Single-Nucleotide Changes in the HIV Rev-Response Element Mediate Resistance to Compounds That Inhibit Rev Function

Abstract

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)....

Limited Nucleotide Changes in the Rev Response Element (RRE) during HIV-1 Infection Alter Overall Rev-RRE Activity and Rev Multimerization

“…Interestingly, the nucleotide changes that occurred between these RRE molecules were located outside the primary Rev binding domain, suggesting that other regions of the RRE contribute to the overall phenotype. These findings are consistent with previous experiments using laboratory-adapted viral clones, where it was demonstrated that one or two nucleotide changes in the RRE could dramatically alter both its structure and activity (23,24). It has now been established that regions of the RRE distant from the primary binding site play a role in multimerization and in modulating the affinity of the primary binding event (22,51).…”

“…Previous studies have shown that the structure and activity of the RRE are sensitive to minimal nucleotide changes (23,24). There- fore, it was of interest to analyze the individual activities of the different patient RREs to determine what role, if any, RRE sequence variability played in mediating Rev-RRE activity differences between patients and time points.…”

“…Some have demonstrated that point mutations in the RRE, outside the primary Rev binding site, can alter the overall RRE structure and have functional effects (22)(23)(24). Similarly, mutations in the Rev coding sequence outside known functional domains can influence the activity (25,26).…”

Identification and Optimization of Thienopyridine Carboxamides as Inhibitors of HIV Regulatory Complexes

“…S5), that the thienopyridine compound 4a specifically targets the Rev-RRE reporter but not the HIV Tat-TAR reporter. The previous screen did not use the HIV LTR, and these investigators further uncovered a thienopyridine resistance mutation in the RRE, consistent with the Rev-RRE interaction as the target of thienopyridines (40). We have also generated this RRE-defective virus and shown that it is resistant to compound 4e (Fig.…”

Importance of codon usage for the temporal regulation of viral gene expression

“…Rev inducibility was lost when 10.5% of the codons were replaced with synonymous codons reflecting the codon usage of RRV gH. While it had been previously known that Rev induction depended on the Rev response element (the RRE) and the presence of a splice donor upstream of the coding sequence (11,18), the dependence on the nature of the skewed codon usage had not been previously known, although publications by Graf et al (14) and Shuck-Lee et al (19) are consistent with it. Similarly, our data show that ORF57 induction of RRV gH protein expression also depends on the nature of the skewed codon use.…”