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 () 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 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 -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.Hepatitis B viruses, or hepadnaviruses, are small, hepatotropic DNA-containing viruses that replicate through an RNA intermediate. Their type member, hepatitis B virus (HBV), is an important human pathogen that causes acute and chronic hepatitis B (6, 33). Because only human and chimpanzee hepatocytes are susceptible to efficient HBV infection, the related animal viruses of woodchucks (woodchuck hepatitis B virus) and of ducks (duck hepatitis B virus [DHBV]) have become important model systems. In all hepadnaviruses, a terminally redundant transcript, the pregenomic RNA (pgRNA), acts as mRNA for core protein and P protein, the reverse transcriptase (RT). Furthermore, this RNA is selectively packaged into viral capsids and reverse transcribed into partially doublestranded circular DNA (22, 23). Both nucleocapsid assembly and replication initiation are crucially dependent on the binding of P protein to a stem-loop structure, ε, close to the 5Ј end of the pgRNA (5, 16, 25); a second copy of ε, in the 3Ј-terminal redundancy, is functionally silent in vivo (27). In contrast to that of retroviruses, hepadnavirus replication is initiated de novo, i.e., without a nucleic acid primer, within a bulged region of ε (Fig. 1B), resulting in the synthesis of a 3-or 4-nucleotide (nt) DNA primer (24, 37, 38) whose 5Ј end becomes covalently linked to P protein (20,41,44)....

Cryptic Protein Priming Sites in Two Different Domains of Duck Hepatitis B Virus Reverse Transcriptase for Initiating DNA Synthesis In Vitro

“…Both TP and RT domains served as a primer to initiate DNA synthesis. Independent TP and RT domains are known to trans-complement each other to carry out the protein priming reaction using the TP domain as a protein primer to initiate DNA synthesis (1,25,27). When the priming reactions were carried out in the presence of Mn 2ϩ , which is known to stimulate priming relative to Mg 2ϩ (27), we found, surprisingly, that the RT domain, as well as TP, acted as a protein primer for initiating DNA synthesis (Fig.…”

“…Both the TP and the RT domains are essential for RT-ε interaction and protein priming but the RNase H domain is dispensable (10,23,33,51). Also, isolated TP and RT domains, when combined together, are able to reconstitute a functional RT protein that is able to carry out protein priming in a trans-complementation reaction (1,23,25,27). Protein priming can be further subdivided into two different stages, requiring distinct RT conformations, the initial covalent attachment of the first nucleotide of the minus-strand DNA (a dGMP in DHBV) to RT (to the primer Y) called initiation and the subsequent addition of the remaining 2 to 3 nucleotides to the initiating nucleotide called polymerization (27,54).…”

Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses

“…A Tyr residue in the terminal protein (TP) domain of P protein acts as acceptor for the first dNTP of a short e-templated oligonucleotide. This "protein-priming" reaction, which for DHBV can be reconstituted completely in vitro (7)(8)(9)(10), establishes a 5′-phosphotyrosyl linkage between P protein and DNA which is maintained during the formation of complete minus-strand DNA (Fig. 1B), RNA-primed synthesis of usually incomplete plus-strand DNA, and circularization into RC-DNA via a short terminal redundancy (r) in the minus strand.…”

Expression of the active human and duck hepatitis B virus polymerases in heterologous system of Pichia methanolica

“…The DHBV P protein has shown RDDP activity without o motif. Recent studies have proposed that some chaperones might be needed for RDDP activity at the start point of DHBV replication (Beck and Nassal, 2001). The RDDP activity of our DHBV P protein was very weak compared with DDDP activity and the HBV P protein did not have RDDP activity.…”

“…The P proteins were purified through Ni-NTA resin (Qiagen, Germany) by His-6X fusion tag and had DNA-dependent DNA polymerase activity compared with Klenow polymerase. A recent study revealed that the viral P protein interacts with some cellular chaperones in the host during the viral life cycle (Beck and Nassal, 2001;Park and Jung, 2001). The present study offers a method measuring possible new chaperone effects on P protein activity.…”