In this study we show that growth of NL4-3 virus in the presence of either 3-amino-5-ethyl-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (103833) or 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile (104366) resulted in selection of resistant virus that had changes in the RRE. In each case, a single nucleotide mutation at the base of stem-loop IIC was sufficient to convey resistance, both to the compound used for selection and to the other compound.
Each of the resistance mutations changes an amino acid in the envelope protein and also allows the formation of an additional base pair at the bottom of RRE stem-loop IIC by substitution of a base either on the 5′ or 3′ side of the stem-loop. Once this base pair forms, it is likely that a G:U base pair directly below it would also form, leading to a lengthening of the stem by two additional base pairs. These structural changes in the RRE appear to be the cause of resistance since our experiments show that each mutated RRE functions better at lower levels of Rev than the wild-type RRE. The mutated RREs also mediate drug resistance in a reporter assay that is independent of the envelope protein. An additional experiment showed that mutations that destabilize the base of stem-loop IIC in the resistant mutants, without making additional changes in the envelope protein, restore sensitivity to the compounds in an infection.
It is interestingly that the RRE with the A7854G change appears to be more active than the one with the C7836U change although both add an additional base pair to the bottom of the stem in stem-loop IIC. Since A7854G forms a G-C base pair while C7836U forms an A-U base pair, it is possible that the increased activity results from the more stable stem structure that is formed.
Previous experiments suggested that the two compounds used in this study do not directly affect Rev-RRE binding since they do not inhibit binding in vitro
). Nevertheless, cells treated with the compounds clearly constitute an environment that compromises Rev function. Thus, any mutation that enables Rev to function better under these circumstances would be expected to cause resistance. In the case of the selected mutations, it would appear that the virus overcomes the block to Rev function by creating an RRE that works more efficiently when Rev levels are low. However, since we do not yet fully understand how the various parts of the RRE interact with themselves and Rev, the basis for this increased efficiency remains unexplained.
The stem-loop IIC region of the RRE that contains the resistance mutations has no known function, but it is immediately adjacent to the stem-loop IIB region of the RRE that functions as the primary Rev binding site (2
). The structure of a modified stem-loop IIB has been determined both alone and in a complex with a peptide derived from the Rev RNA binding domain (3
). Based on these structures, the binding of the initial Rev monomer is thought to involve recognition of a distinct binding pocket in stem-loop IIB that is formed by noncanonical G-G and G-A base pairs. As the structure of these complexes have been determined with only a minimal modified stem-loop IIB sequence and a Rev peptide, they give little insight into why mutations at the base of stem-loop IIC might affect RRE function. It is possible that this region of stem-loop IIC somehow influences the structure of stem-loop IIB and its interaction with Rev, or alternatively stem-loop IIC may interact directly with Rev, contributing to overall Rev function. In fact, recent studies have shown that Rev oligomerizes on the RRE by using different surfaces of its alpha-helical RNA-binding domain to recognize several low-affinity binding sites (13
). This oligomerization appears to involve interactions with the Rev protein dimer interface, as well as regions of the RRE that are distinct from the primary Rev binding site in stem-loop IIB (14
). The RRE resistance mutations that we describe here may increase the efficiency by which Rev is able to bind to such secondary RRE binding sites.
Several studies performed in vivo
using subgenomic reporter constructs have indicated that sequences throughout the RRE are important for function (16
) although others have concluded that stem-loop IIB is the only RRE region required for Rev function (32
). Additionally, studies using RNA aptamers, which have been selected for high binding affinity to Rev, have shown the lack of a strict correlation between the affinity of a primary binding sequence for Rev and its function in vivo
Several lines of evidence specifically point to the stem-loop V region of the RRE as a region important for Rev function although it is distant from stem-loop IIB in the RRE secondary structure model. For example, mutations predicted to disrupt the stem of stem-loop V are nonfunctional in both HIV-1 (17
) and HIV-2 (18
), and oligonucleotides complementary to the stem of stem-loop V are capable of completely disrupting preformed Rev-RRE complexes in vitro
). These oligonucleotides are also 9-fold more active in blocking Rev-RRE function in vivo
than oligonucleotides directed to stem-loop IIB (22
). Additionally, previous work from our own group has shown that single-nucleotide changes in stem-loop V of the RRE can overcome resistance to the transdominant negative rev
allele, RevM10 (27
), by causing a rearrangement of stem-loops III, IV, and V of the RRE into an alternative stable secondary structure (38
). Together, all of these studies, as well as ones presented here, highlight the notion that regions in the RRE outside the primary Rev binding site are important for Rev function. They suggest that the RRE is a dynamic structure with long-range interactions capable of structural rearrangements. Such interactions could be affected by the mutations we have identified.
Although the A7854G change in the RRE clearly mediated resistance in the context of NL4-3, we observed that other viruses which naturally possessed this polymorphism still were at least partially sensitive to the two inhibitors used in this study. In particular, we have previously shown that both compounds effectively inhibited Gag protein production, but not Nef, in U1 cells. The growth of the primary isolate 93BR021 in peripheral blood mononuclear cells (PBMCs) and Rev function in the dual luciferase cell line (DLRev) that was used for secondary screening (46
) were also affected. In each of these cases, the targeted virus had an RRE with a G at 7854 (NL4-3 numbering) as well as multiple other changes relative to NL4-3 over the length of the extended RRE (12/320 nt for 93BR021, 10/320 nt for the U1 cell virus, and 5/320 nt for the virus in the DL Rev cells) (data not shown).
In the present study, replication of virus derived from the HIV proviral clone R7/3 was shown to be inhibited by both compounds tested despite the fact that the R7/3 RRE contained a G at position 7854. The R7/3 RRE also has four additional changes relative to the NL4-3 RRE. This result, together with the observations discussed in the preceding paragraph, strongly suggest that the overall genetic background of any particular virus contributes to the net Rev function of the virus. Factors in the genetic background that are likely to have an influence include the intrinsic activity of the particular Rev allele expressed and the absolute and relative levels of expression of the various classes of mRNA expressed by a particular viral strain. Virtually any variation in the genetic background that would enable the virus to produce its Rev-dependent protein products more efficiently might be expected to be selected in the presence of the compounds since the compounds compromise Rev function and, as a consequence, essential Rev-dependent protein expression.
Growth of the R7/3 viral clone in the presence of compound 103833 led to the selection of two mutations in the envelope open reading frame that conveyed resistance. One of the mutations was synonymous for the envelope protein sequence but is rarely seen in the HIV sequences database (37
). The second mutation changed an amino acid found in R7/3 to the amino acid normally seen in the NL4-3 clone. Neither mutation on its own caused complete resistance although the synonymous change led to partial resistance. While further experiments are needed to understand why the combination of these two mutations cause resistance, the fact that these changes are outside either Rev or the RRE underscores the points discussed above and supports the hypothesis that net Rev function in any given virus is multifactorial. In this particular case, it is tempting to speculate that these changes are operating at the level of RNA structure to create an envelope or Gag-Pol mRNA that can be expressed more efficiently than the wild-type mRNAs in the presence of the inhibitory compound.