Residual BST2 clusters are detected outside virus assembly sites in the presence of Vpu.

<p>MT4, primary CD4+ T cells, SupT1-shortBST2 and SupT1-longBST2 cells were mock-infected (mock) or infected with VSV-G-pseudotyped NL4.3-Ada-GFP WT or dU viruses. <b>(A)</b> Cells were stained with anti-BST2 Abs (blue), fixed, permeabilized and then sequentially stained with anti-p17 Abs (red). Infected cells (GFP+) are marked with a green letter G. An uninfected cell is shown next to WT-infected cells as indicated. Clusters of free BST2 are marked with white open arrows. White bar = 10 μm. <b>(B)</b> The number of residual BST2 clusters not co-localizing with p17 (designated as free BST2) per cell was calculated and expressed as the percentage of the total number of surface BST2 clusters. <b>(C)</b> Quantitative analysis of surface BST2 was determined as described in Materials and Methods. One way ANOVA with Bonferroni’s multiple comparison test was used (*** p<0.001, ** p<0.01, ns not significant (p>0.05)). Error bars indicate the standard error of the mean after analysis of at least 50 distinct cells.</p

Innate sensing of WT or Vpu-defective HIV-infected T cells requires Env-dependent viral fusion and is largely dependent on TLR7.

<p>MT4 cells were mock-infected (m) or infected with GFP-encoding NL4.3 variants (WT or dU) as indicated. <b>(A-B)</b> Cells were left un-treated (no Tx) or were treated with T-20 prior to co-culture with PBMCs. To assess the effect of inhibiting reverse transcription, PBMCs were treated with 3TC prior to co-culture with infected MT4 cells. As a positive control, CpG was added to inhibitor-treated or untreated mock-infected cells. A representative example of absolute levels <b>(A)</b> or relative percentages <b>(B)</b> of IFN-I detected after co-culture of WT or dU HIV-infected MT4 cells with PBMCs in the presence or absence of inhibitors are shown. Results are expressed relative to values obtained in the no-Tx samples (n = 8). <b>(C-F)</b> PBMCs were pre-treated with either TLR9 or TLR7/8/9 antagonists (antag.) or their respective controls (antag. Ctrl) prior to TLR agonist treatment <b>(C-D)</b> or to co-culture with the indicated infected cells <b>(E-F)</b>. A representative example of absolute levels of IFN-I detected after treatment with either TLR9 agonist (CpG-A) <b>(C)</b> or TLR7 agonist (R848) <b>(D)</b> is shown. A representative example of absolute levels <b>(E)</b> or relative percentages <b>(F)</b> of IFN-I produced in the indicated co-cultures in the presence of TLR antagonists or controls are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in the presence of the TLR7/8/9 antagonist control was set at 100% (n = 3). Two-tailed paired <i>t</i>-test was used. (*** p<0.001, * p<0.05, ns not significant (p>0.05)). Error bars represent SD.</p

Vpu-mediated control of IFN-I production by pDCs involves engagement and activation of ILT7 by BST2.

<p><b>(A-C)</b> Vpu-mediated BST2 antagonism enhances activation of ILT7. ILT7+ NFAT-GFP reporter cells were co-cultured with HEK293T (mock) or BST2-expressing HEK293T cells mock-transfected or transfected with the indicated pNL4.3 constructs (WT or dU). <b>(A)</b> Representative example of ILT7 activation as determined by the percentage of NFAT-GFP positive cells measured by flow cytometry. <b>(B)</b> Percentage of ILT7 activation after co-culture with the indicated HIV/BST2 expressing HEK 293T cells relative to WT HIV-producing cells (100%) (n = 4). <b>(C)</b> Percentage of BST2 surface expression in HEK293T cells after co-transfection of BST2 with the indicated HIV provirus relative to dU HIV-producing cells (100%) (n = 4). <b>(D-F)</b> Effect of ILT7 depletion on IFN-I production by pDCs. <b>(D)</b> Non-pDC fraction (BDCA-2-) and siRNA-treated enriched pDCs (CD14-/BDCA-2+) were stained using anti-ILT7 Abs as indicated. A representative example of absolute levels <b>(E)</b> or relative percentages <b>(F)</b> of IFN-I produced after co-culture of control pDCs (pDC-siCTRL) or ILT7-depleted pDCs (pDC-siILT7) with the indicated infected MT4 cells are shown. The amount of IFN-I released by pDC siCTRL in contact with dU HIV-infected cells was set at 100% (n = 4). <b>(G-I)</b> Effect of recombinant soluble ILT7 on IFN-I production by pDCs. <b>(G)</b> Expression of a HA-tagged soluble ILT7 (soILT7-HA) in HEK 293T cells. Cells and supernatants (sup) were analyzed by Western blot (WB) using anti-HA Abs. Purity of secreted soILT7-HA was confirmed by Coomassie staining (sup Coom). A representative example of <b>(H)</b> absolute levels or <b>(I)</b> relative percentages of IFN-I production after co-culture of PBMCs with the indicated infected MT4 cells pre-treated with control (CTRL) or soILT7-HA-containing supernatants are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of CTRL supernatant was set at 100% (n = 6). Two-tailed paired <i>t</i>-test was used in B-C while repeated measures ANOVA with Bonferroni’s multiple comparison test was used in F and I (*** p<0.001, ** p<0.01, ns not significant (p>0.05)). Error bars represent SD.</p

Effect of a BST2 GPI anchor mutant on Vpu-mediated control of IFN-I production by pDCs.

<p><b>(A-B)</b> HEK293T cells were transfected with either an empty plasmid or plasmids expressing BST2 or BST2-dGPI 48 h prior to co-culture with PBMCs. <b>(A)</b> Surface expression of BST2 was evaluated 48 h post transfection by flow cytometry in controls cells (shaded grey histogram), as well as in cell expressing BST2 (solid black histogram), or BST2-dGPI (dashed grey histogram). Mean fluorescence intensity (MFI) values are indicated for each sample <b>(B)</b> After 6 h of co-culture, samples were untreated or treated with Imiquimod (TLR7 agonist) and levels of bioactive IFN-I in supernatants were measured 18 h later. The amount of IFN-I released by PBMCs in contact with HEK293T cells transfected with the empty plasmid in presence of the TLR 7 agonist was set at 100% (n = 4). As a control, transfected HEK293T cells were treated with TLR7 agonist without PBMCs. <b>(C)</b> Percentage of IFN-I released after co-culture of infected SupT1-Empty with PBMCs normalized to the value obtained with dU HIV-infected SuptT1 cells (100%) (n = 4). <b>(D)</b> A representative example of absolute levels of IFN-I produced after co-culture of mock or infected-SupT1,-SupT1-BST2 or-SupT1-BST2-dGPI cells with PBMCs is shown. <b>(E)</b> Relative percentages of IFN-I produced after co-culture of infected-SupT1-BST2 or SupT1-BST2-dGPI cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected SupT1-BST2 cells was set at 100% (n = 4). Two-tailed paired <i>t</i>-test was used in B and C (* p<0.05, ns not significant (p>0.05)). Repeated measures ANOVA with Bonferroni’s multiple comparison test was used in E (*** p<0.001, ns not significant (p>0.05)). Error bars represent SD.</p

Vpu-mediated control of IFN-I production by pDCs requires the presence of BST2 on infected donor cells.

<p><b>(A-C)</b> Control (MT4-shNT) or BST2-depleted (MT4-shBST2) MT4 cells were mock-infected, or infected with GFP-marked NL4.3 WT or dU viruses for 48 h. <b>(A)</b> Surface expression of BST2 on GFP-positive MT4 cells infected with WT (dashed grey histogram) or dU (solid black histogram) was evaluated by flow cytometry. Mean fluorescence intensity (MFI) values are indicated for each sample (staining using pre-immune rabbit serum, PI, shaded grey histograms). <b>(B-C)</b> The indicated MT4 donor cells were co-cultured with PBMCs. After 24 h, levels of bioactive IFN-I were measured in supernatants. A representative example of absolute levels <b>(B)</b> or relative percentages <b>(C)</b> of IFN-I produced after co-culture of the indicated infected MT4 cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected MT4-shNT cells was set at 100% (n = 12). <b>(D-E)</b> MT4-shNT (BST2 +) or MT4-shBST2 (BST2 -) cells were infected with GFP-marked NL4.3 WT or T/F CH077 viruses for 48 h. Similar number of p24+ infected cells were then co-cultured with PBMCs. After 24 h, levels of bioactive IFN-I were measured in supernatants. A representative example of absolute levels <b>(D)</b> or relative percentages <b>(E)</b> of IFN-I produced after co-culture of the indicated infected MT4 cells with PBMCs are shown. The amount of IFN-I released by PBMCs in contact with infected MT4-shBST2 cells was set at 100% (n = 6). Repeated measures ANOVA with Bonferroni’s multiple comparison test was used. (*** p<0.001, ns not significant (p>0.05)). Error bars represent SD.</p

BST2 at the surface of infected cells is required for Vpu-mediated control of IFN-I production.

<p>MT4 cells were mock-infected or infected with GFP-marked NL4.3 WT or dU viruses and pre-incubated with anti-BST2 rabbit polyclonal (Rb BST2 Ab) or pre-immune (Rb PI) Abs or left untreated (No Ab). <b>(A)</b> Mock cells were subsequently stained for surface BST2 using mAb 26F8 and analyzed by flow cytometry. As a positive control, cells were directly stained with mAb 26F8. <b>(B-C)</b> The indicated MT4 cells were co-cultured with PBMCs. After 24 h, levels of IFN-I released in supernatants were measured. A representative example of absolute levels <b>(B)</b> or relative percentages <b>(C)</b> of IFN-I produced after co-culture of PBMCs with infected MT4 cells pre-treated with the indicated Abs are shown. The amount of IFN-I released by PBMCs in contact with dU HIV-infected cells in presence of Rb PI was set at 100% (n = 4). Two-tailed paired <i>t</i>-test was used (** p<0.01, * p<0.05, ns not significant (p>0.05)). Error bars represent SD.</p

BST2 binds and effectively activates ILT7.

<p><b>(A-E)</b> BST2 binds ILT7. <b>(A)</b> Purified GST-BST2 and bacILT7 were analyzed by SDS-PAGE and visualized by Coomassie brilliant blue staining. <b>(B)</b> Recombinant GST-BST2 pre-coated on the surface of Biacore sensor chips, was mixed with the indicated concentrations of bacILT7. The kinetic response data after subtracting the value from a reference cell coated with GST alone are shown. Kinetic constants (K_D = 2.33 μM, k_on = 1.25×10³ M^-1s^-1, k_off = 3.08×10⁻³ s^-1) were derived by fitting the data (dotted lines) to a 1:1 Langmuir model (black lines) using local R_max parameters (chi² = 14). <b>(C)</b> Control (293T) or ILT7-expressing HEK 293T cells (293T-ILT7) were incubated with control supernatant (CTRL) or with BST2-Fc-containing supernatant (BST2-Fc) prior to crosslinking with DTSSP. Cells were then stained for surface BST2-Fc and analyzed by flow cytometry. <b>(D-E)</b> ILT7+ or ILT7- NFAT-GFP reporter cells were incubated with control supernatant (CTRL) or with BST2-Fc-containing supernatant (BST2-Fc). Proximity ligation assay (PLA) was performed using mouse ILT7 mAb and rabbit polyclonal anti-BST2 Abs. A fluorochrome-labeled probe (red) was used to reveal locations of close proximity, and nuclei were highlighted with DAPI staining (blue). <b>(D)</b> Images were acquired by confocal microscopy using a 63Å~ objective. Images shown are representative of multiple fields. A magnification of the section marked in yellow is shown beside the panel. White bar = 10 μm. <b>(E)</b> The percentage of cells with PLA red staining (% positive cells) was calculated from at least 50 cells per condition. <b>(F-K)</b> BST2 effectively activates ILT7. <b>(F)</b> ILT7+ NFAT-GFP reporter cells were cultured in the presence or absence of plate-bound BST2-Fc for 24 h and analyzed for GFP expression using flow cytometry. <b>(G-H)</b> ILT7+ NFAT-GFP reporter cells were cultured in the presence of plate-bound or soluble anti-ILT7_alexa647 Abs (grey shaded or solid black histograms, respectively) or soluble isotype_alexa647 Ab as negative control (dotted lines). Twenty-four hours later, cells were harvested and samples in contact with the isotype Ab were stained for surface ILT7 only, using the above mentioned Abs for 30 min at 4°C (isotype_alexa647: dotted black histogram and aILT7_alexa647: dotted red histogram). All sample were analyzed by flow cytometry to detect <b>(G)</b> the percentage of GFP positive cells and <b>(H)</b> anti-ILT7 Abs (surface or total: surface + internalized). <b>(I-K)</b> ILT7+ NFAT-GFP reporter cells were co-cultured for 24 h with <b>(I)</b> control Hela or BST2-depleted Hela (Hela shBST2) cells or <b>(J-K)</b> HEK293T cells expressing either <b>(J)</b> increasing amounts of BST2 or <b>(K)</b> a fixed amount of BST2 and analyzed by flow cytometry (n = 2). <b>(K)</b> Prior to co-cultures, HEK293T-BST2 cells were incubated with rabbit anti-BST2 Abs (aBST2) or ILT7+ NFAT-GFP reporter cells were incubated with anti-ILT7 Abs (aILT7) for 1h or cells were left untreated as control (noAb). Relative percentage of ILT7 activation was plotted as % of GFP+ cells in each condition relative to the no Ab condition, which was set at 100%. Error bars represent SD.</p

FIX-ΔLUNA virus does not increase viral genome copy number after IL6 induced differentiation of CD14⁺ cells.

<p>Viral DNA was quantified by qPCR. A) Total DNA was extracted from FIX-WT, FIX-ΔLUNA, and FIX-Rev infected HF cells over a 20 d time course. B) Total DNA was extracted from FIX-WT, FIX-ΔLUNA, and FIX-Rev infected CD14⁺ cells at 1 dpi, 3 dpi, 5 dpi, 10 dpi, 20 dpi. Differentiation was induced in CD14⁺ cells at 10 dpi by the addition of IL6 and at 5 days (sample termed as 15dpIL6) or 10 days (samples termed as 20dpIL6) total DNA was collected. All samples were run in triplicate. Statistical analysis was done using a Student <i>t</i> test comparing FIX-WT vs. FIXΔLUNA or FIX-Rev vs. FIX-ΔLUNA. The * indicates a <i>P</i>-value<0.01, and the ** indicates a <i>P</i>-value<0.001.</p

HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14⁺ Cells

<div><p>Human cytomegalovirus (HCMV) is a member of the <em>Herpesviridae</em> family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors <em>in vivo</em>. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.</p> </div

Expression of HCMV US proteins by MSC decreases PBMNC proliferation and correlates with the levels of HLA-I.

<p>(A) Each of the transduced and untransduced MSCs were used as stimulators and were co-cultured with responders human PBMNC. After five days, DNA synthesis was assayed with the BrdU cell proliferation colorimetric ELISA. Data represents mean ± SEM of four independent experiments. In each experiment the specific stimulator-responder co-culture was performed in triplicate (* indicates p<0.01 and were considered statistically significant compared to MSC-E levels). (B) Furthermore a direct correlation was found between the levels of HLA-I MFI and human PBMNC proliferation.</p