title: 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: 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, ε, 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, ε 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.
authors.affiliation: University Hospital Freiburg, Internal Medicine II/Molecular Biology, Freiburg, Germany.
authors.forename: Yong-Xiang
authors.initials: YX
authors.lastname: Wang
authors.forename: Cheng
authors.initials: C
authors.lastname: Luo
authors.forename: Dan
authors.initials: D
authors.lastname: Zhao
authors.forename: Jürgen
authors.initials: J
authors.lastname: Beck
authors.forename: Michael
authors.initials: M
authors.lastname: Nassal
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D004261:DNA Replication; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015611:Hepatitis B Virus, Duck / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D014764:Viral Proteins; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase