Oligonucleotide mapping of the core genomic RNA dimer linkage in human T-cell leukaemia virus type-1

Abstract

We have previously mapped the sequences required for dimerisation of the 5′ leader of the human T-cell leukaemia virus type-1 (HTLV-1) genome. The smallest sequence necessary and sufficient for dimer formation, in vitro, was ascertained to be a 37 nucleotide (nt) region downstream of the splice donor and just upstream of the primer binding site. Deletion of a 32 base-pair sequence encompassing this region within the provirus was associated with a minor decrease in infectivity of the virus in an in vitro system. To further map and help elucidate the nature of the dimer linkage, we used RNA and DNA oligonucleotide competition assays to define the nucleotides involved. These experiments revealed that a 14 nt sequence containing a potential stem loop structure, formed from a palindromic sequence, is important for dimer formation. This was confirmed by the ability of this RNA sequence to form heterodimers with larger RNA transcripts from the same region, while sequences lacking this motif could not. RNA transcripts containing the reverse sequence, the same nucleotides in a random arrangement, and complementary DNA oligos, all failed to form heterodimers with the 14 nt sequence. The primary dimer initiation site of HTLV-1 has thus been located to a 14 nt palindrome containing sequence, and dimerisation is shown to be dependent on specific sense–sense RNA interactions.