tyr residue, and primer synthesis commences. L56 RNA binds to P protein but is unable to undergo this conformational shift, possibly because the loop mutations disturb the apical interaction with the protein. Formation of the initiation complex could provide a specific binding site for core protein dimers (connected circles) and hence could trigger encapsidation.