Sequences involved in the dimerisation of human T cell leukaemia virus type-1 RNA

Abstract

The formation of a genomic RNA dimer appears to be a critical step in the life cycle of all retroviruses. To investigate the site and nucleotide interactions involved in this process, a 531 bp DNA fragment encompassing sequences up- and downstream of the splice donor in human T cell leukaemia virus type 1 (HTLV-1) was inserted into a plasmid vector under the control of the SP6 promoter. RNA transcripts generated in vitro from this template formed dimers which could be dissociated by heating at 60-80°C for 3 min. The physical properties of the dimeric RNA were not consistent with either Watson-Crick base pairing or guanine tetrad formation as being solely responsible for the interaction. Deletion mutagenesis identified a 32 nt sequence required for dimerisation. Computer modelling was carried out in order to identify putative RNA secondary structures within this essential region. A stem-loop structure was identified, the stem of which was conserved among different sequenced isolates of HTLV-1. This sequence also contains a 15 nt palindrome. We sought by disruptive and compensatory mutagenesis to define the possible roles of these two structures in dimer linkage.