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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Generation of replication-competent hepatitis B virus nucleocapsids in insect cells.

The double-stranded DNA genome of human hepatitis B virus (HBV) and related hepadnaviruses is reverse transcribed from a pregenomic RNA by a viral polymerase ( Pol) harboring both priming and RNA- and DNA-dependent elongation activities. Although hepadnavirus replication occurs inside viral nucleocapsids, or cores, biochemical systems for analyzing this reaction are currently limited to unencapsidated Pols expressed in heterologous systems. Here, we describe cis and trans classes of replicative 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 insect 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 minus strands in in vitro endogenous polymerase assays. This reaction strictly required HBV Pol but not the epsilon stem-loop element, although the presence of one epsilon, or better, two epsilons, enhanced minus-strand synthesis up to 10-fold. Compared to unencapsidated Pol enzymes, encapsidated Pol appeared to be (i) highly processive, able to extend minus-strand DNAs of 400 nucleotides from DR1 in vitro, and (ii) more active for HBV plus-strand synthesis. These observations suggest possible contributions to the replication process from the HBV core protein. These novel core reagents should facilitate the analysis of HBV replication in its natural environment, the interior of the capsid, and also fuel the development of new anti-HBV drug screens.[1]

References

  1. Generation of replication-competent hepatitis B virus nucleocapsids in insect cells. Seifer, M., Hamatake, R., Bifano, M., Standring, D.N. J. Virol. (1998) [Pubmed]
 
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