FIG (5-6 and 7 (graphical abstract)) Telomere and ATM dynamics in CD4 T cell depletion in active and virus-suppressed HIV-1 infection

Fig.5. DNA damage response in HIV-infected SupT1 and CD4 T cells with or without RAL treatment. A-B) Confocal microscopy analysis of 53BP1 and TRF1 co-localization as a marker for dysfunctional telomere-induced foci (TIF) in SupT1 and primary CD4 T cells with HIV-1 infection. C-D) Representative overlaid histograms and summary data of mean fluorescence intensity (MFI) of telomere lengths in SupT1 cells or primary CD4 T cells, with or without HIV-1 infection and RAL treatment, as measured by Flow-FISH at the indicated times.

Fig.6. The PI3K/ATM pathways are dysregulated in HIV-infected T cells and promote DDR and cell apoptosis. A) The microarray results of PI3K/ATM-related gene expressions in SupT1 cells with or without HIV-1 infection at day 3. B) RT-PCR analysis of p24 and ATM/AKT-related gene expressions in SupT1 cells with or without HIV-1 infection at day 3 and day 6 after HIV-1 infection. C) Array results of PI3K/ATM-related gene expressions in primary CD4 T cells with or without HIV-1 infection at day 5. D) RT-PCR analysis of p24 and ATM/AKT-related gene expressions in primary CD4 T cells at day 5 after HIV-1 infection. E) Western blot analysis of p24, ATM, CHK2, AKT, γH2AX, and PARP-1 expressions in SupT1 cells at the early (day 3) and late (day 6) phase of HIV-1 infection. F) Western blot analysis of p24, ATM, CHK2, AKT, γH2AX and PARP-1 expressions in primary CD4 cells at the early (day 3) and late (day 5) phase of HIV-1 infection. G) Western blot analysis of p24, ATM, pATM, CHK2, pCHK2, AKT, pAKT, γH2AX and PARP-1 expressions in SupT1 cells at day 6 after HIV infection and RAL treatment. H) Western blot analysis of p24, ATM, CHK2, pCHK2, AKT and pAKT expressions in primary CD4 T cells at day 5 after HIV infection and RAL treatment.

Fig.7. Model of HIV-1 infection in CD4 T cells. The PI3K/ATM pathways are initially activated and then become deficient, causing telomeric DNA damage and erosion, CD4 T cell apoptosis and depletion, during HIV-1 infection. ART can partially reverse this process by inhibiting HIV-1 replication; however, PI3K/ATM pathways will remain dysregulated in these virus-suppressed, ART-treated CD4 T cells, and thus, the cells will survive but in an early apoptotic state in this setting of latent HIV-1 infection and/or inflammation.

图5　经或不经雷替拉韦（RAL）处理的HIV感染SupT1细胞与原代CD4 T细胞的DNA损伤应答。A-B：对HIV-1感染的SupT1细胞及原代CD4 T细胞中作为功能失调端粒诱导灶（TIF）标志物的53BP1与TRF1共定位进行共聚焦显微镜分析。C-D：在指定时间点通过流式荧光原位杂交（Flow-FISH）检测的、经或不经HIV-1感染与RAL处理的SupT1细胞或原代CD4 T细胞的端粒长度平均荧光强度（MFI）代表性叠加直方图与汇总数据。

图6　HIV感染T细胞中磷脂酰肌醇3-激酶/毛细血管扩张性共济失调突变激酶（PI3K/ATM）通路失调，并可促进DNA损伤应答（DDR）与细胞凋亡。A：感染后第3天的SupT1细胞经或不经HIV-1感染时的PI3K/ATM相关基因表达基因芯片（microarray）结果。B：感染后第3天与第6天的SupT1细胞经或不经HIV-1感染时的p24与ATM/AKT相关基因表达逆转录聚合酶链反应（RT-PCR）分析。C：感染后第5天的原代CD4 T细胞经或不经HIV-1感染时的PI3K/ATM相关基因表达基因芯片结果。D：感染后第5天的原代CD4 T细胞的p24与ATM/AKT相关基因表达RT-PCR分析。E：HIV-1感染早期（第3天）与晚期（第6天）的SupT1细胞中p24、ATM、CHK2、AKT、γH2AX及PARP-1表达的蛋白质印迹（Western blot）分析。F：HIV-1感染早期（第3天）与晚期（第5天）的原代CD4 T细胞中p24、ATM、CHK2、AKT、γH2AX及PARP-1表达的蛋白质印迹分析。G：经HIV感染并经RAL处理后第6天的SupT1细胞中p24、ATM、磷酸化ATM（pATM）、CHK2、磷酸化CHK2（pCHK2）、AKT、磷酸化AKT（pAKT）、γH2AX及PARP-1表达的蛋白质印迹分析。H：经HIV感染并经RAL处理后第5天的原代CD4 T细胞中p24、ATM、CHK2、pCHK2、AKT及pAKT表达的蛋白质印迹分析。

图7　CD4 T细胞HIV-1感染模型。HIV-1感染过程中，PI3K/ATM通路先被激活后发生功能缺陷，进而引发端粒DNA损伤与端粒耗竭、CD4 T细胞凋亡及耗竭。抗逆转录病毒治疗（ART）可通过抑制HIV-1复制部分逆转该过程；但在病毒被抑制且接受ART治疗的CD4 T细胞中，PI3K/ATM通路仍处于失调状态，因此在潜伏HIV-1感染及/或炎症背景下，这些细胞虽可存活，但会处于早期凋亡状态。