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. 2008 Sep 23;105(38):14365-70.
doi: 10.1073/pnas.0804461105. Epub 2008 Sep 5.

Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element

Michal Legiewicz  1 , Christopher S Badorrek, Kevin B Turner, Daniele Fabris, Tiffany E Hamm, David Rekosh, Marie-Louise Hammarskjöld, Stuart F J Le Grice
Affiliations

Resistance to RevM10 inhibition reflects a conformational switch in the HIV-1 Rev response element

Michal Legiewicz et al. Proc Natl Acad Sci U S A. .

Abstract

Nuclear export of certain HIV-1 mRNAs requires an interaction between the viral Rev protein and the Rev response element (RRE), a structured element located in the Env region of its RNA genome. This interaction is an attractive target for both drug design and gene therapy, exemplified by RevM10, a transdominant negative protein that, when introduced into host cells, disrupts viral mRNA export. However, two silent G->A mutations in the RRE (RRE61) confer RevM10 resistance, which prompted us to examine RRE structure using a novel chemical probing strategy. Variations in region III/IV/V of mutant RNAs suggest a stepwise rearrangement to RevM10 resistance. Mass spectrometry was used to directly assess Rev "loading" onto RRE and its variants, indicating that this is unaffected by RNA structural changes. Similarity in chemical footprints with mutant protein implicates additional host factors in RevM10 resistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NMIA footprinting of the WT HIV-1 RRE and RRE61. (A) 2′-O adduct formation for region III/IV/V of each RNA. (B) Histograms depicting absolute SHAPE reactivities. The shaded region indicates where the RNAs differ in NMIA reactivity. Positions of high (>21%) reactivity are in red, those of moderate (10–21%) reactivity are in green, those of low (5–10%) reactivity are in blue, and those of nonreactive (<5%) reactivity are in black. Asterisks indicates where NMIA reactivity was affected by background.
Fig. 2.
Fig. 2.
Secondary structure model for the RRE RNA. The structure is shown with color-coded nucleotides defining NMIA reactivity as indicated in the insert and is consistent with Fig. 1 coding. The G:A164 and G:A245 mutations are boxed.
Fig. 3.
Fig. 3.
Secondary structure model for the RRE61 RNA. Color coding of nucleotide reactivity follows the legend to Fig. 2. The two G:A mutations are now incorporated into the structure and are highlighted with open circles.
Fig. 4.
Fig. 4.
Replication of HIV with altered RREs in CEM/14M10B cells. Supernatants from 293 T cells transfected with NL4–3 proviral clones containing WT or variant RREs were used to infect CEM/14M10B cells. Replication was determined by measuring supernatant RT activity. Input virus was standardized using the RT activity of the supernatant. Data are from two separate experiments.
Fig. 5.
Fig. 5.
SHAPE analysis of RREs with individual G:A245 and G:A164 mutations. (A and B) Quantitative comparison step plots of RRE61 vs. G:A245 and RRE vs. G:A164, respectively. (C and D) Secondary structures of region III/IV/V of the G:A245 and G:A164 mutants, respectively. NMIA reactivity is color-coded as in Fig. 2 and 3.
Fig. 6.
Fig. 6.
Effects of Rev binding on the native and RRE61 RNAs. (A and B) ESI-MS analysis of RRE and RRE61, respectively. (C and D) NMIA footprinting. Nucleotides that are constrained or flexible on Rev binding are indicated by open and closed circles, respectively. The shaded box is the primary Rev binding site.
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