Phylogenetic tree of HIV-1 C2V5 nucleotide clonal sequences of subject #53 during DAA therapy.

<p>The viral sequences at the various time points are indicated by different colours, see table insert. Bootstrap values ≥80% are indicated. Bars indicate p distance scale. In the insert mean diversity (number of nucleotide substitutions/site) of the sequences detected at different time points is reported.</p

The scatterplot of baseline HIV-1 DNA levels and change between EOT and baseline.

<p>The scatterplot of baseline HIV-1 DNA levels and change between EOT and baseline.</p

Factors associated with increase of HIV-1 DNA (≥0.5 log₁₀ copies/million PBMC) at EOT when compared to baseline at univariable and multivariable analysis.

<p>Factors associated with increase of HIV-1 DNA (≥0.5 log₁₀ copies/million PBMC) at EOT when compared to baseline at univariable and multivariable analysis.</p

Median PBMC-associated total HIV-1 DNA at baseline, EOT and 3 months after DAA in OVC and SVC.

<p>Median PBMC-associated total HIV-1 DNA at baseline, EOT and 3 months after DAA in OVC and SVC.</p

Effect of the ATM knockdown or pharmacological ATM inhibition on the Env-elicited apoptotic signal transduction cascade.

<p>HeLa CD4 and HeLa Env cells were separately transfected with the indicated siRNAs and 36 h later the cells were cocultured for further 36 h, followed by the immunoblot detection of the indicated proteins and phoshoproteins (A) or, alternatively, by immunofluorescence detection (B, C) of the indicated parameters (X±SD, n = 5). Alternatively, the cells were not transfected and rather treated with the chemical ATM inhibitor KU-55933 (C), at a dose that did not inhibit karyogamy (100 nM or 1 µM), followed by the determination of the indicated parameters by immunofluorescence analyses (X±SD, n = 3). Moreover, the two cell lines (HeLa CD4 and HeLa Env) were both transfected with a control siRNA or two distinct siRNAs targeting NBS1 (D,E), followed by immunoblot detection of NBS1 after admixture at a 1∶1 ratio (D) or coculture for 36 h and determination of the indicated parameters by immunofluorescence (E, F). Representative fluorescence microphotographs are shown in E and quantitative data (X±SD, n = 3) are summarized in F. Asterisks mark significant inhibitory effects of the ATM-specific siRNA (as compared to a control siRNA in B), of KU-55933 (as compared to untreated cells in C) or of the two NBS1-specific siRNAs (as compared to a control siRNA in F).</p

Involvement of ATM and NBS1 in syncytial apoptosis observed in HIV-1 infected human lymphoblasts.

<p>PHA/IL-2 lymphoblasts obtained from healthy donors were infected with HIV-1 WT and HIV1 IN^D64V at a MOI of 10 for 48 h and then stained for the indicated markers. A. DNA damage foci containing ATMS1981P in syncytia were identified by immunofluorescence after infection of PHA/IL-2 lymphoblasts with HIV-1 WT and HIV1 IN^D64V. B. Comparative quantification of DNA damage foci induced by infection with HIV-1 WT and HIV1 IN^D64V at a MOI of 10 for 48 h. The phosphorylation status of ATM, H2AX and apoptosis were quantified (X±SD, n = 3 experiments). C. Effect of the ATM inhibitor KU-55933 on the apoptosis of HIV-1 infected primary T lymphoblasts. PHA/IL-2 lymphoblasts from healthy donors (n = 3) were infected with the indicated HIV-1 strain (MOI 5, 48 h), followed by Hoechst 33342 staining and quantification of syncytia exhibiting apoptotic chromatin condensation. D. Reduced syncytial apoptosis of Herpes virus saimiri (HVS)-transformed T cells from A-T patients. CD4⁺ T lymphoblasts from an A-T patient and a healthy donor (Co.) were infected for 24 hours with HIV-1 WT or HIV1 IN^D64V at a MOI of 10, followed by determination of the frequency of nuclear apoptosis among syncytia by Hoechst 33342 staining. E,F. Comparison of T lymphoblasts from healthy and A-T patients. PHA/IL-2 lymphoblasts from controls or A-T patients (n = 3) were infected by HIV-1 WT, and the frequency of syncytia with Tunel⁺ nuclei was assessed. Representative syncytia are shown in E and quantitative data (X±SD, n = 3) are reported in F. G. Decreased HIV-1-induced apoptosis of immortalized T cells obtained from a patent with Nijmegen breakage syndrome (NBS). Cells from an NBS patient and a healthy donor (Co.) were infected for 48h hours with HIV-1 WT at a MOI of 10, and the frequency of syncytia bearing Tunel-positive nuclei was evaluated. H. Comparison of primary T lymphoblasts from healthy and NBS donors. PHA/IL-2 lymphoblasts from controls (n = 2) or NBS patients (n = 2) were infected. The frequency of syncytial apoptosis was determined by TUNEL assay. Asterisks indicate significant (p<0.01) effects of KU-55933, NBS or A-T-mutations as compared to the respective untreated or unmutated control cells.</p

DNA damage foci in karyogamic syncytia elicited by HIV-1 Env.

<p>A. ATM and histone H2AX phosphorylation. Syncytia were generated by coculture of HeLa CD4 and HeLa Env cells (36 h), followed by immunofluorescence double staining for the detection of ATMS1981P and γ-H2AX. Representative pre-karyogamic (pre-KG), karyogamic (KG), and apoptotic cells are shown. The inserts depict the staining with the chromatin-specific dye Hoechst 33342. B. Biochemical evidence for ATM and H2AX phosphorylation. Extemporaneous 1∶1 mixtures of HeLa Env and HeLa CD4 single cells (SC) or syncytia (48h, which are mixtures of pre-KG and KG cells) were subjected to immunoblot detection of total ATM, ATMS1981, total H2AX and γ-H2AX. C. Quantitation of ATM and H2AX phosphorylation in distinct categories of syncytia, as determined in A. D. Time course of ATM, H2AX and caspase activation in syncytia, as determined by immunofluorescence stainings with anti-ATMS1981P, anti-γ-H2AX (as in A) and an antibody only recognizing active caspase-3. In addition, karyogamy and apoptotic chromatin condensation was assessed (X±SD, n = 3). E. Role of cell fusion in ATM activation. Single cell (SC) or HeLa Env/HeLa CD4 cocultures were performed in standard conditions (as in A), in the absence or presence of the fusion inhibitor AMD3100. Alternatively, HeLa CD4 and HeLa Env cells were subjected to polyethylene glycol (PEG)-enforced cell fusion, either separatedly or in mixed cultures, maintained in the absence or presence of AMD3100. Twenty four hours and 36 hours later, karyogamy, nuclear apoptosis, ATM phosphorylation was assessed by immunostaining. F,G. Inhibition of ATM activation by cell cycle arrest. Hela CD4 and HeLa Env cells were exposed twice overnight to thymidine (1 mM), which leads to an accumulation (synchronization) of cells in the early S-phase. Then, synchronized (“T-blocked”) or non-synchronized cells were cocultured in the absence or in the presence of thymidine (to leave or maintain the cell cycle blockade). Note that continuous blockade prevents BrdU incorporation (F), karyogamy as well as the phosphorylation of ATM (G). Results are means±SD of three independent experiments, each performed in triplicate.</p

DNA damage foci in vivo, in HIV-1-infected patients.

<p>A. Phosphorylation of ATM and H2AX in giant multinuclear cells present in the frontal cortex of patients with HIV-1-associated encephalitis (HAE). Frontal cortex sections from control subjects or untreated HIV-1 carriers without opportunistic infections at the time of death were subjected to the immunohistochemical detection of ATMS1981P or the ATM substrate γ-H2AX (insert), using the same antibodies as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002458#pone-0002458-g001" target="_blank">Fig. 1</a>. Arrows indicate syncytia and inserts show higher magnifications. Control brains from HIV-1⁻ subjects did not contain syncytia. The percentage of syncytia (X±SEM) staining positively for the indicated antigens were determined for six different HIV-1 carriers (A). Note that <5% of mononuclear cells were positive for any of the indicated antigens. B. DNA damage foci in lymph nodes from HIV-1 carriers. Lymph node biopsies from control subjects or untreated HIV-1 carriers were stained with the indicated antibodies Arrows and inserts show bona-fide syncytia. C, D. Correlation between ATM activation, H2AX phosphorylation, viral status and HAART treatment. PBMC from untreated patients with distinct levels of viremia were subjected to ATMS1981P and γ-H2AX staining. p values refer to the correlation coefficients (C). In addition, a cohort of uninfected donors, untreated HIV-1 carriers, and HAART-treated subjects with undetectable viral titers were analyzed for ATM activation and γ-H2AX positivity (D). Note that that some but not all of the patient samples could be analyzed for all indicated parameters.</p

Hypothetical signaling cascade leading to syncytial apoptosis.

<p>Sequential events leading to syncytial cell death in HIV-1 infection are shown. All inhibitory agents used throughout the paper are listed.</p

Activation and contribution of ATM to syncytial apoptosis of HIV-1-infected CD4⁺cell lines.

<p>A–D. CD4⁺ HeLa cells were infected with HIV-1 WT, HIV-1 Env^VSV and HIV1 IN^D64V at a MOI of 10 for 38 h and then stained for ATMS1981P (A) or all other markers indicating syncytial apoptosis. The percentages (X±SD, n  = 3) of syncytia exhibiting the indicated characteristics were quantified at an MOI of 10 (B), and the percentage of nuclei with apoptotic chromatin condensation (usual within syncytia) was quantified at different MOI (C). CD4⁺ HeLa cells were either transfected with specific siRNAs depleting ATM or treated with the ATM inhibitor KU-55933. Then, cells were infected with HIV-1 WT or HIV1 IN^D64V at a MOI of 10 for 48 h, and the expression of the viral protein p24, the activation of ATM or p53, and apoptosis were quantified (D). Results are plotted as the percent inhibition of the indicated parameters determined for syncytia (excluding single cells) compared to control siRNA transfected or untransfected cells. E. Effect of NBS1 knockdown on syncytial apoptosis of HIV-1 infected CD4⁺ HeLa cells. CD4⁺ HeLa cells were transfected with NBS1-specific or control siRNAs, and infected for 48h with HIV-1 WT (MOI of 10), followed by quantification of the indicated parameters by immunofluorescence. F, G. Effect of ATM inhibition on syncytial apoptosis in HIV-1 infected CEM T cells. Cells were infected with wild type HIV-1 in the presence (or in the absence) of the ATM inhibitor KU-55933, and nuclear apoptosis was determined 72 h later (F). Moreover, the number of infectious viruses contained in the culture supernatant was determined (G). Asterisks in F and G indicate significant inhibitory effects of KU-55933.</p