사람 B형 간염 바이러스 복제효소의 활성에 관한 연구
Studies on the polymerase activities of the hepatitis B virus
iv, 30 p.
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Hepadnavirus polymerases initiate reverse transcription in a protein primed reaction that involves the covalent linkage of the first deoxyribonucleotide to the polymerase polypeptide. Analysis of the initial steps in this reaction as well as certain details of genome replication has been hampered by the difficulties encountered in the expression of functional hepadnavirus polymerase in the heterologous system. We have expressed human hepatits B virus (HBV) polymerase (pol) in insect cells, using the recombinant baculovirus system. The purified pol was also active in the invitro polymerase assay. Incubation of pol with labeled deoxyribonucleotide triphosphates resulted in the labeling of pol polypeptide in a reaction of the in vitro nucleotide priming. In vitro nucleotide priming was confirmed by the appearance of ^32P-labled phosphotyrosine on pol following in vitro reaction with ^32P-labled deoxyribonucleotide triphosphates. The ability to purify significant quantities of HBV pol will be facillitated with the functional and physical analysis of this enzyme as well as the search for novel inhibitors of HBV replication. Some herbal extracts as well as nucleoside analog, lamivudine, were tested for the inhibitory activities in the priming reaction of pol protein. In addition we previously described a transcomplementation assay for analysis of the roles of the TP and RT domains of HBV reverse transcriptase in the priming reaction. Independently expressed TP and RT domains form a functional complex for the in vitro priming reaction wheras TP protein expressed in E. coli was not functional in the protein-primed reaction with RT protein expressed in the baculovirus/Sf9 expression system. These studies suggest that the complex of protein-protein interaction is required in HBV genome replication. such cellular factors will be further identified by the transcomplementation assay for the nucleotide priming reaction.