MDMs Constitutively Express NFAT5, Which Is Required for Replication of HIV-1 Representative Isolates from Subtypes B, C, and E

<div><p>(A) NFAT5 expression was detected in human MDMs using real-time PCR. NFAT5 mRNA expression was significantly inhibited 6 d after transfection with NFAT5 siRNA (NFAT5#1) compared with cells transfected with GFP siRNA or mock transfected (<i>p</i> < 0.02).</p><p>(B) Flow cytomety analysis of intracellular NFAT5 levels in MDMs. A representative experiment is shown of a flow cytometry analysis of MDM using a monoclonal antibody to NFAT5. Intracellular NFAT5 protein levels were lower in the cells transfected with NFAT5 siRNA than in those transfected with GFP siRNA or those mock transfected. The experiment is representative of five independent experiments.</p><p>(C) Flow cytometry analysis of intracellular p24 levels in HIV-1_BAL–infected MDMs. Mock- or siRNA-transfected MDMs were infected with HIV-1_Bal, and intracellular p24 levels were determined on day 4 post-infection with flow cytometry using the p24-RD1 conjugated antibody. Intracellular virus levels were 26.9% lower in the cells transfected with NFAT5 siRNA than in GFP siRNA–transfected cells.</p><p>(D) Knockdown of NFAT5 RNA inhibits replication of HIV-1 representative isolates from subtypes B, C, and E. Cell-free virus replication was determined by p24 ELISA at day 3, 6, 9, and 13 post-infection of MDMs by subtype B (HIV-1_BAL), C (HIV-1_98IN22), or E (HIV-1_93TH64) viruses as indicated. Free virus levels were lower in the cells transfected with NFAT5 siRNA compared with the cells transfected with GFP siRNA (control) in cellular supernatants at all the time points monitored and indicated in the figure. Notably, on day 13 post-infection, inhibition of free virus levels in the NFAT5 siRNA–transfected cultures was on average 1.9-fold lower for HIV-1_BAL, 2-fold lower for HIV-1_98IN22, and 2.1-fold lower for HIV-1_93TH64 compared with GFP siRNA–transfected cultures.</p></div

NFAT5 Binds Distinctly to a Motif Conserved in HIV-1 Subtype B, C, and E LTR Promoter Regions

<div><p>(A) NF-κβ, NFATp, and NFAT5 bind to distinct and overlapping sequences in the HIV-1 LTR enhancer/promoter region of HIV-1 subtypes B, C, and E. Quantitative DNase I footprinting analysis is shown using HIV-1 LTR fragments (−208 to +64 nt relative to the transcription start site) from representative subtype B, C, and E viral isolates and increasing concentrations of recombinant NF-κB (p50/p65), NFATp (20 ng, 100 ng, 400 ng, and 20 μg), or NFAT5 (10 μg, 50 μg, 200 μg, and 1 mg) as shown. The low DNA-binding affinity of NFAT5 relative to NFATp has been noted previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020130#ppat-0020130-b060" target="_blank">60</a>]. The regions that are protected from DNase cleavage by the binding of NF-κB, NFATp, and NFAT5 are indicated with bars. We note that in the case of NFAT5, distinct cleavage patterns (including a hypersensitive site 5′ to the region of DNase I protection) are evident at 200 μg.</p><p>(B) Summary of DNase footprinting results with NF-κβ, NFATp, and NFAT5, and alignment of HIV-1 LTR nucleotide sequences from representative isolates from subtypes B, C, and E. The diagram illustrates the sequences protected from DNase cleavage by the binding of NF-κβ (blue), NFATp (green), and NFAT5 (red). The NF-κβ, NFATp, and NFAT5 binding sites are boxed. The broken boxes indicate sites that have sequence similarity to NF-κβ, NFATp, and NFAT5 sites, but which do not bind the proteins in the footprinting assay displayed in (A). The GGA repeats in the NFATp core dimer binding region are shown in bold.</p></div

Alignment of LTR Promoter and Enhancer Sequences in Representative Isolates of Subtypes B, C, and E

<p>Sequence variation within the NF-κβ/NFATp, SP1, and TATA box regions that distinguish the C and E subtypes from the B subtype are indicated in the boxes. Sequences were aligned and analyzed by using ClustalW (<a href="http://www.ebi.ac.uk/clustalw" target="_blank">http://www.ebi.ac.uk/clustalw</a>).</p

The NFAT5 Binding Motif Is Conserved in HIV-1, HIV-2, and SIV LTRs

<p>Sequence analysis of HIV-1 Group M subtypes A through G and HIV-1 Group O as well as HIV-2 and selected SIV LTR enhancer/promoter regions. Numbering is based on B subtype (HIV-1_LAI) sequences. The conserved NFAT5 binding motif is highlighted in yellow; the sequence corresponding to the canonical NF-κB site is in red, and the unique 3′ terminal adenine is in blue. Sequences were obtained from the Los Alamos HIV Sequence Database (<a href="http://hiv-web.lanl.gov/content/hiv-db/mainpage.html" target="_blank">http://hiv-web.lanl.gov/content/hiv-db/mainpage.html</a>) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020130#ppat-0020130-b017" target="_blank">17</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020130#ppat-0020130-b056" target="_blank">56</a>] and aligned using HIV-1 subtype B sequences spanning −123 to −68 nt relative to the viral transcription start site as a reference.</p

NFAT5 Plays an Important Functional Role in HIV-1 Replication in HeLa-CD4 Cells

<div><p>(A) Real-time PCR showing that HeLa-CD4 cells constitutively express NFAT5 mRNA. Using siRNA-targeting NFAT5, we were able to reduce NFAT5 mRNA levels significantly (<i>p</i> < 0.01) compared with that of GFP siRNA or mock-transfected cells. To confirm that NFAT5 knockdown was specific, we used two NFAT5 siRNAs (NFAT5#1 and NFAT#2) targeting two different exons of the NFAT5 gene. We chose the sequence siRNA to GFP because this sequence has been validated and shown to have no nonspecific effects upon HIV replication [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020130#ppat-0020130-b046" target="_blank">46</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020130#ppat-0020130-b058" target="_blank">58</a>]. Results show the average of four independent experiments.</p><p>(B) Flow cytomety analysis of intracellular NFAT5 levels in HeLa-CD4 cells. A representative experiment of a flow cytometry analysis using a monoclonal antibody to NFAT5 demonstrated that intracellular NFAT5 protein levels were lower in the cells transfected with NFAT5 siRNA than those that were transfected with GFP siRNA or mock transfected. The experiment is representative of four independent experiments.</p><p>(C) Flow cytometry analysis of intracellular p24 levels in HIV-1_LAI infected HeLa-CD4 cells. Intracellular HIV-1 levels were determined using a p24-RD1 conjugate in a representative experiment. Three days following virus infection, intracellular p24 was lower in cells transfected with NFAT5 siRNA targeting two different exons of NFAT5 (NFAT5#1 and NFAT5#2; 33.3% and 36.27% inhibition, respectively) than in cells transfected with a GFP control siRNA or mock transfected. This experiment is representative of four independent experiments.</p><p>(D) Cell-free p24 production in supernatants of HIV-1_LAI infected HeLa-CD4 cells. HIV-1 replication as assessed by p24 ELISA is significantly (<i>p</i> < 0.04) lower in cellular cultures transfected with NFAT5 siRNA targeting two different exons of NFAT5 (NFAT5#1 and NFAT5#2) than in cells mock transfected or transfected with the GFP control siRNA.</p></div

Specific Disruption of NFAT5 Binding Impairs LTR–Reporter Gene Activity in Monocytic THP-1 Cells

<div><p>(A) Disruption of NFAT5 binding does not impair NF-κβ (p50/p65) binding. An EMSA is shown using recombinant NFAT5 and NF-κB proteins with an oligonucleotide matching the NFAT5/NF-κβ site from the HIV-1_LAI (wild-type [WT]) or an oligonucleotide with a mutation in the nt predicted to be required for NFAT5 binding (N5-Mut), but not NF-κB binding (shown at the bottom of the figure).</p><p>(B) Mutation of the NFAT5 binding site significantly reduces HIV-1_LAI LTR reporter activity in THP-1 cells. HIV-1_LAI LTR-Luc reporter constructs that contained −208 to +64 nt relative to the transcriptional initiation site of HIV-1_LAI, and that were isogenic except for the specific N5 mutation, were transfected into THP-1 cells with the <i>Renilla</i> luciferase (pRL-TK) control plasmid. Luciferase activity was determined 26 h later. The LTR mutation, which abolishes NFAT5 binding to the promoter/enhancer region, significantly (**, <i>p</i> < 0.01) suppressed LTR-dependent activity of transcription compared with WT in THP-1 cells.</p></div