Mapping of the hepatitis B virus reverse transcriptase TP and RT domains by transcomplementation for nucleotide priming and by protein-protein interaction.
Hepadnavirus polymerases initiate reverse transcription in a protein-primed reaction. We previously described a complementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase (pol) in the priming reaction. Independently expressed TP and RT domains form a complex functional for in vitro priming reactions. To map the minimal functional TP and RT domains, we prepared baculoviruses expressing amino- and carboxyl-terminal deletions of both the TP and RT domains and analyzed the proteins for the ability to participate in transcomplementation for the priming reaction. The minimal TP domain spanned amino acids 20 to 175; however, very little activity was observed without a TP domain spanning amino acids 1 to 199. The minimal RT domain spanned amino acids 300 to 775; however, little activity was observed unless the carboxyl end of the RT domain extended to amino acid 800. Thus, most of the RNase H domain was required. In previous studies, we observed a TP inhibitory domain between amino acids 199 and 344. The current analysis narrowed this domain to residues 300 to 334, which is a portion of the minimal RT domain. In addition, the ability of TP and RT deletion mutants to form stable TP-RT complexes was examined in coimmunoprecipitation assays. The minimal TP and RT domains capable of protein-protein interaction were considerably smaller than the domains required for functional interaction in the transcomplementation assays, and unlike priming activity, TP-RT interaction did not require the epsilon RNA stem-loop. These studies help to further define the complex protein-protein interactions required in HBV genome replication.
Lanford
R E
RE
Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227, USA. rlanford@icarus.sfbr.org
Kim
Y H
YH
Lee
H
H
Notvall
L
L
Beames
B
B
D000818:Animals; D002460:Cell Line; D005816:Genetic Complementation Test; D006515:Hepatitis B virus / Q000201:enzymology*; D010449:Peptide Mapping; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D018411:Spodoptera