Distinct requirement for two stages of protein-primed initiation of reverse transcription in hepadnaviruses

Abstract

Reverse transcription in hepadnaviruses is primed by the viral reverse transcriptase (RT) (protein priming) and requires the specific interaction between the RT and a viral RNA signal termed epsilon, which bears the specific template sequence for protein priming. The product of protein priming is a short oligodeoxynucleotide which represents the 5' end of the viral minus-strand DNA and is covalently attached to the RT. We have now identified truncated RT variants from the duck hepatitis B virus that were fully active in the initial step of protein priming, i.e., the covalent attachment of the first nucleotide to the protein (RT deoxynucleotidylation), but defective in any subsequent DNA polymerization. A short sequence in the RT domain was localized that was dispensable for RT deoxynucleotidylation but essential for the subsequent DNA polymerization. These results have thus revealed two distinct stages of protein priming, i.e., the initial attachment of the first nucleotide to the RT (RT deoxynucleotidylation or initiation of protein priming) and the subsequent DNA synthesis (polymerization) to complete protein priming, with the second step entailing additional RT sequences. Two models are proposed to explain the observed differential sequence requirement for the two distinct stages of the protein priming reaction.

FIG. 1.

RT sequence requirement for the initiation and polymerization stage of protein priming. (A) GST-MiniRT1 (lanes 7 to 12) and GST-MiniRT2 (lanes 1 to 6) were purified from bacteria and assayed for in vitro protein priming activity, with (lanes 2, 4, 6, 8, 10, and 12) or without (lanes 1, 3, 5, 7, 9, and 11) reconstitution with the reticulocyte lysate (RL). In addition, MiniRT2 (without the GST fusion) was expressed in the RL by in vitro translation and assayed for protein priming activity (lanes 13 to 15). As indicated, different ³²P-labeled nucleotide precursors (dATP, dA; dGTP, dG; TTP, T) were used; unlabeled dNTP mixtures (without the nucleotide corresponding to the labeled one) were also added to the reaction mixtures. Labeled RT proteins, as indicated by the arrows, were then detected by resolving the reactions by SDS-PAGE and autoradiography. Three distinct labeled products were detected in the reactions using GST-MiniRT1, as indicated by the numbers 1, 2, and 3. The top band (band 1) represents the intact GST-MiniRT1 protein, whereas the middle (band 2) and bottom (band 3) bands represent degradation products from GST-MiniRT1. Note that product 3 from GST-MiniRT1 migrated almost exactly as GST-MiniRT2. (B) C-terminal truncations of MiniRT1 were expressed in the RL by in vitro translation from templates linearized at the indicated restriction sites (Pml, PmlI; Nsp, NspI) (see Fig. 6 for a schematic diagram of the RT structure and the positions of these restriction sites). The truncated RT proteins were then tested for in vitro protein priming activity in the presence of either ³²P-labeled dGTP (lanes 1 and 2) or dATP (lanes 3 and 4). Unlabeled dNTP mixtures (without the nucleotide corresponding to the labeled one) were also added to the reactions.

FIG. 2.

Protein priming by both MiniRT1 and MiniRT2 required ɛ binding and RT catalytic activity. GST-MiniRT1, its mutant derivative GST-MiniRT1/YMHA, GST-MiniRT2, and its mutant derivative GST-MiniRT2/CA29 were purified from bacteria and assayed for in vitro protein priming activity. All reactions were carried out in the presence of reticulocyte lysate and [α-³²P]dGTP. The ɛ RNA was added to all reactions except those shown in lanes 2 and 5. The labeled RT proteins are indicated as in the legend to Fig. 1.

FIG. 3.

Association of MiniRT2 with Hsp90 and p23 in mammalian cells. Plasmid DNA expressing the GST-tagged MiniRT2 (pEBG-MiniRT2) or GST alone (pEBG vector) was transfected into 293T cells. Transfected cells were lysed, and the GST proteins were purified by using GSH affinity beads. Bound proteins were eluted with GSH, resolved by SDS-PAGE, and detected by Western blot analyses using MAbs against Hsp90 (Anti-Hsp90) or p23 (Anti-p23) or by Coomassie blue staining (top panel). Hsp90, p23, GST-MiniRT2, and GST are indicated. The star to the left of the top panel denotes a nonspecific band associated with the GSH beads.

FIG. 4.

GST-MiniRT2 purified from mammalian cells was active in the initiation of protein priming but defective in DNA polymerization. GST-MiniRT2 was expressed in 293T cells following transient transfection of pEBG-MiniRT2 and purified using GSH affinity resin. (A) Purified GST-MiniRT2 was assayed for in vitro protein priming activity, supplemented with buffer alone (lanes 1 and 2), with an ATP regenerating system (ATP RS [lanes 3 and 4), or with reticulocyte lysate (RL [lanes 5 and 6]). The ɛ RNA was added to the indicated reaction mixtures only (lanes 2, 4, and 6). [α-³²P]dGTP was used as the nucleotide precursor. (B) Purified GST-MiniRT2 was assayed for in vitro protein priming, all reactions being supplemented with reticulocyte lysate. Either [α-³²P]dGTP (lanes 1 and 2) or [α-³²P]dATP (plus unlabeled dGTP and TTP) (lanes 3 and 4) was used as the labeled nucleotide precursor. The ɛ RNA was added to the mixtures for reactions 1 and 3 only.

FIG. 5.

Protein priming in the presence of variant ɛ RNA template. GST-MiniRT2 purified from bacteria was assayed for in vitro protein priming activity reconstituted by reticulocyte lysate, using as template either the wild-type ɛ RNA (with the bulge sequence UUAC as the template residues [lanes 1 to 3]) or a variant ɛ with the 3′ nucleotide of the template sequence changed from C to U (UUAU [lanes 4 to 6]). As indicated, either [α-³²P]dGTP (dG [lanes 1 and 4]) or [α-³²P]dATP (dA) was used as the labeled nucleotide precursor (lanes 2, 3, 5, and 6). Unlabeled dNTP mixture (without dATP) was also added to the reactions shown in lanes 2 and 5.

FIG. 6.

Summary of RT sequences required for the initiation and polymerization stages of protein priming. Shown on the top is a schematic diagram of the DHBV RT domain structure (TP, spacer, RT, and RNase H). The primer tyrosine residue and the double aspartate residues at the RT active site are indicated. The RT domain is further divided into the “finger” (F), “palm” (P), and “thumb” (T) subdomains (with the approximate boundaries marked), based on alignment with the RT structure of the human immunodeficiency virus (8, 25). Summarized below the diagram are the activities of various RT deletion/truncation variants in the initiation and polymerization steps of the protein priming reaction, which were measured by dGTP and dATP incorporation, respectively, into the RT proteins. The truncations at the C terminus are indicated by the corresponding restriction sites.

FIG. 7.

Multistage, protein-primed initiation of reverse transcription in hepadnaviruses. Multiple functional and conformational states of the viral RT. The viral RT, without assistance of host cell factors, is in an inactive state (RT¹) unable to bind to ɛ RNA or carry out protein priming. Upon association with the cellular Hsp90 chaperone complex, the RT undergoes a conformational maturation and adopts an ɛ binding-competent state (RT²) (11, 13, 15, 16). Binding of ɛ then induces another conformational change in the RT (RT³) that may be required for the RT to gain its enzymatic activity (29). The initiation of protein priming then leads to the covalent attachment of the first nucleotide of the viral minus-strand DNA (a dGMP residue in the case of DHBV) to the RT (RT deoxynucleotidylation), using ɛ as the template. Following this initiation reaction, a conformational change of the RT (RT⁴) may be required for further DNA synthesis to produce the 4-nucleotide nascent minus-strand DNA oligomer (dGTAA in DHBV), still templated by ɛ (30, 31). Clearly, the putative RT thumb subdomain is dispensable for the initiation of protein priming (step 3) but required for its completion (step 4). Following the completion of protein priming, the RT undergoes another major conformational change (RT⁵), dissociates from the ɛ RNA and the RT-nascent minus-strand DNA complex, and then translocates to the 3′ end of the pgRNA (minus-strand template switch) to continue DNA synthesis (elongation mode) (30, 31).