Previously we described the identification of two compounds (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b] pyridine-2-carboxamide [103833] and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile[104366]) that interfered with HIV replication through the inhibition of Rev function. We now describe resistant viral variants that arose after drug selection, using virus derived from two different HIV proviral clones, NL4-3 and R7/3. With HIV NL4-3 , each compound selected a different single point mutation in the Rev response element (RRE) at the bottom of stem-loop IIC. Either mutation led to the lengthening of the stem-loop IIC stem by an additional base pair, creating an RRE that was more responsive to lower concentrations of Rev than the wild type. Surprisingly, wild-type HIV R7/3 was also found to be inhibited when tested with these compounds, in spite of the fact this virus already has an RNA stem-loop IIC similar to the one in the resistant NL4-3 variant. When drug resistance was selected in HIV R7/3 , a virus arose with two nucleotide changes that mapped to the envelope region outside the RRE. One of these nucleotide changes was synonymous with respect to env, and one was not. The combination of both nucleotide changes appeared to be necessary for the resistance phenotype as the individual point mutations by themselves did not convey resistance. Thus, although drug-resistant variants can be generated with both viral strains, the underlying mechanism is clearly different. These results highlight that minor nucleotide changes in HIV RNA, outside the primary Rev binding site, can significantly alter the efficiency of the Rev/RRE pathway.
Cells infected with human immunodeficiency virus (HIV)produce stable species of unspliced and incompletely spliced mRNAs that require the Rev protein for their nuclear-cytoplasmic export and expression (for a review, see reference 29). Rev is a small basic protein of about 116 amino acids that is encoded in the HIV genome (for a review, see reference 44). One well-defined function of Rev in HIV infection is to bind (7,10,11,30,31,41,53) to the Rev response element (RRE) (15,26,45) that is found in these unspliced and incompletely spliced mRNAs and promote their nucleo-cytoplasmic export (21,28,40) by interacting with the Crm1 cellular export receptor and Ran-GTP (1).Many previous studies identified stem-loop IIB of the RRE as the primary binding site for a Rev protein monomer and suggested that 6 to 10 subunits of Rev can subsequently bind to the RRE (2, 9, 32, 33, 36, 50). Oligomerization of Rev requires the Rev multimerization domain that has also been shown to be necessary for Rev function (12,34,39,42,54). Additionally, the molecular details of the interaction of the Rev monomer and its primary binding site have been well characterized through the use of an isolated Rev peptide containing only the arginine-rich binding motif (ARM) and the stem IIB RNA hairpin (3,4,48,49).Recently, Rev has been shown to form a cooperative highaffinity oligomeric complex with the RRE (13)....