A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein

Abstract

Hepadnaviruses are the only known viruses that replicate by protein-primed reverse transcription. Beyond the conserved reverse transcriptase (RT) and RNase H domains, their polymerases (P proteins) carry a unique terminal protein (TP) domain that provides a specific Tyr residue, Tyr96 in duck hepatitis B virus (DHBV), to which the first nucleotide of minus-strand DNA is autocatalytically attached and extended by three more nucleotides. In vitro reconstitution of this priming reaction with DHBV P protein and cellular chaperones had revealed strict requirements for the D RNA stem-loop as a template and for catalytic activity of the RT domain plus RNA-binding competence of the TP domain. Chaperone dependence can be obviated by using a truncated P protein (miniP). Here, we found that miniP with a tobacco etch virus (TEV) protease cleavage site between TP and RT (miniP TEV ) displayed authentic priming activity when supplied with ␣-32 P-labeled deoxynucleoside triphosphates; however, protease cleavage revealed, surprisingly, that the RT domain was also labeled. RT labeling had identical requirements as priming at Tyr96 and originated from dNMP transfer to a unique Tyr residue identified as Tyr561 in the presumed RT primer grip motif. Mutating Tyr561 did not affect Tyr96 priming in vitro and only modestly reduced replication competence of an intact DHBV genome; hence, deoxynucleotidylated Tyr561 is not an obligate intermediate in TP priming. However, as a first alternative substrate for the exquisitely complex protein-priming reaction, dNMP transfer to Tyr561 is a novel tool to further clarify the mechanism of hepadnaviral replication initiation and suggests that specific priming inhibitors can be found.Hepadnaviruses, with human hepatitis B virus (HBV) as their prototypic member and a most important pathogen, are small hepatotropic DNA viruses that replicate their ϳ3-kb relaxed circular DNA (rcDNA) genome through proteinprimed reverse transcription. Because of numerous experimental restrictions with HBV, the related duck HBV (DHBV) is widely used as a model (39). Crucial for protein priming is the terminal protein (TP) domain which P proteins carry in addition to the reverse transcriptase (RT) and RNase H (RH) domains that are conserved in all RTs (Fig. 1A). TP provides the specific Tyr residue (Tyr96 in DHBV P and Tyr63 in HBV P) to whose phenolic OH-group the first DNA nucleotide of minus-strand DNA is covalently attached (24,55,62).Studies in HBV-or DHBV-transfected cells have provided a general outline of the hepadnaviral replication cycle (reviewed in references 8 and 27). Like core protein, P protein is translated from the pregenomic RNA (pgRNA) and then binds to the 5Ј proximal ε (Dε for HBV) stem-loop on pgRNA, triggering RNA encapsidation and replication initiation. In this priming reaction a bulged region within ε templates the synthesis of a 3-or 4-nucleotide-long, TP-linked DNA oligonucleotide. Its translocation to a 3Ј-proximal RNA element, DR1*, and extension from there yields minus-strand DNA. Concomit...

Carbonyl J Acid Derivatives Block Protein Priming of Hepadnaviral P Protein and DNA-Dependent DNA Synthesis Activity of Hepadnaviral Nucleocapsids

“…Priming inhibition by KM-1 and its relatives could be due to interactions with P protein, with Dε RNA, or with another factor in the reaction that is required for priming. In the miniP system, the only such factors are bivalent metal ions, which are crucial for P activity (5,29). However, these were present at millimolar concentrations whereas the IC 50 for KM-1 inhibition was around 1 M, ruling out metal ion sequestration.…”

“…Bacterial expression and purification of miniDP, MBP-TP, and MBP-RT fusion proteins were performed in a manner analogous to that previously reported (5). In brief, lysates from induced cultures of appropriately transformed E. coli BL21(DE3) cells were cleared by centrifugation.…”

“…Soluble proteins from the supernatants were purified by immobilized metal ion affinity chromatography (IMAC) with an Ni-nitrilotriacetic acid Superflow column (Qiagen), followed by size exclusion chromatography using a Superdex 75 HiLoad 16/60 column (GE Healthcare). Insoluble proteins from the pellets were dissolved in 7 M guanidinium hydrochloride containing buffer and refolded by rapid dilution into refolding buffer (5).…”

“…MiniP proteins can support Dε RNA-dependent in vitro priming at alternative ("cryptic") residues to Y96, including at Y561 in the RT domain (5,13). Exploiting the ability of separate TP and RT domains to trans complement each other, we generated MBP fusions carrying either the WT TP domain or a mutant form with a defective natural acceptor site (TP-Y96D), or a WT RT domain or an enzymatically disabled mutant form (YMDD ¡ YMHA).…”

“…1A). According to homology-based modeling for HBV (3,16) and DHBV (5,55), the latter part forms a thumb subdomain similar to that in HIV-1 RT. Structural similarity between the two hepadnaviral P proteins is further supported by their similar sensitivities against various NAs in the context of viral infection or transfection.…”