Impact of the Ku Complex on HIV-1 Expression and Latency

Ku, a cellular complex required for human cell survival and involved in double strand break DNA repair and multiple other cellular processes, may modulate retroviral multiplication, although the precise mechanism through which it acts is still controversial. Recently, Ku was identified as a possible anti-human immunodeficiency virus type 1 (HIV-1) target in human cells, in two global approaches. Here we investigated the role of Ku on the HIV-1 replication cycle by analyzing the expression level of a panel of non-replicative lentiviral vectors expressing the green fluorescent protein in human colorectal carcinoma HCT 116 cells, stably or transiently depleted of Ku. We found that in this cellular model the depletion of Ku did not affect the efficiency of (pre-)integrative steps but decreased the early HIV-1 expression by acting at the transcriptional level. This negative effect was specific of the HIV-1 promoter, required the obligatory step of viral DNA integration and was reversed by transient depletion of p53. We also provided evidence on a direct binding of Ku to HIV-1 LTR in transduced cells. Ku not only promotes the early transcription from the HIV-1 promoter, but also limits the constitution of viral latency. Moreover, in the presence of a normal level of Ku, HIV-1 expression was gradually lost over time, likely due to the counter-selection of HIV-1-expressing cells. On the contrary, the reactivation of transgene expression from HIV-1 by means of trichostatin A- or tumor necrosis factor α-administration was enhanced under condition of Ku haplodepletion, suggesting a phenomenon of provirus latency. These observations plead in favor of the hypothesis that Ku has an impact on HIV-1 expression and latency at early- and mid-time after integration.Ku蛋白（Ku）作为人类细胞存活所必需的细胞复合物，参与DNA双链断裂修复及多种其他细胞生理过程，或可调控逆转录病毒的增殖，但其具体作用机制仍存在争议。近期两项全局研究已证实，Ku可作为人类细胞中潜在的抗人类免疫缺陷病毒1型（HIV-1）靶点。本研究通过分析表达绿色荧光蛋白（GFP）的非复制型慢病毒载体在稳定或瞬时敲低Ku的结直肠癌HCT 116细胞中的表达水平，探究了Ku在HIV-1复制周期中的作用。实验结果显示，在该细胞模型中，Ku敲低并未影响HIV-1的整合前步骤效率，但通过转录层面的调控降低了HIV-1的早期表达。该抑制效应具有HIV-1启动子特异性，且依赖于病毒DNA整合的必需步骤，同时可通过瞬时敲低p53得以逆转。本研究还证实，Ku可在转导细胞中直接结合HIV-1长末端重复序列（LTR）。Ku不仅可促进HIV-1启动子介导的早期转录，还可抑制病毒潜伏的建立。此外，在Ku正常表达水平下，HIV-1的表达会随时间逐渐消失，这可能源于表达HIV-1的细胞被反向选择清除。与之相反，在Ku单倍体敲低（haplodepletion）的条件下，通过曲古抑菌素A（trichostatin A）或肿瘤坏死因子α（TNF-α）处理以重新激活HIV-1转基因表达的效果会显著增强，这提示Ku参与了前病毒潜伏过程。上述研究结果支持如下假说：Ku可在病毒整合后的早期及中期阶段对HIV-1的表达与潜伏产生调控作用。