Identification oftheprimary site ofthehumanimmunodeficiency virus type1RNA dimerization invitro

Thediploid genomeofallretroviruses is madeoftwohomologous copies ofRNAintimately associated neartheir 5'end,inaregion called thedimerlinkage structure. Dimerizatlon ofgenomic RNA isthought tobe important forcrucial functions oftheretroviral life cycle (reverse transcription, translation, encapsidation). Previous invitro studies mappedthedimer linkage structure ofhuman immunodeficiency virus type1(HIV-1) inaregion down- stream ofthesplice donorsite, containing conserved purine tracts that werepostulated tomediate dimerization, through purine quartets. However, werecently showed thatdimer- ization ofHIV-1RNAalso involves sequences upstream ofthe splice donor site. Here, weusedchemical modification inter- ference toidentify nucleotides that arerequired inunmodi- fied formfordimerization ofaRNA fragment containing nucleotides 1-707ofHIV-1RNA.Thesenucleotides map exclusively inarestricted areaupstream ofthesplice donor site anddownstream oftheprimer binding site. Theyare centered arounda palindromic sequence (GUGCAC279) located inahairpin loop. Ourresults support amodelin whichdimerformation isinitiated bytheannealing ofthe palindromic sequences, possibly byaloop-oop interaction between thetwo, monomers. Further experiments showthat thedeletion ofthestein-loop orbase substitutions intheloop abolish dimerization, despite thepresence ofthepreviously postulated dimerlinkage structure. On theotherhand, deletions ofthepurine tracts downstream ofthesplice donor site donotprevent dimerization. Therefore, weconclude that thepalindromic region represents thedimerization Initiation site ofgenomic RNA.