GENERATION OF REPLICATION-COMPETENT HEPATITIS-B VIRUS NUCLEOCAPSIDS IN INSECT CELLS

The double-stranded DNA genome of human hepatitis B virus (HBV) and re lated hepadnaviruses is reverse transcribed from a pregenomic RNA by a viral polymerase (Pol) harboring both priming and RNA-and DNA-depende nt elongation activities, Although hepadnavirus replication occurs ins ide viral nucleocapsids, or cores, biochemical systems for analyzing t his reaction are currently limited to unencapsidated Pols expressed in heterologous systems, Here, we describe cis and trans classes of repl icative HBV cores, produced in the recombinant baculovirus system via coexpression of HBV core and Pol proteins from either a single RNA (i. e,, in cis) or two distinct RNAs (in trans), Upon isolation from insec t cells, cis and trans cores contained Pol-linked HBV minus-strand DNA with 5' ends mapping to the authentic elongation origin DR1 and also plus-strand DNA species, Only trans cores, however, were highly active for the de novo priming and reverse transcription of authentic HBV mi nus strands in in vitro endogenous polymerase assays, This reaction st rictly required HBV Pol but not the epsilon stem-loop element, althoug h the presence of one epsilon, or better, two epsilon s, enhanced minu s-strand synthesis up to 10-fold, Compared to unencapsidated Pol enzym es, encapsidated Pol appeared to be (i) highly processive, able to ext end minus-strand DNAs of 400 nucleotides from DR1 in vitro, and (ii) m ore active for HBV plus-strand synthesis. These observations suggest p ossible contributions to the replication process from the HBV core pro tein. These novel core reagents should facilitate the analysis of HBV replication in its natural environment, the interior of the capsid, an d also fuel the development of new anti-HBV drug screens.