Structural investigation of HIV-1 genomic RNA dimerization process reveals a role for the Major Splice-site Donor stem loop.

ABSTRACT

The 5'UnTranslated Region (5'UTR) of HIV-1 genomic RNA, which precedes the Gag coding sequence, fulfills several roles during the lentivirus life cycle. This 335 nucleotides leader contains many stable structures that are crucial for the regulation of genetic expression at the level of transcription, splicing, and translation. In the late steps of the virus cycle, i.e. virions formation, the genomic RNA serves as propagated genome and its encapsidation in new particles relies on its ability to form non-covalent dimers. Dimerization is proposed to be initiated by the intermolecular pairing of a self-complementary sequence located in the apical loop of the DIS hairpin (Dimer Initiation Sequence). The regulation of this phenomenon and the extraordinary stability of the dimers imply that structural elements other than this kissing complex remain to be identified. Here, we show that swapping the Gag open reading frame (ORF) by reporter genes interferes with dimers formation efficiency. Importantly, the nature of the ORF alters specific structures of the 5'UTR. By using a systematic "SHAPE" approach, we pointed out that sequences within the Major Splice Site are involved in the dimerization process. Furthermore, by the use of an antisense oligonucleotide specific for the MSD associated to a SHAPE analysis of the 5'UTR structure, we demonstrated that interfering with the MSD results both in an impaired dimerization and in modifications of the 5'UTR structure. All together these data support a recently proposed model in which intramolecular base pairings are important determinants for the dimerization process. We further conclude that much care should be taken when comparing translation activity of reporter constructs with the viral situation.