Background:  The rev response element (RRE) contains the gp41 region (amino acids 36 to 45) in which enfuvirtide (T-20) -resistance mutations are selected. Therefore, the RRE structure and RRE-rev interactions, which are critical in the HIV replication cycle, might be influenced by T-20-resistance changes. The genetic variability of RRE and rev sequences was examined in patients harbouring different patterns of T-20-resistance mutations.

Methods:  We identified 10 patients who began T-20 salvage therapy and subsequently showed virological failure. Clonal analysis of gp41 was performed on a total of 23 plasma samples collected on longitudinal follow-up (range, 8 to 264 weeks on T-20). Sequences generated were analyzed for both the RRE and the gp41 open reading frame. RRE secondary structures were predicted using the RNA-fold program. The rev protein (exons 1 and 2) was sequenced at baseline and upon selection of specific T-20-resistance mutations.

Results:  A total of 421 clones were generated from 23 samples harboring distinct T-20-mutation patterns:  1 G36V, 1 G36V+L44V, 3 G36D+L44V, 1 G36D+L40V, 1 V38E, 1 V38A/V+N43D/N, 1 Q39H+N43D, 1 Q39R+N43D, 1 Q40H, 2 N42T+N43K, 2 N42K+N43D, 2 N43D+Q40H, 4 N43D, 1 N43D+L44M, and 1 L45W. Selection of double mutations was observed in 6 patients during weeks 16 and 212 of T-20 therapy. Mutations at codons 36, 38, and 43 were always present in separate clones. Conversely, changes Q39H/R, N42T/K, or L44M/V always appeared together with mutations at positions 36, 38, or 43. The RRE motif 5’-CACTATGGG-3’, known as the “high affinity rev binding site,” was highly conserved in all clones. The predicted RRE secondary structure showed a minimal effect of T-20-resistance mutations at positions 36, 39, 40, 42, 43, or 44 on the critical region for RRE-rev interactions. However, mutations V38A and V38E significantly altered the secondary structure of the “high affinity rev binding site.” The rev protein was examined in 24 samples before and upon selection of T-20-resistance changes. No evolution of amino acid substitutions was observed, even in patients harboring changes at codon 38 in gp41. 

Conclusions:  The “high affinity rev binding site” of RRE is highly conserved in T-20-resistant clones. V38A and V38E mutations are predicted to alter the critical region for RRE-rev interactions. T-20-resistance mutations do not seem to have any effect on the rev protein.