A non-canonical base pair within the human immunodeficiency virus Rev-responsive element is involved in both Rev and small molecule recognition

Background: Human immunodeficiency virus (HIV) replication depends on the interaction of an HIV regulatory protein, Rev, with a viral RNA element (the Rev-responsive element, RRE). The high affinity RRE core region contains a non-canonical base pair (G48:G71) that is important for Rev recognition. Aminoglycoside antibiotics, specifically neomycin B, bind to the RRE and selectively block Rev-RRE interactions in vivo and in vitro. We attempted to generate an in vitro model for the establishment of HIV-1 resistance to neomycin B. Results: We have used in vitro genetic selection to evolve RRE variants that bind to Rev in the presence of neomycin B. Most of the RRE variants selected in the presence of 10 mu M neomycin B contain a G48:G71 to A48:A71 substitution. Those selected in 100 mu M neomycin B contain either C:A or A:A substitutions at this position. Binding constants for the interaction of neomycin B with the wild-type RRE and a subset of the selected RRE variants were determined using a novel ultrafiltration procedure. Conclusions: A purine-purine base pair within the bulge region of the RRE core element is critical for neomycin B binding as well as Rev binding. RRE variants that survive in high concentrations of neomycin do so either by binding Rev better than wild-type (this correlates with the sequence A48:A71) or by binding neomycin poorly (correlating with the sequence C48:A71). Other sequences must also influence both Rev and neomycin B binding.