Identification of an Essential Molecular Contact Point on the Duck Hepatitis B Virus Reverse Transcriptase

Abstract

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.Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription (reviewed in reference 9). It has a lipid envelope studded with viral glycoproteins that surrounds an icosahedral core particle composed of the core protein. Within the core particle are the viral nucleic acids and reverse transcriptase (P). Other hepadnaviruses infect woolly monkeys, woodchucks, ground squirrels, ducks, geese, and herons (6,17,29,31,32). Significant differences exist among the hepadnaviruses, but they share a high degree of hepatotropism, follow the same replication cycle, and have a nearly identical genetic organization.Hepadnaviral reverse transcription (34) occurs within cytoplasmic capsid particles. Reverse transcription begins with binding of P to an RNA stem-loop (ε) on the pregenomic RNA, and then this complex is encapsidated. Reverse transcription is primed by P itself, so minus-strand DNA is covalently linked to P (36, 40). Complexes containing P and the viral nucleic acids must be dynamic because three strand transfers are required to produce the mature circular viral DNA (21,22,38,41).The binding of P to ε requires the active participation of a molecular chaperone complex (13). In vitro reconstitution studies with recombinant duck hepatitis B virus (DHBV) P revealed that P-ε binding requires HSP90, HSP70, HSP40, HSP23, and HOP (2,11,14), although under certain circumstances only HSC70 and HSP40 are necessary (3). Because chaperones mod...

Chaperones Activate Hepadnavirus Reverse Transcriptase by Transiently Exposing a C-Proximal Region in the Terminal Protein Domain That Contributes to ε RNA Binding

“…Five MAbs with approximately known epitope locations in the DHBV P protein TP domain (8) were kindly provided by J. Tavis (St. Louis University). Using the PepScan technique (13) and cellulose membrane immobilized TPderived 15-mer peptides, each overlapping the previous one by 13 amino acids, we confirmed and refined the mapping data previously obtained by using a set of truncated DHBV P protein derivatives (8).…”

“…Five MAbs with approximately known epitope locations in the DHBV P protein TP domain (8) were kindly provided by J. Tavis (St. Louis University). Using the PepScan technique (13) and cellulose membrane immobilized TPderived 15-mer peptides, each overlapping the previous one by 13 amino acids, we confirmed and refined the mapping data previously obtained by using a set of truncated DHBV P protein derivatives (8). Operationally, we defined an epitope as the common sequence present in usually three to five overlapping peptides that reacted strongly with the respective MAb (the previously reported mapping data are given in parentheses): MAb9 and MAb11, aa 53 to 59 (aa 46 to 77); MAb5, aa 141 to 147 (aa 138 to 160); and MAb6 and MAb10, aa 191 to 197 (aa 182 to 202).…”

“…Primed P-ε complexes were generated and subjected to RNase A and V8 treatment as described above. The reactions were split and incubated overnight at 4°C with MAb9 or MAb6 or an irrelevant anti-green fluorescent protein (GFP) MAb immobilized on Gammabind PlusSepharose beads suspended in 0.5 ml of radioimmunoprecipitation assay (RIPA) buffer (8). Beads were washed four times with 1 ml each of RIPA buffer.…”

“…Because of the large size of intact antibody molecules, the inhibitory effects of the MAbs described above did not necessarily derive from a direct competition with Dε RNA for a common binding site. As a more sitespecific tool we used GrpDP with point mutations in the center of the chaperone-exposed region, encompassing the so-called T3 motif, a short stretch of amino acids (A 177 GILYKR 183 ) that is highly conserved between the TP sequences of avian and mammalian hepadnaviruses (8). In addition to K182 and R183, there are four more basic (three K and one R) amino acids, but only one acidic (E176), in close proximity.…”

“…The in vitro reconstitution system allowed us to test the effects of defined combinations of purified chaperones, and ATP, on P-protein structure and to correlate them with functional P activation; moreover, the initial chaperone activation step could directly be monitored in the absence of Dε RNA. Second, several monoclonal antibodies (MAbs) against different regions in the TP domain (8) and site-specifically mutated P proteins allowed us to assign proteolytic fragments to specific sequence segments. Third, the known location of the priming Y residue allowed us to identify TP-associated changes in enzymatically active, primed P protein.…”