IDENTIFICATION OF THE PRIMARY SITE OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA DIMERIZATION IN-VITRO

The diploid genome of all retroviruses is made of two homologous copie s of RNA intimately associated near their 5' end, in a region called t he dimer linkage structure. Dimerization of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (rever se transcription, translation, encapsidation). Previous in vitro studi es mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, contai ning conserved purine tracts that were postulated to mediate dimerizat ion, through purine quartets. However, we recently showed that dimeriz ation of HIV-1 RNA also involves sequences upstream of the splice dono r site. Here, we used chemical modification interference to identify n ucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides 1-707 of HIV-1 RNA. These nucleoti des map exclusively in a restricted area upstream of the splice donor site and downstream of the primer binding site. They are centered arou nd a palindromic sequence (GUGCAC279) located in a hairpin loop. Our r esults support a model in which dimer formation is initiated by the an nealing of the palindromic sequences, possibly by a loop-loop interact ion between the two monomers. Further experiments show that the deleti on of the stem-loop or base substitutions in the loop abolish dimeriza tion, despite the presence of the previously postulated dimer linkage structure. On the other hand, deletions of the purine tracts downstrea m of the splice donor site do not prevent dimerization. Therefore, we conclude that the palindromic region represents the dimerization initi ation site of genomic RNA.