Vol. 189, No. 2, 1992 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION! December 15, 1992 Pages 1170-1171 DUCK HEPATITIS B VIRUS POLYMERASE PRODUCED BY IN VITRO TRANSCRIPTION AND TRANSLATION POSSESSES DNA POLYMERASE AND REVERSE TRANSCRIPTASE ACTIVITIES Anita Y.M. Howe, John F. Elliott and David L.J. Tyrrell Department of Medical Microbiology and Infectious Diseases 1-41 Medical Sciences Building, University of Alberta Edmonton. Canada T6G 2H7 Received November 2, 1992 Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promotor. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35 S-Methionine labelled 79 Kd band on SDS - polyacrylamide gel electrophoresis.The translation product showed DNA polymerase and reverse uanscriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases. 0 1992 Academic Press, Inc. Hepatitis B virus (HBV) is a partially double-stranded DNA virus with a genome size of about 3200 nucleotides. Persistent infection with this virus is often associated with hepatitis and progression to hepatocellular carcinoma. The life cycle of Hepatitis B virus can be roughly divided into four stages: (i) attachment of a virion to the cell surface, followed by uncoating and transport of the genomic DNA into the nucleus (1); (ii) formation of a closed circular DNA (ccc DNA) and its transcription into viral RNAs in the nucleus (1); (iii) formation of replicative complexes followed by progeny DNA replication in the cytoplasm (2); and (iv) assembly into mature virions, or transport of the replicative complexes into the nucleus for amplification of the cccDNA (3). Hepatitis B virus DNA synthesis begins with the replication of a minus strand DNA from a pregenomic RNA template. This process is mediated by the virus encoded polymerase which is a multifunctional protein. Studies on the functions of the polymerase 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved. 1170