Defective Replication UnitsofHepatitis B Virus

Thehepatitis Bvirus (HBV)genome isapartially doublestranded DNA circle withneither strand covalently closed. Thefull-length minusstrand isabout3.2kilobases andhas defined 5'and3'ends. Theplusstrand isincomplete owing tovariable 3'ends. Thecircular structure ofthegenome is maintained bycohesive overlaps atthe5'endsofthetwo strands. Thecohesive endregion isbracketed byan 11base-pair (bp)direct repeat, whichisassumed toplay arole inthereplication ofthegenome, whichinvolves reverse transcription ofa terminally redundant RNA pregenome. Thispregenome istranscribed froma completely doublestranded, covalently closed circular DNA whichisformed following entryofthegenome ofan infecting virus intothe nucleus ofthehostcell (7). Thenextstepsinthereplication oftheviral genome, i.e., theformation ofthelongandshort strands, areclosely associated withvirus particle formation. Transfection ofcells withhead-to-tail repeats ofcloned HBV DNA hasbeensuccessfully usedtoinitiate replicative processesleading tothetransient formation ofmaturevirus particles (1,3,16,21).Inthistypeofexperiment, the transfecting DNA substitutes forthecovalently closed circular HBV DNA. Inthis paperwe report on defective HBV DNA constructs whichserve as templates forthesynthesis ofdefective pregenomes.Various partsoftheHBV genome exceptfor thecohesive endregion withtheflanking 11-bpdirect repeats, DRIandDRII,were replaced bynonviral DNA. Deletions introduced inthis way constituted upto80%ofthe total viral DNA sequences.Included intothestudy was a dimerofa defective HBV DNA unitthathadbeenderived fromtheintegrated state inahepatocellular carcinoma. This unitcarried a deletion comprising theviral enhancer element.Following cotransfection ofcells withdefective and replication-competent dimeric wild-type (wt)DNA,defective pregenomesbecameincluded inthepoolofreplicating viral nucleic acids. Toidentify viral DNA sequenceswhicharedispensable forhelper-dependent replication, were constructed three defective HBV DNA replication unitsinwhichextended partsoftheHBV DNA genome were replaced bynonviral sequences.Thestructures oftheseplasmids, pddl,pdml, andpdd3, areoutlined inFig.1A,2A,and3A,respectively.