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PMID: 7539509 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Nucleotide priming and reverse transcriptase activity of hepatitis B virus polymerase expressed in insect cells.

Journal of virology ·Vol. 69 ·No. 7 ·1995-07-00 ·Pages 4431-9

Lanford RE, Notvall L, Beames B

Abstract

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 polymerases in heterologous systems. We have expressed human hepatitis B virus (HBV) polymerase (pol) in insect cells, using the recombinant baculovirus system. Analysis of immunoaffinity-purified pol indicated that (i) a portion of pol had initiated minus-strand DNA synthesis within infected insect cells; (ii) the pol mRNA appeared to be the template for reverse transcription; (iii) the products were small (100 to 500 nucleotides); (iv) only minus-strand DNA was synthesized; (v) the products were covalently bound to protein; and (vi) the 5' end of the minus-strand DNA mapped to DR1 by primer extension. The purified pol was also active in an in vitro polymerase assay. Analyses suggested that a different fraction of pol was active in the in vitro assays. Incubation of pol with labeled deoxyribonucleotide triphosphates resulted in the labeling of the pol polypeptide in a reaction that appeared to represent in vitro nucleotide priming. In vitro nucleotide priming was confirmed by the appearance of 32P-labeled phosphotyrosine on pol following in vitro reactions with 32P-labeled deoxyribonucleotide triphosphates. The ability to purify significant quantities of HBV pol will facilitate functional and physical analysis of this enzyme as well as the search for novel inhibitors of HBV replication.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Cells, Cultured DNA, Viral/analysis,biosynthesis DNA-Directed DNA Polymerase/isolation & purification,metabolism Hepatitis B virus/enzymology,genetics Molecular Sequence Data Molecular Weight Nucleotides/metabolism RNA-Directed DNA Polymerase/metabolism Recombinant Proteins/metabolism Spodoptera
Chemicals
DNA, Viral Nucleotides Recombinant Proteins RNA-Directed DNA Polymerase DNA-Directed DNA Polymerase
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Lanford R E
Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas 78227, USA.
Notvall L
Beames B
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Article Info
Journal
Journal of virology
Abbr.
J Virol
ISSN
0022-538X
DOI
10.1128/JVI.69.7.4431-4439.1995
Published
1995-07-00
Pages
4431-9
Language
English
Region
United States
NLM ID
0113724
PMCID
PMC189185
Subset
IM
External Links
PubMed source DOI: 10.1128/JVI.69.7.4431-4439.1995 PMC Full Text View journal details
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