Genetic consequences ofpackaging twoRNA genomesinone retroviral particle: Pseudodiploidy andhighrateof genetic recombination

Retroviruses contain twocomplete viral ge- nomic RNAsineachvirion. Asystem tostudy inasingle round ofreplication theproducts ofvirions with twodifferent genomic RNAswasestablished. Aspleen necrosis virus-based splicing vector containing boththeneomycin-resistance gene(neo) and thehygromycin Bphosphotransferase gene(hygro) wasused. Twoframeshift mutants werederived fromthis vector such that theneoandthehygro genes wereinactivated inseparate vectors. Thus,eachvector confers resistance toonlyone selection. Thevectors withframeshift mutations weresepa- rately propagated andwerepooled toinfect DSDhhelper cells. Doubly resistant cell clones wereisolated, andviruses produced fromthese clones wereusedtoinfect D17cells. Thisprotocol allowed virions containing twodifferent genomic RNAs(het- erozygotes) tocomplete oneround ofretroviral replication. The molecular nature ofprogeny that conferred resistance tosingle ordouble selection andtheir ratio weredetermined. Ourdata demonstrate that eachinfectious heterozygous virion produces only oneprovirus. Therate ofretroviral recombination is=2% perkilobase perreplication cycle. Recombinant proviruses are progeny ofheterozygous virions. Theretroviral life cycle requires DNAmolecules tobecopied fromviral RNAandtointegrate into thehost genome toform theprovirus (1). However, aunique feature ofretroviruses is that twoRNAgenomes arepackaged inonevirion (2-7). It hasbeensuggested that oneprovirus isformed fromthetwo