Functional Analyses Reveal Extensive RRE Plasticity in Primary HIV-1 Sequences Selected under Selective Pressure

<div><p>Background</p><p>HIV-1 Rev response element (RRE) is a functional region of viral RNA lying immediately downstream to the junction of gp120 and gp41 in the <i>env</i> coding sequence. The RRE is essential for HIV replication and binds with the Rev protein to facilitate the export of viral mRNA from nucleus to cytoplasm. It has been suggested that changes in the predicted secondary structure of primary RRE sequences impact the function of the RREs; however, functional assays have not yet been performed. The aim of this study was to characterize the genetic, structural and functional variation in the RRE primary sequences selected <i>in vivo</i> by Enfuvirtide pressure.</p><p>Results</p><p>Multiple RRE variants were obtained from viruses isolated from patients who failed an Enfuvirtide-containing regimen. Different alterations were observed in the predicted RRE secondary structures, with the abrogation of the primary Rev binding site in one of the variants. In spite of this, most of the RRE variants were able to bind Rev and promote the cytoplasmic export of the viral mRNAs with equivalent efficiency in a cell-based assay. Only RRE45 and RRE40-45 showed an impaired ability to bind Rev in a gel-shift binding assay. Unexpectedly, this impairment was not reflected in functional capacity when RNA export was evaluated using a reporter assay, or during virus replication in lymphoid cells, suggesting that <i>in vivo</i> the RRE would be highly malleable.</p><p>Conclusions</p><p>The Rev-RRE functionality is unaffected in RRE variants selected in patients failing an ENF-containing regimen. Our data show that the current understanding of the Rev-RRE complex structure does not suffice and fails to rationally predict the function of naturally occurring RRE mutants. Therefore, this data should be taken into account in the development of antiviral agents that target the RRE-Rev complex.</p></div

Functional analyses of RRE with changes at positions 40 and 45 in lymphoid cells.

<p>The lymphoid cell line MT-4 was co-electroporated with the NL4-3 hemigenomic plasmids p83-2 and with the p83-10 constructed variants, which only differed in their RRE region. A) Fold increase of cytosolic unspliced RNA in infected cells. Levels of unspliced RNA in the cytoplasmic fractions were determined by qRT-PCR. The fold change was calculated by the relative quantitation method 2(^−ΔΔCt). GAPDH was used for normalization and RREWT (40Q-45L) as a calibrator. Data (mean +/− SEM) is derived from three independent experiments, with triplicate samples in each PCR. B) Quantification of the transcriptional activity per infected cell. The ratio between the levels of unspliced HIV RNA and total HIV DNA (2(^−ΔΔCt) Unspliced HIV-1 RNA/2(^−ΔΔCt) Total HIV-1 DNA) was calculated. Total HIV DNA content was determined in cells by qPCR using the same approach as described with the unspliced RNA levels. Data (mean +/− SEM) is derived from three independent experiments, with triplicate samples in each PCR. C) P24 protein production in cell-free supernatants. The p24 present in the supernatant of the cultures was quantified by ELISA from the same time-point that the RNA and DNA levels were determined. Data (mean +/− SEM) is derived from two independent experiments. D) Normalized p24 protein production. Raw p24 values were normalized to copies of total HIV-1 DNA to correct for differences in electroporation efficiency and gp41 function. Data (mean +/− SEM) is derived from two independent experiments.</p

<i>In vitro</i> Rev-RRE binding assay.

<p>Electro-mobility shift assay (EMSA) in the absence or in the presence of 20 ng, 40 ng or 80 ng of Rev protein with the different RRE RNA variants (RREWT, sRRE40, sRRE45 and RRE40-45). The reaction products were separated in a polyacrylamide gel and the quantification of the gel shifts are displayed.</p

Predicted secondary structures of RRE variants constructed by site-directed mutagenesis.

<p>RRE variants with nucleotide substitutions at position 40 sRRE40 (Q40H), at position 45 sRRE45 (L45M) or without any, WT (40Q-45L), were generated by site-directed mutagenesis using as a template a RRE40-45 clone (Q40H-L45M). The sequences of these constructed clones were subjected to RNA fold analyses as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106299#pone-0106299-g002" target="_blank">Figure 2</a>. Nucleotides that were substituted are highlighted in the filled red circles.</p

Alignment of patient-derived RRE variants used for functional analysis.

<p>Multiple nucleotide alignment of the full-length patient-derived RRE variants used for prediction of secondary structures and functional analyses. Several full-length RREs derived from two patients (patient 10: P10; and patient 5: P5) were amplified from plasma samples, cloned and sequenced. Boxes highlight the nucleotides coding ENF resistance mutations G36V/D, V38A and N43D for patient 10; and Q40H and L45M for patient 5. The characteristic five stem-loop regions of the RRE are identified on top of the sequence. Variants were designated according to the patient number, amino acid change in gp41 and clone number. The shaded areas indicate the reported high-affinity binding site of Rev located in the stem II. The NL4-3 RRE sequence is included for comparison.</p

Predicted secondary structures of patient-derived RRE variants.

<p>The prediction of the secondary structure of our RRE sequences was generated including the 329-nucleotide sequence of the patient-derived RREs in the RNA Fold Web server. The five well-defined stem-loop structures, including the branched stem-loop IIB that is critical for the binding of the Rev protein, were identified for most of the sequences. A) Predicted secondary structure of RRE from patient 10. Predicted secondary structure of the complete RRE of a BL clone from patient 10 is shown. The nucleotides encoding the amino acid changes associated with ENF resistance are underlined and a dotted box encloses the high-affinity Rev binding site. Stems loops II-III-IV and V for representative samples for each substitution present in Patent 10 are shown. Nucleotide changes present in the RRE variants with regard to the BL clone are marked with filled red circles. B) Representative RRE structures obtained from samples of patient 5.</p

Rev-dependent RNA transport.

<p>293T cells were co-transfected with the constructed RRE variants, pDM628 or pDM628ΔRRE with or without pCMV-Rev. The export levels of the different variants were quantified by luminescence and corrected by the background signal from the luminometer noise or due to Rev-independent transport. This correction was performed to each sample by subtracting the luminescence that was measured when the cells were transfected without Rev (replaced with pcDNA 3.0). And finally, corrected luminescence values were calculated as the fold-change increase, which was performed by dividing the corrected luminescence of each plasmid by the corrected luminescence of the pDM628 plasmid. A) Rev-RRE mediated export from RNA variants containing changes at positions 36, 38 and 43, evaluated in the presence of Rev (ratio 1:5, Rev:RRE). B) Cytoplasmic export of RRE variants with changes at positions 40 and 45. Rev-dependent transport of the pDM628-based RRE variants: WT (40Q-45L), sRRE40 (Q40H), sRRE45 (L45M) and the double mutant RRE40-45 (Q40H-L45M); in the presence of three different concentrations of pCMV-Rev (200 ng, 20 ng or 2 ng per well). Data represent the mean +/− SEM of 3 independent experiments performed in triplicate transfections.</p

Additional file 1: of Increased ex vivo cell death of central memory CD4 T cells in treated HIV infected individuals with unsatisfactory immune recovery

Figure S1. Gating strategy followed to identify T-cell maturation stages. Panel A. CD4 and CD8 T cells were gated and analyzed for the expression of CD27 and CD28. For CD4 T cells, terminally differentiated (TTD) cells were defined as CD28–CD27−, while effector memory (TEM) cells were CD28+CD27−. Double positive cells were further analyzed for CCR7 and CD45RA expression to identify naïve (TN) cells (CCR7+CD45RA+), central memory (TCM) cells (CCR7+CD45RA−) and transitional Memory (TTM) cells (CCR7–CD45RA−). Panel B. For CD8 T cells, the general strategy was similar, unless for the definition of TEM cells that was CD27+CD28– cells. The expression of CD57 was analyzed in the whole CD4 or CD8 T-cell population (lower left dot plot in each panel) to define replicative senescence (CD28–CD57+ cells). In addition the expression of CD57 in each subset was also evaluated (lower dot plots in each panels)

Additional file 3: of Increased ex vivo cell death of central memory CD4 T cells in treated HIV infected individuals with unsatisfactory immune recovery

Figure S2. A model for CD4 homeostasis in Immunodiscordant individuals. Immunoconcordant individuals show a full recovery of CD4 T-cell counts with a high representation of naïve cells, and balanced frequencies of different memory subsets (upper panels). The profile of CD4 T-cell maturation in immunoconcordant individuals (green line) overlaps with that of HIV uninfected individuals (blue line in upper right plot) in which CCR7− CD4 T cells show increased turnover and short live [22]. Conversely,immunodiscordant individuals (lower panels) show a reduced naïve subset with no signs of altered turnover [14] that limits the generation of new memory cells. Among memory subsets, central memory cells are also reduced and subjected to homeostatic pressure to generate new cells [14] increasing TCM→TTM transition (and lowering TCM/TTM ratios) and increasing TCM cell death. TTM and TEM cells emerging from TCM cells also show higher sensitivity to cell death (Figure 2D), explaining the lack of accumulation of terminally differentiated cells in these subjects. This scenario results in a shifted profile of CD4 T-cell maturation (red line) compared to healthy individuals (blue line in lower right plot)

Analysis of the NKp44L induction by cloned envelopes.

<p>(A) Graphic representation of sequence variability in gp41 regions surrounding the 3S epitope. The picture has been generated using the weblogo software (<a href="http://weblogo.berkeley.edu/" target="_blank">http://weblogo.berkeley.edu/</a>), representing polar amino acids in red, basic in blue, acidic in green and hydrophobic in black. (B) NKp44L expression in CD4 T cells from representative donors after incubation with Env-defective pseudoviruses (pSG3, negative control), with the 3S consensus peptide V, or with pseudoviruses bearing the following HIV Env: NL4-3, BaL (positive controls). The effect of selected Env clones from VNPs (upper panels) or RPs (lower panels is shown) Empty peaks correspond to control staining of untreated CD4 T cells. (C) The effect of a polyclonal anti 3S antibody on NKp44L expression induced by several Env clones is shown. Values represent the relative expression of NKp44L in the surface of CD4 T cells normalized to the effect of the 3S synthetic peptide (100%). Data show the effect of NL4-3 and BaL envelopes (Ctrl-Env) and a total of 8 patient-derived Env clones.</p