Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein.
Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication.
zu Putlitz
J
J
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts 02129, USA.
Lanford
R E
RE
Carlson
R I
RI
Notvall
L
L
de la Monte
S M
SM
Wands
J R
JR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D015153:Blotting, Western; D002460:Cell Line; D018604:Epitope Mapping; D019084:Fluorescent Antibody Technique, Indirect; D015684:Gene Products, pol / Q000276:immunology*; D006510:Hepatitis B Antibodies / Q000096:biosynthesis / Q000276:immunology*; D006515:Hepatitis B virus / Q000201:enzymology*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D012194:RNA-Directed DNA Polymerase / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015684:Gene Products, pol; D006510:Hepatitis B Antibodies; C066027:P protein, Hepatitis B virus; D012194:RNA-Directed DNA Polymerase