Synthesis ofinfectious poliovirus RNA bypurified T7 RNA polymerase

Plasmids containing theentire cDNA se- quence ofpoliovirus type1(Mahoney strain) undercontrol of apromoter forT7RNA polymerase havebeenconstructed. Purified T7RNApolymerase efficiently transcribes theentire poliovirus cDNAineither direction toproduce full-length poliovirus RNA ((+)RNA) oritscomplement ((-)RNA). The (+)RNAproduced initially had60nucleotides onthe5'side of thepoliovirus RNAsequence, including astring of18consec- utive guanine residues generated intheoriginal cloning andan additional 626nucleotides ofpBR322sequence beyond the poly(A) tract atthe3'end.SuchRNA,while muchmore infectious thantheplasmid DNA,isonlyabout0.1%as infectious asRNAisolated fromthevirus. Subsequently, aT7 promoter wasplaced only2basepairs ahead ofthepoliovirus sequence, sothatT7RNA polymerase synthesizes poliovirus RNAwithonly 2additional guanine residues atthe5'endand nomorethanseven nucleotides past thepoly(A) tract atthe3' end.SuchRNAhasmuchhigher specific infectivity, about 5% thatofRNA isolated fromthevirus. Theability tomake infectious poliovirus RNAefficiently fromcloned DNAmakes itpossible toapply techniques ofinvitro mutagenesis tothe analysis ofpoliovirus functions andtheconstruction ofnovel andperhaps useful derivatives ofpoliovirus. A source of variant RNAsshould also allow detailed study ofthesynthesis andprocessing ofpoliovirus proteins invitro. RNA isolated frompoliovirus caninitiate infection ofcul- tured mammalian cells (1). Virion RNAnormally hasasmall protein, VPg,covalently attached toits 5'end, butVPgisnot needed forinfectivity ofpolio RNA (2,3).Unfortunately, techniques thatwouldallow invitro mutagenesis oflarge RNA molecules without introducing lethal damage arenot welldeveloped, andithasnotbeenpossible togenerate useful variants ofpoliovirus bychemical alteration ofthe virion RNA.Recently, however, complete cDNAclones of poliovirus type1RNA havebeenmade(4-6), andtheDNA ofsuchclones isinfectious. Thismakesitpossible toapply thewidevariety oftechniques available forinvitro mutagenesis ofDNA tothestudy ofpoliovirus. Themechanism by whichcells produceinfectious poliovirus fromcDNAclones isnotknown. Presumably, the cloned DNA istranscribed inthecell nucleus toproduce RNAsthataretransported tothecytoplasm, wherethey serveasmRNAs forprotein synthesis andtemplate for replication. However, during anormal infection poliovirus RNA doesnotenter thenucleus andisnotspliced, butit carries adventitious splicing andpolyadenylylation signals that could berecognized inthenuclear transcripts produced fromthecDNA(R.J.KuhnandE.W.,unpublished results). Splicing orpolyadenylylation atsuchsites might beexpected toinactivate mostofthenuclear transcripts. Furthermore, thepoliovirus RNAproduced fromavailable cDNAvectors would beembedded inlonger primary transcripts that might havetobeprocessed toproduce biologically active mole- cules. Therefore, itisnotsurprising thattheefficiency of infection bycDNAclones isverylow.Theefficiency canbe increased byuptothree orders ofmagnitude iftheplasmid iscapable ofreplicating (5), butatbest itremains twoorthree orders ofmagnitude lower thanthat ofvirion RNA. Ifinfectious RNAcould beproduced bytranscription ofa polio cDNAclone invitro, theeaseofmanipulation ofDNA could becombined with theefficiency ofinfection ofRNAto systematically explore thebiological function ofthepoliovi- rusgenome invivo andinvitro. T7RNApolymerase isideal forthis because itishighly selective forits ownpromoters (7), itefficiently makescomplete transcripts ofverylong DNAs fromavariety ofsources (unpublished results), anditiseasily prepared fromthecloned gene(8). Wehavenowplaced the polio cDNAunder control ofaT7promoter andhaveshown that purified T7RNApolymerase canproduce large amounts ofinfectious poliovirus RNA.A similar approach, using Escherichia coli RNA polymerase, hasbeensuccessfully applied tobromemosaic virus byAlquist etal.(9).