Ethics statement

De-identified human monocytes from healthy blood donors were obtained from the University of Pennsylvania's Human Immunology Core (operating under the supervision of the University of Pennsylvania's Institutional Review Board). We did not have any interaction with human subjects or protected information, and therefore no informed consent was required. All studies were approved and supervised by Drexel University's Institutional Review Board.

Cells and cell culture

MDMs were generated by culturing monocytes in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (ΔFBS; BenchMark FBS, Gemini Bio-Products, West Sacramento, CA; certified for low endotoxin and hemoglobin levels), antibiotics, and 16 U/ml macrophage colony-stimulating factor (eBioscience, San Diego, CA) for 7 d. Human embryonic kidney 293T cells were cultured in DMEM supplemented with 10% ΔFBS and antibiotics. Human osteosarcoma cells stably expressing CD4 and CCR5 (HOS-CD4-CCR5; obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH [ARRRP] from Dr. Nathaniel Landau) [46], and human glioblastoma U87 cells stably expressing CD4 and CCR5 (U87-CD4-CCR5; obtained through the ARRRP from Drs. HongKui Deng and Dan Littman) [47], were cultured as previously described [48].

Production of viruses and pseudoviruses for infection

We produced a fully-infectious, firefly luciferase-reporter recombinant virus by transfecting 293T cells by calcium phosphate precipitation method (ProFection Mammalian Transfection System; Promega, Madison, WI) with the pNL-ADA-Fluc provirus, which we constructed by introducing the envelope glycoprotein of the CCR5-using, macrophage-tropic isolate HIV-1_ADA [49] into the pNL-HxB-Fluc provirus [50]. Work with fully-infectious virus was performed in a Bio-safety level 3 laboratory under standard operating procedures. Production of single-round infectious, luciferase-reporter pseudotypes was performed in 293T cells as previously described [48], [51], using the envelope-expression vectors of CCR5-using, macrophage-tropic BaL [52] or BR [48], [51] HIV-1 envelope glycoproteins, or a VSV-G expression vector. Supernatants containing viral stocks were collected 2 d post transfection, clarified, aliquoted, and stored at −80°C.

For infections, a 1∶2 dilution of viral stocks was added to target cells for 5–6 h. Cells were then washed twice with PBS and incubated with fresh media for 5 d (fully-infectious viruses) or 2–3 d (pseudoviruses), before quantitating infectivity by measuring luciferase activity in cell lysates (Luciferase assay system, Promega), as previously described [48], [51].

TLR stimulation

Fully differentiated MDMs were incubated for 14–16 h in medium containing selected TLR ligands (all from InvivoGen, San Diego, CA; used at the manufacturer's recommended optimal concentrations), as follows: TLR2, heat-killed Listeria monocytogenes (10⁸ PFU/ml); TLR3, dsRNA analogue poly(I∶C) of a high molecular weight (average size of 1.5–8 kb) (10 µg/ml); TLR4, LPS (10 µg/ml); TLR7, Imiquimod (5 µg/ml); and TLR9, unmethylated CpG (10 µg/ml). After incubation, supernatants were collected, clarified and stored, and cells were washed and used for infection or additional tests.

Immunofluorescence microscopy

MDMs were cultured in glass slides for 16 h in medium alone or with TLR ligands, and immediately after stimulation, stained with goat anti-human CD4 (AF-379-NA, R&D Systems, Minneapolis, MN) and mouse anti-human CCR5 (CTC8, R&D Systems) antibodies (Abs), followed by Alexa Fluor 488-conjugated anti-goat IgG and Alexa Fluor 594-conjugated anti-mouse IgG Abs (Invitrogen, Carlsbad, CA). DAPI (4–6′-diamidino-2-phenylindole) was used for nuclear staining. Images were obtained using an Olympus 1×81 spinning disk deconvolution fluorescent microscope and SlideBook 5.0 software (Intelligent Imaging Innovations, Denver, CO).

Flow cytometry

For assessment of CD4 and CCR5 expression, MDMs were cultured in 6-well plates for 16 h in medium alone or with TLR ligands, and immediately after stimulation, gently collected from the plates using cold PBS, washed and stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CCR5 Abs (eBioscience), or with the appropriate isotype control Abs. For analysis of TLR expression, unstimulated MDMs were collected as above and stained either under non-permeabilizing conditions (for surface staining) with PE-conjugated, anti-human TLR2 or TLR4 (both from eBioscience), or under permeabilizing conditions (for intracellular staining) with PE-conjugated, anti-human TLR3 or TLR9 (both from eBioscience), or PE-conjugated anti-human TLR7 (R&D Systems), or with the appropriate isotype control Abs. Immunofluorescence intensity was evaluated using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) with CellQuest software (BD Biosciences), and data were analyzed using FlowJo flow cytometry analysis software (Tree Star, Ashland, OR).

Enzyme-linked immunosorbent assays

Supernatants from unstimulated and TLR-stimulated MDMs were used for enzyme-linked immunoassays for quantitation of type I IFNs IFNα (PBL InterferonSource, Piscataway, NJ; and Thermo Scientific, Rockford, IL) and IFNβ (Thermo Scientific), pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (both from eBioscience), and β-chemokines macrophage inflammatory protein-1α (MIP-1α; SABiosciences, Frederick, MD), MIP-1β (Mabtech, Mariemont, OH) and regulated upon activation, normal T-cell expressed and secreted (RANTES; Peprotech, Rocky Hill, NJ), following manufacturer's instructions.

MDM supernatant transfer experiment

Supernatants from untreated or TLR-stimulated MDMs were collected and added to fresh “naïve” macrophages for specified duration. After treatment with the conditioned supernatants, the MDMs were washed twice with PBS and infected with BAL pseudotypes for 48 h. Cells were then lysed and processed for luciferase activity.

miRNA microarray

miRNA microarray analysis was performed in total RNA isolated using TRIzol (Invitrogen, Life Technologies Corp, Carlsbad, CA), following manufacturer's instructions, from macrophages from one donor cultured in the absence or in the presence of the specific ligands for TLR3, TLR4 and TLR7. miRNA expression profiles were evaluated at GenoSensor Corp. (Tempe, AZ) using GenoExplorer miRNA proprietary arrays with over 1300 miRNA probes (each in triplicate) for all described mature and most precursor human miRNA sequences (miRBase v13), in addition to multiple positive and negative control probes. Validated data were normalized according to the intensity of the signal of the positive control probes on each array chip. Signal intensity thresholds were defined as the array background plus 2 standard deviations, and at least two of the three repeating probes for each miRNA had to be above the signal intensity threshold for that miRNA to be further considered. Microarray data were deposited in the National Center for Biotechnology Information's Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo) under accession number GSE34428.

Quantitative RT-PCR

For quantitation of miR-155 levels, total RNA isolated from MDMs and cell lines (50 ng) was used for cDNA synthesis using TaqMan miRNA Reverse Transcription kit, followed by real-time PCR analysis using the miR-155-specific TaqMan miRNA Assay [53] (both from Applied Biosystems, Life Technologies Corp), performed in an Applied Biosystems 7300 Real Time PCR System. Results were calculated as relative quantitation compared to the levels of the U6 snRNA, using the comparative cycle threshold (Ct) method (also referred to as the 2^−ΔΔCt method) [54], [55], and were expressed as fold-change (mean ± SD) with respect to the appropriate biological control.

For relative quantitation of mRNA levels of IFNα1, IFNα2, IFNβ1 and several confirmed or potential HDFs, total RNA (500 ng) isolated from MDMs under different conditions was used for reverse transcription with Superscript III reverse transcriptase kit (Invitrogen), followed by real-time PCR using Taqman Gene Expression assays (Applied Biosystems), as per manufacturer's instructions. Results were calculated as relative quantitation compared to the levels of the 18S rRNA as endogenous control, using the comparative Ct method, and were expressed as fold-change with respect to the appropriate biological control.

miR-155 inhibition

The Ambion Anti-miR miR-155 inhibitor that specifically binds to and inhibits human miR-155 (anti-miR-155), or the Cy3 dye-labeled Anti-miR scrambled negative control (anti-miR-scr, which does not target any known human miRNA) (Applied Biosystems), were transfected at an optimized concentration of 30 nM into MDMs using Lipofectamine RNAiMax transfection reagent (Invitrogen). At 24 h post-transfection, MDMs were washed with PBS and replaced with media alone or containing poly(I∶C) or Imiquimod for TLR3 and TLR7 stimulation, respectively, for 14–16 h. After stimulation, cells were used for infection or for RNA isolation, as described above. In order to estimate transfection efficiency of MDMs, images of cells transfected with Cy3 dye-labeled anti-miR-scr were obtained using an Olympus 1×81 spinning disk deconvolution fluorescent microscope and SlideBook 5.0 software.

miR-155 over-expression in MDMs and cell lines

Over-expression of human miR-155 was achieved by transiently transfecting the non-viral plasmid miExpress Precursor microRNA pEZX-MR04 (Product ID: HmiR0358) containing the precursor sequence of hsa-miR-155 (pEZX-miR-155), or a scrambled sequence as negative control (pEZX-scrambled) (GeneCopoeia, Rockville, MD). Both plasmids encode for GFP expression that can be used for estimation of transfection efficiency. Plasmid DNA stocks were prepared using EndoFree plasmid maxi kit (Qiagen, Valencia, CA). HOS/CD4/CCR5 and U87/CD4/CCR5 cells were transfected with various amounts of pEZX-miR-155 or pEZX-scrambled plasmids (1–10 µg of DNA for 10⁶ cells per well in 6-well plates) using calcium precipitation, as indicated above. For MDMs, 6×10⁵ cells per well in 6-well plates were transfected with 6 µg of pEZX-miR-155 or pEZX-scrambled plasmids using jetPEI-Macrophage transfection reagent (PolyPlus-transfection, Illkirch, France), as per manufacturer's instructions. At 48 h post-transfection, cells were either infected or lysed for protein or RNA isolation. In addition, to estimate transfection efficiency of MDMs, images of cells untransfected or transfected with pEZX-miR-155 or pEZX-scrambled were obtained using an Olympus 1×81 spinning disk deconvolution fluorescent microscope and SlideBook 5.0 software.

Quantitative PCR analysis of viral DNA products

Total DNA was isolated at 48 h post-infection from MDMs or cell lines using DNeasy Blood and Tissue kit (Qiagen). Detection of the early (RU5) and late (U5Ψ) RT products of HIV-1, and of 2-LTR circles (a short-lived, nuclear dead-end product), was performed by quantitative PCR as previously described [56], [57]. Primers and probes are listed in Table S1. As control for the amplification of 2-LTR circles, we treated MDMs with 5 µM or 20 µM Raltegravir (an HIV-1 integrase inhibitor; a gift from Dr. Simon Cocklin) for 24 h immediately after infection, and before extraction of DNA. For an absolute quantitation of early and late RT products, a serially diluted HIV-1 backbone plasmid pNL4.3 with known copy numbers, and no-template controls, were used in each PCR procedure to generate a standard curve, as previously described [58].

Integration of HIV-1 viral sequences was detected using a two-step Alu-LTR PCR as previously described [59], with minor modifications. Briefly, an initial non-kinetic amplification step was performed with 100 ng total DNA using Tth DNA polymerase (Promega) with previously described primers (Table S1) and the following conditions: 3 min hot-start at 94°C, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min and 40 s, and a single step final extension at 72°C for 3 min. The second-round, quantitative PCR was performed using 25 µl of the first amplification with the previously described conditions and primers, but with a modified probe (Table S1). For absolute quantitation of integrated pro-viral copy numbers, a known standard of serially diluted CEM cells infected with VSV-G pseudotyped, HIV-Δenv-hygromycin-GFP viruses (plasmid kindly provided by Dr. Robert Siliciano) was used, as previously described by Liszewski et al. [58].

To estimate the viral copy number per cell, we simultaneously performed detection of the housekeeping gene porphobilinogen deaminase (PBGD; accession number M95623) as endogenous control, in standards of known concentration of serially diluted PBMC DNA and in all samples, as previously described [56], allowing the estimation of the number of cells per well, and subsequently the calculation of copy number per cell. Relative quantitation of 2-LTR circles was determined by using CCR5 as endogenous control, as previously described [56]. PBGD and CCR5 primers and probes are listed in Table S1. All real-time PCR reactions were performed using TaqMan Universal Master Mix (Applied Biosystems), and were run in an Applied Biosystems 7300 Real Time PCR System.

3′ UTR target reporter assays

To validate potential miR-155 targets, 293T cells (which do not have detectable endogenous miR-155) were co-transfected in 6-well plates by calcium precipitation with: (i) 3 µg DNA of pEZX-miR-155 or the pEZX-scrambled control; and (ii) 3 µg DNA of miTarget microRNA 3′ UTR Target Sequence (pEZX-MT01) expression clones (GeneCopoeia), which contain the 3′ UTR sequence of interest (HmiT001349: LEDGF; HmiT000372: ADAM10; HmiT054432: TNPO3; HmiT023438: Nup153; and CmiT000001: a negative control 3′ UTR) inserted downstream of the firefly luciferase gene, and an independently-controlled Renilla luciferase gene to be used as normalization control, allowing functional validation of predicted miRNA targets. At 48 h post-co-transfection of 293T cells, cells were lysed and the regulatory effect of miR-155 on its potential target was assessed in cell lysates with a dual luciferase assay (Luc-Pair miR Luciferase assay; GeneCopoeia) as per manufacturer's instructions. Results were calculated as [(firefly/Renilla)_miR-155/(firefly/Renilla)_scrambled]×100, for each of the pEZX-MT01 clones, and are expressed as mean ± SD from four independent wells.

Silencing of selected HDFs

MDMs were either untransfected or transfected for 48 h (using a 1∶1 ratio with LipoRNAiMax, Invitrogen) with Silencer Negative Control #2 siRNA (which has no significant sequence similarity to human gene sequences and is validated for use in human cells), or with Silencer Select siRNAs (two pre-validated siRNAs per target) for LEDGF, ADAM10 and Nup153 (all from Ambion), alone or in various combinations, following manufacturer's recommendations. Final concentration of each siRNA was 5 nM. Subsequently, cells were either lysed for western-blot analyses of protein levels, or infected and lysed for quantitation of infection by luciferase activity, or infected and lysed for DNA isolation and quantitation of viral RT products and integrated pro-virus.

Western-blots

Whole cell lysates were prepared using RIPA buffer (Pierce Thermo Scientific) in MDMs at 48 h post TLR stimulation, post pEZX-miR-155 or -scrambled plasmid transfection, and post siRNA transfection. Protein amounts were estimated using a BCA assay (Pierce Thermo Scientific). Subsequently, 30–50 µg of total protein was loaded in each lane of a SDS-PAGE gel (BioRad) in denaturing conditions and then transferred to nitrocellulose membrane. Blots were probed with rabbit anti-human ADAM10 (1∶500 dilution) (Abcam, Cambridge, MA; ab84595), mouse anti-human LEDGF (1∶500 dilution) (Abcam; ab110023), mouse anti-human Nup153 (1∶300) (Abcam; ab24700), and mouse anti-human TNPO3 (1∶100 dilution) (Abcam; ab54353). All blots were also probed with rabbit anti-human GAPDH Ab (1∶5,000 dilution) (Cell Signaling, Danvers, MA) as loading control. All primary Abs were incubated overnight at 4°C as per manufacturer's instructions. Goat anti-mouse and goat anti-rabbit poly-HRP secondary Abs (Pierce Thermo Scientific) were used at 1∶5,000 dilution, as per manufacturer's instructions. Western blotting Chemiluminescence Luminol reagent (Santa Cruz Biotechnology; Santa Cruz, CA) was used as substrate and an Alpha Innotech FluorChem SP digital imaging system was used for capturing images and for the spot densitometry analyses. Chemoluminescent signals were then normalized with the loading control, and were expressed as percent with respect to unstimulated or untransfected controls.

Statistical analyses

Statistical significance of experimental data was evaluated using SPSS Statistics software, with a p value equal to or less than 0.05 considered statistically significant.

HIV-1 infection is suppressed in MDMs stimulated through TLR3 or TLR4, but not TLR7 or TLR9

Macrophages are usually susceptible to HIV-1 infection in vivo and in vitro after their differentiation from blood monocytes [49], [60], [61], and the MDM model is widely used to address the regulation of HIV-1 infection in macrophages. Using this model, modulation of receptor expression, as well as increases in β-chemokines or IFNα production have been proposed, among others, as mechanisms that contribute to the reduced HIV-1 infection in LPS-stimulated MDMs [12]–[18], while no clear mechanism has been reported for poly(I∶C)-induced restriction of HIV-1 infection in MDMs [22]–[24]. We found that stimulation of MDMs from various donors with poly(I∶C) or LPS consistently suppressed HIV-1 infection, while Imiquimod and CpG reduced infectivity to varying extent depending on the donor, but never to levels similar to those achieved with poly(I∶C) and LPS (Figure 1A). In addition, we found abrogation – or almost complete suppression – of infection by pseudoviruses containing the BaL or BR envelope glycoproteins in MDMs stimulated through TLR3 or TLR4, with only partial reduction observed in cells stimulated through TLR2, TLR7 or TLR9 (Figure 1B). Remarkably, identical results were found when MDMs were infected with VSV-G-pseudotyped virions, suggesting that the differential effects observed did not involve alterations in the process of viral entry, but were rather due to post-entry events. This is in agreement with some previous studies that had also reported inhibition of infection of VSV-G-pseudotyped HIV-1 virions by TLR agonists (poly(I∶C) and LPS) in primary human myeloid cells [62], [63].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. TLR3 and TLR4 stimulation abrogates susceptibility to HIV-1 infection of primary macrophages.

(A) MDMs were untreated or incubated for 16 h with specific ligands for TLR3 (10 µg/ml poly(I∶C)), TLR4 (10 µg/ml LPS), TLR7 (5 µg/ml Imiquimod) or TLR9 (10 µg/ml CpG), and, after washing, infected with a fully-infectious, luciferase-reporter recombinant virus containing the macrophage-tropic ADA envelope glycoprotein. Inocula were removed after 4–6 h and cells were maintained for 5–6 days before quantitating infection through luciferase (luc) activity in cell lysates. Results with cells from four normal donors are shown as relative infection compared to untreated (mean ± standard deviation [SD]). (B) MDMs were untreated or incubated overnight with specific ligands for TLR2 (10⁸ PFU/ml heat-killed Listeria monocytogenes, HKLM), TLR3, TLR4, TLR7 or TLR9 (as indicated above), and then infected with luc-reporter, lentiviral particles pseudotyped with the macrophage-tropic BaL or BR HIV-1 envelope glycoproteins, or with VSV-G. Luc activity was measured 2–3 days post-infection and results from a representative experiment are shown as actual luc activity in cell lysates measured as relative light units per second (mean ± SD).

https://doi.org/10.1371/journal.ppat.1002937.g001

Expression of TLRs and viral receptors and cytokine/chemokine production

Despite the fact that we observed the same modulation of infection by HIV-1 envelope- and VSV-G-pseudotyped virions, we wanted to investigate whether the TLR stimulation had any effect on CD4 and/or CCR5 expression in MDMs. Using immunofluorescence microscopy, we did not observe any difference in the expression of CD4 or CCR5 between untreated and poly(I∶C)-, LPS- or Imiquimod-treated MDMs (Figure S1A–D). We also utilized flow cytometry with staining performed under non-permeabilizing conditions, and found again an absence of differential CD4 or CCR5 expression among unstimulated and TLR ligand-stimulated MDMs (Figure S1E). In addition, flow cytometry analysis under non-permeabilizing (TLR2, TLR4) or permeabilizing (TLR3, TLR7, TLR9) conditions demonstrated that all TLRs investigated had detectable expression in primary human MDMs (Figure S2), albeit, as expected, with some donor-to-donor variability.

We next investigated the potential role of type I IFNs, pro-inflammatory cytokines and β-chemokines in the reported effects of TLR agonists in HIV-1 infection of MDMs. We first measured the production of type I IFNs and found that neither IFNα nor IFNβ were consistently produced to high levels, with robust increases in IFNα or IFNβ production dispersedly observed in a ligand- and donor-dependent manner (Figure 2A–B). Next, we measured MDM production of pro-inflammatory cytokines TNF-α and IL-6 and β-chemokines CCL3–5 (MIP-1α, MIP-1β and RANTES, respectively), and found that all ligands induced robust increases in the production of all of them, confirming that the MDMs were indeed responding to the TLR stimulation. However, despite small variations between ligands, their levels did not correlate with the observed differences in susceptibility to infection (Figure 2C–G). In addition, we quantitated the levels of type I IFN mRNAs induced by the ligands. In agreement with the lack of consistent robust production of IFNα or IFNβ into the culture supernatants, we found that IFNα1, IFNα2 and IFNβ mRNAs increased, although in a donor-dependent manner, in response to selected TLR stimulation (Figure S3).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Production of type I IFNs, pro-inflammatory cytokines and β-chemokines after TLR stimulation in macrophages.

MDMs were cultured in quadruplicate for 16 h unstimulated or with the ligands for TLR2, TLR3, TLR4, TLR7 or TLR9, and then supernatants were collected, clarified, aliquoted and stored at −80°C until use. Commercial enzyme-linked immunoassays were used for quantitation of type I IFNs IFNα (PBL InterferonSource and Thermo Scientific) (A) and IFNβ (Thermo Scientific) (B), pro-inflammatory cytokines TNF-α (C) and IL-6 (D) (both from eBioscience), and β-chemokines MIP-1α (CCL3) (E) (SABiosciences), MIP-1β (CCL4) (F) (Mabtech) and RANTES (CCL5) (G) (Peprotech). All samples were analyzed in duplicate. Multiple donors are shown for type I IFNs and one representative donor is shown for pro-inflammatory cytokines and β-chemokines. Results are shown as mean ± SD of duplicate cultures. Dashed lines indicate the limit of detection of the assays for quantitation of type I IFNs.

https://doi.org/10.1371/journal.ppat.1002937.g002

Finally, we found that culture supernatants from MDMs stimulated with poly(I∶C), LPS or Imiquimod all induced a similar reduction in susceptibility to HIV-1 infection in fresh “naive” macrophages (Figure S4), ranging between 60 and 88% of infection observed in the same cells exposed to supernatants from unstimulated MDMs.

Differential miRNA expression in TLR-stimulated MDMs

To gain an insight into the potential differences in miRNA expression profiles between unstimulated and poly(I∶C)-, LPS- and Imiquimod-stimulated MDMs, we performed miRNA microarray analyses using GenoExplorer miRNA Expression Analysis (GenoSensor). Validated data (Gene Expression Omnibus database accession number GSE34428) showed that TLR3-, 4- and 7-stimulated MDMs displayed overlapping but distinct changes in miRNA expression profiles compared to unstimulated cells (Figure S5A–B). We identified a subset of four miRNAs that were significantly up-regulated in cells stimulated through TLR3 and TLR4 but not through TLR7 and could potentially play a role in modulating susceptibility to HIV-1 infection in this system. Among them, we focused on miR-155 since it displayed the highest up-regulation in confirmatory quantitative PCR (not shown), and because of its roles in macrophage inflammatory and immune responses.

We then determined the relative infection and miR-155 levels upon TLR stimulation in MDMs from a large cohort of normal donors. Despite the expected variability in their response to stimulation among primary cells from multiple donors, poly(I∶C)- and LPS stimulation in MDMs consistently resulted in abrogation or almost complete suppression of infection, which was never observed with Imiquimod stimulation (Figure 3A; Table S2). In addition, poly(I∶C) and LPS stimulation of MDMs generally resulted in robust increases in the levels of miR-155, mostly absent in Imiquimod-stimulated cells, as compared to unstimulated MDMs (Figure 3B; Table S2). Although a similar miR-155 increase in response to poly(I∶C) and LPS was observed in cells from some donors, a more robust induction with poly(I∶C) than with LPS was found in most of them, resulting in a statistically significant difference in miR-155 levels, as determined by paired Student's t test (Table S3). Taking together all infection and miR-155 data from poly(I∶C)-, LPS- and Imiquimod-stimulated MDMs, we performed curve estimation using a logarithmic regression model with miR-155 levels as independent variable and relative infection data as dependent variable, and found that the model is a good fit for the data (R² = 0.48, p<0.001) (Figure S6A), which suggests that susceptibility to infection is determined, at least in part, by miR-155 levels.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. Stimulation through TLR3 and TLR4, but not TLR7, increases miR-155 levels in primary macrophages.

MDMs from 12 different donors were cultured for 16 h unstimulated or with the ligands for TLR3, TLR4 or TLR7, and then used for infection with BaL pseudotypes (A) or for RNA isolation with Trizol (Invitrogen) for miR-155 quantitation (B). Infection was evaluated in 8 replicates as percent luc activity compared to unstimulated MDMs (mean ± SD). Relative miR-155 levels were evaluated in duplicate in total RNA using TaqMan miRNA Reverse Transcription kit and Universal PCR Master Mix, and the miR-155-specific TaqMan miRNA Assay (Applied Biosystems), and results were calculated relative to the U6 snRNA levels using the comparative Ct method (also referred to as the 2^−ΔΔCt method), and expressed as fold-change (mean ± SD) with respect to the unstimulated control. ND, not determined.

https://doi.org/10.1371/journal.ppat.1002937.g003

These results suggested that poly(I∶C)- and LPS-induced suppression of HIV-1 infection in MDMs is related with their ability to induce miR-155.

miR-155 contributes to the anti-HIV-1 effects of poly(I∶C)

To experimentally define whether the poly(I∶C)-induced increase in miR-155 levels contributed to the observed anti-HIV-1 effects upon TLR3 stimulation, we transfected MDMs with a specific anti-miR-155 inhibitor or a scrambled negative control (anti-miR-scr, which does not target any known human miRNA) before TLR stimulation. In general, we observed a very good efficiency of transfection of MDMs with the Ambion Cy3 dye-labeled anti-miR-scr (used as suggested by the manufacturer at an optimized concentration of 30 nM with Lipofectamine RNAiMax transfection reagent) (Figure S7). Remarkably, anti-miR-155 pre-treatment resulted in a statistically significant increase in relative infection of poly(I∶C)-stimulated MDMs from multiple donors (poly(I∶C), 4.7±1.7%; anti-miR-155+poly(I∶C), 26.0±10.2%; paired Student's t test, p<0.01), while anti-miR-scr pre-treatment had no effect (6.6±2.6%) (Figure 4A). The augmented susceptibility to infection induced by anti-miR-155 was concurrent with an abolishment of the increase in miR-155 levels observed in poly(I∶C)-stimulated MDMs (Figure 4B; poly(I∶C), 15.3±6.9 fold change; anti-miR-155+poly(I∶C), 1.9±1.0 fold change; paired Student's t test, p<0.01), while anti-miR-scr pre-treatment again had no effect (13.9±9.1 fold change). In addition, the anti-miR-155 pre-treatment in Imiquimod-stimulated MDMs failed to induce any changes (Figure 4C–D), except in MDM12 cells, in which a small increase in miR-155 level had been observed (Figure 3B), and anti-miR-155 pre-treatment resulted in a slightly increased infection concurrently with an abolishment of the small miR-155 induction.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. Inhibition of miR-155 in TLR3-stimulated macrophages increases their susceptibility to HIV-1 infection.

MDMs from seven different donors were transfected for 24 h (LipoRNAiMax, Invitrogen) with 30 nM anti-miR-155 antagomir or a scrambled control that doesn't target any known human miRNA (anti-miR-scr) (Applied Biosystems), and then stimulated with poly(I∶C) (A and B) or Imiquimod (C and D) for 16 h, before either infecting with BaL pseudotypes (A, C) or isolating RNA for miR-155 quantitation (B, D). Infection was evaluated in 8 replicates as percent luc activity compared to untreated, unstimulated MDMs (mean ± SD). Relative miR-155 levels were evaluated as in Figure 3 and results are shown as fold-change (mean ± SD) with respect to the untreated, unstimulated MDMs.

https://doi.org/10.1371/journal.ppat.1002937.g004

Finally, treatment of MDMs with anti-miR-155 did not result in an alteration in the levels of type I IFN mRNAs induced by TLR3 stimulation (Figure S3), suggesting that type I IFN induction and miR-155 increase are independent effects of poly(I∶C) in MDMs.

Ectopic expression of miR-155 reduces HIV-1 infection in MDMs and cell lines

To confirm that an increase in the levels of miR-155 in primary macrophages can have an effect on their susceptibility to HIV-1 infection, we performed over-expression experiments by transfecting MDMs with a miR-155-expressing plasmid (pEZX-miR-155) or a scrambled control (pEZX-scrambled). MDMs transfected with pEZX-miR-155 showed a remarkably lower HIV-1 infection than that observed in untransfected or pEZX-scrambled-transfected cells in 10 out of the 12 donors investigated (Figure 5A), with between 60 and 96% inhibition of infection. In addition, the reduced infectivity correlated with an increase in miR-155 levels in pEZX-miR-155-transfected cells from the same 10 donors, which was absent in untransfected or pEZX-scrambled-transfected cells (Figure 5B). MDM2 and MDM6 cells failed to show a decrease in HIV-1 infection but also lacked an increase in miR-155 levels upon transfection with pEZX-miR-155, suggesting that the absence of an effect on susceptibility to infection was due to inefficiency of transfection of MDMs from these two donors.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. miR-155 over-expression in primary macrophages and cell lines decreases their susceptibility to HIV-1 infection.

MDMs from 12 different donors were transfected with miR-155 expression plasmid (pEZX-miR-155) or scrambled control (pEZX-scrambled) (GeneCopoeia) using jetPEI-Macrophage transfection reagent (Polyplus), following manufacturer's instructions. After 48 h., cells were either infected with BaL pseudotype (A) to evaluate infection through luciferase activity as indicated above; or total RNA was isolated for miR-155 quantitation by real-time PCR (B) as indicated above. HOS-CD4-CCR5 cells were also transfected with various amounts of the miR-155 or scrambled plasmids using calcium precipitation, and then used for infection with BaL or VSV-G pseudotypes (C) or RNA isolation for miR-155 quantitation (D). Infection was evaluated in 8 replicates as percent luc activity compared to untransfected cells (mean ± SD). Relative miR-155 levels were evaluated as in Figure 3 and results are shown as fold-change (mean ± SD) with respect to the untransfected control.

https://doi.org/10.1371/journal.ppat.1002937.g005

Since primary cells in general, and macrophages in particular, are known to be partially refractory to transfection, we evaluated GFP expression by fluorescent microscopy in MDMs transfected with pEZX-miR-155 and pEZX-scrambled control (Figure S8), confirming that the large biological effects observed in cells from a majority of donors are probably related with a generally high efficiency of transfection. Curve estimation using a logarithmic regression model of miR-155 levels (independent variable) and relative infection data (dependent variable) indicated a good fit of the model (R² = 0.514, p<0.01) (Figure S6B), despite the presence of an outlier that resulted in a moderate multiple correlation coefficient (R = 0.717). Removal of the outlier increased even further the goodness of the model (R² = 0.804, p<0.001), suggesting that susceptibility to infection in transfected MDMs is determined, in large part, by the miR-155 levels achieved upon transfection.

Furthermore, transfection of HOS-CD4-CCR5 cells with increasing amounts of pEZX-miR-155 plasmid, but not with pEZX-scrambled control plasmid, resulted in a dose-dependent decrease in infection with both HIV-1 envelope- and VSV-G-pseudotyped viruses, which correlated with a dose-dependent increase in miR-155 levels (Figure 5C–D). Similar effects were observed in U87-CD4-CCR5 cells (data not shown). These results suggest that miR-155-induced anti-HIV-1 effects are cell type-independent, observed in immune and non-immune system cell types, and are mediated by alteration of one or more post-entry steps in the viral life cycle.

miR-155 induction leads to an accumulation of late viral transcripts and reduced integration

To define the potential mechanism(s) involved in anti-HIV-1 effects induced by higher levels of miR-155 both in the context of TLR3 engagement and during over-expression experiments, we performed absolute quantitation of early and late RT products (RU5 and U5Ψ, respectively), and integrated pro-viral DNA by real-time PCR. No differences in the copy number per cell of early RT products were observed between unstimulated and poly(I∶C)-, LPS- or Imiquimod-stimulated MDMs of multiple donors (Figure 6A). However, poly(I∶C)-stimulated MDMs consistently displayed an accumulation of late RT products compared to unstimulated or Imiquimod-stimulated cells (Figure 6B), while LPS-stimulated cells did not consistently show this increase in the copy number per cell of late RT product. Moreover, integrated pro-viral genomes were readily detected in unstimulated and Imiquimod-stimulated MDMs but were undetectable in 2 out of 3 poly(I∶C)-stimulated MDMs (with a 10-fold decrease in copy number per cell in the remaining donor), and in 1 out of 3 LPS-stimulated MDMs (with reduced copy numbers per cell in the other two) (Figure 6C). The greater consistency observed in these effects at the level of viral DNA detection with poly(I∶C), as compared to with LPS, could relate with the more consistent and robust effect of poly(I∶C) in increasing miR-155 levels across the large number of donors investigated. Finally, while anti-miR-scr pre-treatment of poly(I∶C)-stimulated MDMs did not alter detection of early RT, late RT or integrated viral DNA forms with respect to poly(I∶C) alone, the specific anti-miR-155 antagomir completely reverted the poly(I∶C)-induced accumulation of late RT products in all three donors investigated, and also remarkably increased the amounts of integrated pro-viruses in all three donors (to similar copy numbers to those found in unstimulated MDMs; Figure 6B–C). Therefore, inhibition of the poly(I∶C)-induced miR-155 increase restored the capacity of the virus to complete nuclear import and integration, and prevented the accumulation of late RT product.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 6. Increase in miR-155 leads to late RT products accumulation and greatly reduced integration.

Total DNA was isolated at 48 h post-infection from: (i) BaL pseudotype-infected MDMs (from three different donors) that were unstimulated, or stimulated with poly(I∶C), LPS or Imiquimod, and in the case of poly(I∶C), untransfected or transfected (prior to stimulation) with anti-miR-155 or anti-miR-scr (A–C); (ii) BaL pseudotype-infected MDMs (from three different donors) that were untransfected or transfected (prior to infection) with miR-155 expression plasmid (pEZX-miR-155) or scrambled control (pEZX-scrambled) (D–F). Real-time PCR was performed in total DNA for the absolute quantitation of early RT product (RU5) (A,D), late RT product (U5Ψ) (B,E) and integrated pro-virus (C,F), as described in Materials and Methods. Copy numbers per cell were calculated using PBGD as endogenous control to normalize for DNA recovery and number of cells, and are shown as mean ± SD from two independent quantitations. Arrows indicate lack of amplification in real-time PCR, implying levels below the limit of detection of the technique.

https://doi.org/10.1371/journal.ppat.1002937.g006

Similarly, in the context of ectopic expression of miR-155, no change was observed in detection of early RT products; however, we found that miR-155 induced substantial increases in the copy number per cell of late RT products and large decreases (up to over 2 orders of magnitude) in the copy number per cell of integrated pro-virus (Figure 6D–F). None of these effects were observed in MDMs transfected with the pEZX-scrambled control plasmid, which were indistinguishable from the untransfected control. Lastly, an accumulation of late RT product and absence of detection of integrated pro-virus, with no change in early RT product, were found as well in pEZX-miR-155-transfected U87-CD4-CCR5 cells, while no change in detection of any of the viral products was observed in pEZX-scrambled control-transfected cells (data not shown). Thus, ectopic expression of miR-155 in MDMs and cell lines led to an accumulation of late RT product, in the presence of greatly decreased amounts of integrated pro-virus, resulting in an identical phenotype to that found in poly(I∶C)-stimulated MDMs.

To determine whether the large reduction in, or absence of integration and accumulation of late RT products indicated an absence of nuclear import of viral DNA, we performed real-time PCR for detection of 2-LTR circles, a short-lived, dead-end product of viral DNA that forms in the nucleus and serves as a marker of recent infection, completion of reverse transcription and successful nuclear import of viral DNA. 2-LTR circles were either undetectable or detected at very low levels in most of our MDM samples, regardless of stimulation or transfection. However, we treated MDMs with the integrase inhibitor Raltegravir and demonstrated the formation of 2-LTR circles under those conditions (Figure S9). Hence, it seems that higher levels of miR-155 induced by poly(I∶C) stimulation or ectopic expression may prevent nuclear import of viral DNA, resulting in an accumulation of late RT products in the cytoplasm of infected cells.

miR-155 may target mRNAs of HDFs involved in trafficking/nuclear import of the pre-integration complex

To identify potential miR-155 targets that could be mediating the effects reported above, we used: miRGen (www.diana.pcbi.upenn.edu/miRGen) [64], an interface that provides access to unions and intersections of widely used target prediction programs (e.g., TargetScan [65]–[68] and MicroCosm Targets [69]–[72]), and to experimentally-supported targets; and miRWalk (www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) [73], which features a newly developed algorithm that evaluates potential targets on the complete sequence of all known human genes, and also provides information from 8 established, 3′ UTR-based, miRNA prediction programs and validated targets. These in silico analyses suggested that several confirmed or potential HDFs [74], [75] (due to the cellular processes that they are involved in), may be targets for miR-155. Specifically, we identified importins α3 and α5 (KPNA4 and 1, respectively), various nucleoporins from the nuclear pore complex (Nup107, 153 and 358 [or RanBP2]), and the cellular transcriptional co-activator lens epithelium-derived growth factor (LEDGF)/p75 (or PSIP1), all of which may participate in trafficking and nuclear import of the viral pre-integration complexes (PICs).

Therefore, we used total RNA from five donor MDMs over-expressing miR-155 to quantitate mRNAs for the identified potential targets, and for some additional HDFs such as importins α1 and β1 (KPNA2 and KPNB1, respectively), transportin-3 (TNPO3) and ADAM10 (a member of a disintegrin and metalloprotease family of proteins), which also seem to participate in the trafficking and/or nuclear import of PICs, although they were not predicted at all (KPNA2, KPNB1, TNPO3), or only by one algorithm (ADAM10), to be potential targets for miR-155. As expected, we found reduced mRNA levels of two validated miR-155 targets, inositol polyphosphate-5-phosphatase/145 kDa (INPP5D or SHIP1) [76] and suppressor of cytokine signaling 1 (SOCS1) [77], [78], which were evaluated as controls (Figure 7A). More importantly, ADAM10, TNPO3, NUP153 and LEDGF/p75 (PSIP1) all showed lower mRNA levels in miR-155-transfected than in untransfected MDMs, with a reduction larger than 2-fold in 3–5 of the MDMs investigated, while no changes were observed in total RNA from MDMs transfected with the pEZX-scrambled control (Figure 7A). Other mRNAs tested (KPNA1, KPNA2, KPNA4, KPNB1, Nup107, RanBP2) were either unchanged or not consistently reduced by at least 2-fold in at least three of the MDMs examined (data not shown).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 7. Increased miR-155 levels result in reduced mRNA levels of several HIV-1 dependency factors.

We isolated total RNA at: (i) 48 h post-transfection of MDMs from five different donors with either pEZX-miR-155 or pEZX-scrambled control (A); and (ii) 24 h post-stimulation of MDMs from two different donors with poly(I∶C) or LPS (B); and then performed relative quantitation of mRNAs by real-time RT-PCR of two experimentally-demonstrated miR-155 targets (SHIP1 and SOCS1), and four not previously described, potential miR-155 targets (ADAM10, TNPO3, LEDGF and Nup153), using TaqMan Gene Expression assays (Applied Biosystems). Results were calculated using 18S rRNA as internal control and the comparative Ct method (also known as the 2^−ΔΔCt method), and are shown as fold-change (mean ± SD from two independent quantitations) with respect to the untransfected control in (A), or as fold-change (calculated from a single experiment) with respect to unstimulated in (B).

https://doi.org/10.1371/journal.ppat.1002937.g007

Subsequently, we measured the mRNA levels of ADAM10, TNPO3, LEDGF and Nup153, and SHIP1 and SOCS1 as positive controls, in total RNA isolated from unstimulated, poly(I∶C)- and LPS-treated MDMs from two donors. As shown in Figure 7B, mRNA levels of LEDGF and TNPO3 were reduced greater than 2-fold by both poly(I∶C) and LPS, compared to unstimulated control, in the two donor MDMs, while the reduction was slightly less than 2-fold in one donor with poly(I∶C) for Nup153, and for one donor with poly(I∶C) and for the other donor with LPS, for ADAM10. Overall, the magnitude of the changes in mRNA levels of HDFs observed upon increased expression of miR-155, as well as upon TLR3 or 4 stimulation, are comparable to or larger than those reported in the literature for validated miRNA targets, e.g., such as in the studies describing SHIP1 and SOCS1 as miR-155 targets [76], [77], [79]. Thus, it seems possible that increased levels of miR-155 induced by either ectopic expression or TLR3 or TLR4 stimulation can reduce cellular susceptibility to HIV-1 infection by decreasing the expression levels of one or more HDFs, leading to the generation of an intracellular environment that is less conducive to productive HIV-1 infection.

The 3′UTRs of LEDGF, ADAM10, TNPO3 and Nup153 are targeted by miR-155

To functionally validate that the 3′UTRs of candidate HDFs identified above contain a miR-155 target site that could mediate the miR-155-induced effects, we utilized a dual luciferase reporter expression system in 293T cells, which do not contain any detectable endogenous miR-155. In this system, the presence of the 3′UTR of interest downstream of the firefly luciferase will result in a modulation of expression only if that 3′UTR contains a miR-155 target site, while the independently-controlled Renilla luciferase expression will be unaffected, allowing the validation of the target and the quantitation of the inhibitory effect. Specificity was assessed by co-transfecting the pEZX-miR-155 with a pEZX-MT01 control reporter vector (which does not contain any known miRNA target site) and, as expected, no suppressive effect on firefly luciferase expression was observed, when compared to the co-transfection of pEZX-scrambled and pEZX-MT01 control (Figure 8). Firefly luciferase expression was remarkably reduced by miR-155, relative to that observed with the scrambled control, in the presence of the 3′UTR of LEDGF, and to a lower extent, with the 3′UTRs of ADAM10, TNPO3 and Nup153. To a large extent, these data seem to agree with the relative strength of the in silico prediction studies reported above, which strongly suggested LEDGF as a miR-155 target, and had a less robust prediction for ADAM10 and Nup153 as miR-155 targets.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 8. The 3′UTRs of LEDGF, ADAM10, TNPO3 and Nup153 are targeted by miR-155.

293T cells were co-transfected by calcium precipitation with: (i) pEZX-miR-155 or the pEZX-scrambled control; and (ii) with miTarget microRNA 3′UTR Target Sequence (pEZX-MT01) expression clones (GeneCopoeia), which contain the 3′UTR sequence of interest (LEDGF, ADAM10, TNPO3, Nup153 and a negative control 3′UTR) inserted downstream of the firefly luciferase, and an independently-controlled Renilla luciferase gene to be used as normalization control, allowing functional validation of predicted miRNA targets. At 48 h post-co-transfection of 293T cells, cells were lysed and the regulatory effect of miR-155 on its potential target was assessed in cell lysates with a dual luciferase assay. Results were calculated as [(firefly/Renilla)_miR-155/(firefly/Renilla)_scrambled]×100, for each of the pEZX-MT01 clones, and are expressed as mean ± SD from four independent wells.

https://doi.org/10.1371/journal.ppat.1002937.g008

miR-155 increase in MDMs leads to reduced protein levels of selected HDFs

To confirm the functional validation of LEDGF, ADAM10, Nup153 and TNPO3 as targets of miR-155, we investigated their levels in MDMs both in the context of TLR stimulation and ectopic expression of miR-155. Western-blot analyses of cell lysates from unstimulated, poly(I∶C)- and LPS-treated MDMs from 3 donors demonstrated that both ligands resulted in robust decreases in the levels of LEDGF, and that poly(I∶C) reduced the levels of ADAM10 and Nup153 to a larger extent than LPS (Figure 9A–B), while both ligands had a less robust but detectable effect on the levels of TNPO3. Similarly, western-blot analyses of cell lysates from mock transfected, pEZX-miR-155- and pEZX-scrambled-transfected MDMs from 5 donors demonstrated that ectopic expression of miR-155 induced remarkable decreases in the levels of LEDGF, Nup153 and ADAM10, while the levels of TNPO3 were unaffected (Figure 9C–D). In addition, transfection of the scrambled control did not alter the level of expression of any of the HDFs investigated. These results demonstrate that increased levels of miR-155 not only lead to reduced mRNA levels of several HDFs, but also, and more relevantly, to reduced protein levels, confirming, at least for LEDGF, Nup153 and ADAM10, that they are bona-fide targets of miR-155 in primary macrophages. Regarding TNPO3, although TLR3/4 stimulation led to reductions both at the mRNA and protein levels and the 3′UTR reporter assay showed an effect of miR-155 on TNPO3's 3′UTR, ectopic miR-155 expression in macrophages only reduced its mRNA but not the protein levels, which could suggest that TNPO3 is also a miR-155 target but with a lower efficiency (perhaps due to imperfect complementarity) than the other HDFs. It is thus possible that this could relate with the fact that in silico analyses had not predicted TNPO3 as a target of miR-155, as well as with a greater stability or longer half-life of the protein that would require an extended period of time to reflect mRNA changes at the protein level.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 9. Increased miR-155 levels result in reduced protein levels of several HIV-1 dependency factors.

We generated cellular lysates at: (i) 48 h post-stimulation of MDMs from three different donors with poly(I∶C) or LPS (A,B); and (ii) 48 h post-transfection of MDMs from five different donors with either pEZX-miR-155 or pEZX-scrambled control (C,D); and then performed western-blots using antibodies for ADAM10, LEDGF, Nup153 and TNPO3 (Abcam), and GAPDH (Cell Signaling) as loading control. Intensity of the chemoluminiscent signals was quantitated by densitometry analysis and normalized with the loading control, and is expressed as percent with respect to unstimulated (A,B) or untransfected (C,D) controls. (A) Poly(I∶C) and LPS decreased the levels of ADAM10, LEDGF, Nup153 and TNPO3 (not shown) to a different extent and with some donor-to-donor variation. (B) Effects of poly(I∶C) and LPS on expression of HDFs in MDMs from three donors, expressed as mean ± SD. (C) Ectopic expression of miR-155 led to decreased levels of ADAM10, LEDGF and Nup153, but not TNPO3 (not shown), with some donor-to-donor variation; three out of a total five donors are shown. (D) Effects of ectopic expression of miR-155 on expression of HDFs in MDMs from five donors, expressed as mean ± SD.

https://doi.org/10.1371/journal.ppat.1002937.g009

Silencing of selected HDFs in MDMs replicates the miR-155-induced phenotype

Finally, in order to firmly demonstrate that the modulation of the levels of LEDGF, Nup153 and ADAM10 by miR-155 mediates the reduced susceptibility to infection in macrophages, through an accumulation of late RT products and absence or large reduction in integrated pro-virus, we performed a silencing experiment in which the expression of the above mentioned HDFs was reduced individually, or in various combinations, in a TLR- and miR-155-independent context, using Silencer Select siRNAs (Ambion). The siRNAs directed to each HDF of interest decreased its levels, whether applied individually or in combination, approximately to a similar extent (Figure 10A). Remarkably, the relative HDFs levels achieved, compared to those in untransfected MDMs, were similar to the levels obtained both in the context of TLR stimulation (relative to unstimulated) and ectopic expression of miR-155 (relative to untransfected) (Figure 9). Regarding infectivity, silencing of LEDGF decreased infection to a greater extent than silencing of ADAM10 or Nup153 (Figure 10B). Co-silencing of LEDGF with ADAM10 resulted in a slightly more robust reduction of infection than with Nup153, but both combinations were more efficient than silencing LEDGF alone, and co-silencing of ADAM10 and Nup153 also resulted in lower infectivity than silencing of each one individually. Finally, infectivity when co-silencing all three HDFs was not reduced further with respect to the co-silencing of LEDGF and ADAM10.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 10. Silencing of selected HDFs replicate the miR-155-induced effects.

MDMs from one donor were either untransfected or transfected for 48 h (LipoRNAiMax, Invitrogen) with Silencer Negative Control #2 siRNA (which has no significant sequence similarity to human gene sequences and is validated for use in human cells), or with Silencer Select siRNAs (two per target) for LEDGF, ADAM10 and Nup153 (all from Ambion), alone or in various combinations. Subsequently, cells were either lysed for western-blot analyses of protein levels (A) (as in Figure 9), or infected and lysed for quantitation of infection by luciferase activity (B), or infected and lysed for DNA isolation and quantitation of early (C) and late (D) viral RT products and integrated pro-virus (E) (as in Figure 6). Arrows indicate lack of amplification in real-time PCR, implying levels below the limit of detection of the technique.

https://doi.org/10.1371/journal.ppat.1002937.g010

Subsequently, we performed quantitative real-time PCR to measure early and late RT products and integrated pro-virus, to determine if we could find the same pattern of viral cDNA observed in TLR3 stimulated and in miR-155 over-expressing MDMs. As shown in Figure 10C-E, no change was observed in detection of early RT products; however, we found that silencing of LEDGF led to some increase in the amount of late RT product, which was more robust in the co-silencing of LEDGF and Nup153 and when co-silencing all three HDFs. In addition, silencing of all HFDs led to a reduction in integrated pro-virus, becoming undetectable when co-silencing LEDGF and ADAM10 and when co-silencing all three HDFs (Figure 10E). In summary, co-depletion of LEDGF, ADAM10 and Nup153 replicated the effects, in terms of modulation of susceptibility to infection and viral DNA detection, observed through increased levels of miR-155 both in the context of TLR3 stimulation and ectopic expression in primary human MDMs.