Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

Abstract

c Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ⑀, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for this notion is limited. Exploiting assays available for duck HBV (DHBV) but not the HBV P protein, we assessed the functional consequences of numerous mutations in box E, which forms the DNA primer grip in human immunodeficiency virus type 1 (HIV-1) RT. This substructure coordinates primer 3=-end positioning and RT subdomain movements during the polymerization cycle and is a prime target for nonnucleosidic RT inhibitors (NNRTIs) of HIV-1 RT. Box E was indeed critical for DHBV replication, with the mutations affecting the folding, ⑀ RNA interactions, and polymerase activity of the P protein in a position-and amino acid side chain-dependent fashion similar to that of HIV-1 RT. Structural similarity to HIV-1 RT was underlined by molecular modeling and was confirmed by the replication activity of chimeric P proteins carrying box E, or even box C to box E, from HIV-1 RT. Hence, box E in the DHBV P protein and likely the HBV P protein forms a primer grip-like structure that may provide a new target for anti-HBV NNRTIs. Hepadnaviruses are small hepatotropic DNA viruses that infect humans and select mammals and birds. Hepatitis B virus (HBV), one of the most relevant viral pathogens of humans (20), is their prototypic member. All hepadnaviruses replicate their ϳ3.0-kb genomes by chaperone-assisted protein-primed reverse transcription (10), executed by their P proteins. These are unusual reverse transcriptases (RTs), which, beyond the common RNA-dependent and DNA-dependent DNA polymerase and RNase H (RH) domains, contain a unique terminal protein (TP) domain at their N termini (Fig. 1A). To initiate reverse transcription, the phenolic OH group of a specific Tyr residue in TP fills the role that conventionally is taken by the 3=-hydroxyl end of a nucleic acid primer (30).P proteins are translated from a greater-than-genome-length transcript, the pregenomic RNA (pgRNA), which also acts as mRNA for the viral core protein. The interaction of the P protein with an RNA stem-loop, ⑀, on the pgRNA is crucial for viral replication; it triggers the coencapsidation of pgRNA and the P protein into newly forming nucleocapsids and the synthesis of a short DNA oligonucleotide, which is templated by the bulge in ⑀ and, via its 5=-terminal nucleotide, becomes covalently attached to the Tyr residue in TP ("protein priming"). Upon transfer to a 3=-proximal acceptor site on pgRNA, the oligonucleotide is extended into fulllength minus-strand DNA, and the pgRNA template is concurrently degraded by the P protein's RH activity. Some 15 to 18 residues fro...

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

“…Furthermore, the bulge of Dε specifying the sequence of the DNA primer and the protein-priming Y96 residue in the TP domain must occupy positions relative to the active site and dNTP pocket equivalent to those of the template and primer strands during DNA elongation (5,55). A visual impression of the large surface area occluded by bound nucleic acid is given by the homology-based, mutagenesis-supported (11,55) structural model of the DHBV P protein RT domain with bound primer/template DNA and dTTP (Fig. 11A).…”

“…In contrast, E401/E402 (cyan space fill models in the background), well accessible in free miniDP, would become shielded by nucleic acid, as observed. E499 (blue stick model on the left) is predicted to be part of a long ␣-helix comprising residues G483 to R503 (55), equivalent to the ␣-helix formed by residues G155 to K173 in HIV-1 RT. Within such a stable secondary structure, the C-terminal peptide bond of E499 would not be a protease substrate, consistent with the absence of corresponding cleavage products, regardless of the conditions.…”

“…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.…”

Hepatitis B virus molecular biology and pathogenesis

“…[68] In fact, portions of HBV RT can be replaced by homologous portions of HIV RT; this can generate an active RT that can function to produce mature HBV virions. [69] …”

Unveiling the Roles of HBV Polymerase for New Antiviral Strategies

“…Although box D does not directly affect dNTP binding, it likely contributes to overall structure. Box E contains the primer grip which aids polymerase-ε binding and DNA synthesis [59]. Box F was reported to facilitate interactions between the incoming dNTP and its template nucleotide [55].…”