Selection and Characterization of Human Immunodeficiency Virus Type 1 Variants Resistant to Compounds that Inhibit Rev-RRE Function

The Human Immunodeficiency Virus Type-1 (HIV-1) uses the RevRRE pathway to export its genome and mRNAs containing introns. The interaction between the Rev protein and the Rev Responsive Element (RRE) presents a target for anti-HIV therapeutics. We studied the RevRRE pathway by utilizing small heterocyclic compounds shown in previous research to inhibit Rev function. By identifying and characterizing mutations in resistant NL4-3 variants, we were able to propose and test resistance mechanisms. Analysis of the resistant variants revealed mutations in the RRE. These mutations caused structural changes in RRE stem loop IIC, and also changed the amino acid sequence of gp41. To separate the possibilities contributing to resistance, we utilized site mutagenesis to create point mutations that conserved the amino acid sequence conferred by the resistance mutations, but disrupted the structure of the RRE. The disrupted RREs caused the viruses to become sensitive to the compounds. Therefore the cause of the resistance was the structural changes in the RRE, not the changes in gp41. We also tested the wild type and mutant RREs in transient transfections both in provirus and pCMVGagPol constructs. These experiments indicated that the mutant RREs function at lower Rev levels than the wild type RRE. We then performed selection studies using a HXB2 derivative virus, R73, with a differing genetic background from NL4-3. Instead of RRE mutations, we recovered changes in gp120 Env that conferred resistance. 3 Further selection studies with NL4-3 using both enfuvirtide (T-20), and 103833 or 104366, created variants with changes in RRE stem loop IIC and stem loop V. The change in stem loop IIC, one of our previously identified Rev-RRE compound resistance mutations, did not confer T-20 resistance. A silent mutation in stem loop V caused T-20 resistance. We hypothesized this altered the expression of the envelope message, overcoming the competitive inhibitor T-20 at the tested concentration. Together the data suggest that different HIV-1 viruses function with optimized Rev levels for interaction with their RRE; and that they are able to adapt Rev-RRE interaction to maintain viral replication. Also additional mutations in vpu or env can contribute to resistance through separate mechanisms.

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