Identification of an essential molecular contact point on the duck hepatitis B virus reverse transcriptase.
The hepadnaviral polymerase (P) functions in a complex with viral nucleic acids and cellular chaperones. To begin to identify contacts between P and its partners, we assessed the exposure of the epitopes of six monoclonal antibodies (MAbs) to the terminal protein domain of the duck hepatitis B virus P protein in a partially denaturing buffer (RIPA) and a physiological buffer (IPP150). All MAbs immunoprecipitated in vitro translated P well in RIPA, but three immunoprecipitated P poorly in IPP150. Therefore, the epitopes for these MAbs were obscured in the native conformation of P but were exposed when P was in RIPA. Epitopes for MAbs that immunoprecipitated P poorly in IPP150 were between amino acids (aa) 138 and 202. Mutation of a highly conserved motif within this region (T3; aa 176 to 183) improved the immunoprecipitation of P by these MAbs and simultaneously inhibited DNA priming by P. Peptides containing the T3 motif inhibited DNA priming in a dose-dependent manner, whereas eight irrelevant peptides did not. T3 function appears to be conserved among the hepadnaviruses because mutating T3 ablated DNA synthesis in both duck hepatitis B virus and hepatitis B virus. These results indicate that (i) the conserved T3 motif is a molecular contact point whose ligand can be competed by soluble T3 peptides, (ii) the occupancy of T3 obscures the epitopes for three MAbs, and (iii) proper occupancy of T3 by its ligand is essential for DNA priming. Therefore, small-molecule ligands that compete for binding to T3 with its natural ligand could form a novel class of antiviral drugs.
Cao
Feng
F
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.
Badtke
Matthew P
MP
Metzger
Lisa M
LM
Yao
Ermei
E
Adeyemo
Babatunde
B
Gong
Yunhao
Y
Tavis
John E
JE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis; D000939:Epitopes; D015611:Hepatitis B Virus, Duck / Q000201:enzymology*; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004247:DNA; D012194:RNA-Directed DNA Polymerase