FIG. 3.

Analysis of HBV DNA products generated in in vitro polymerase reactions. (A) Agarose gel analysis of endogenously radiolabeled DNA products from nucleocapsids. Sf9 cells were harvested 90 h postinfection with the recombinant baculoviruses ECPE, EC+PE, EC, and PE. 2.2.15 cells were collected after 10 days of culture. Core particles were immunoprecipitated from clarified cell extracts and subjected to EPAs as described in Materials and Methods. Samples of isolated nucleic acids were analyzed on a 1% agarose gel and then visualized by autoradiography. The approximate size range (in kilobase pairs) of the various products is indicated on the left. (B) SDS-PAGE analysis of Pol-DNA complexes after in vitro polymerase reactions. HBV nucleocapsids were generated either in trans (lanes 1 to 4) or in cis (lanes 5 to 8) by infection of Sf9 insect cells with the baculovirus constructs indicated above each lane. The immunoprecipitated capsids were subjected to EPA and then boiled. Following radioimmunoprecipitation, the covalently linked minus-strand DNA-Pol complexes were examined by SDS-PAGE and autoradiography as described in Materials and Methods. The Pol protein band is indicated on the right; marker proteins (sizes in kilodaltons) are indicated on the left.