Guendel Irene, Meltzer Beatrix W, Baer Alan, Dever Seth M, Valerie Kristoffer, Guo Jia, Wu Yuntao, Kehn-Hall Kylene
National Center for Biodefense & Infectious Diseases, School of Systems Biology, George Mason University, Biomedical Research Lab, 10650 Pyramid Place, MS 1J5, Manassas, VA, 20110, USA.
Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA, 23298, USA.
Virol J. 2015 Mar 6;12:40. doi: 10.1186/s12985-015-0266-8.
Viruses have naturally evolved elegant strategies to manipulate the host's cellular machinery, including ways to hijack cellular DNA repair proteins to aid in their own replication. Retroviruses induce DNA damage through integration of their genome into host DNA. DNA damage signaling proteins including ATR, ATM and BRCA1 contribute to multiple steps in the HIV-1 life cycle, including integration and Vpr-induced G2/M arrest. However, there have been no studies to date regarding the role of BRCA1 in HIV-1 transcription.
Here we performed various transcriptional analyses to assess the role of BRCA1 in HIV-1 transcription by overexpression, selective depletion, and treatment with small molecule inhibitors. We examined association of Tat and BRCA1 through in vitro binding assays, as well as BRCA1-LTR association by chromatin immunoprecipitation.
BRCA1 was found to be important for viral transcription as cells that lack BRCA1 displayed severely reduced HIV-1 Tat-dependent transcription, and gain or loss-of-function studies resulted in enhanced or decreased transcription. Moreover, Tat was detected in complex with BRCA1 aa504-802. Small molecule inhibition of BRCA1 phosphorylation effector kinases, ATR and ATM, decreased Tat-dependent transcription, whereas a Chk2 inhibitor showed no effect. Furthermore, BRCA1 was found at the viral promoter and treatment with curcumin and ATM inhibitors decreased BRCA1 LTR occupancy. Importantly, these findings were validated in a highly relevant model of HIV infection and are indicative of BRCA1 phosphorylation affecting Tat-dependent transcription.
BRCA1 presence at the HIV-1 promoter highlights a novel function of the multifaceted protein in HIV-1 infection. The BRCA1 pathway or enzymes that phosphorylate BRCA1 could potentially be used as complementary host-based treatment for combined antiretroviral therapy, as there are multiple potent ATM inhibitors in development as chemotherapeutics.
病毒自然进化出了巧妙的策略来操纵宿主的细胞机制，包括劫持细胞DNA修复蛋白以帮助自身复制的方式。逆转录病毒通过将其基因组整合到宿主DNA中诱导DNA损伤。包括ATR、ATM和BRCA1在内的DNA损伤信号蛋白在HIV-1生命周期的多个步骤中发挥作用，包括整合和Vpr诱导的G2/M期阻滞。然而，迄今为止尚无关于BRCA1在HIV-1转录中作用的研究。
在此，我们通过过表达、选择性缺失以及用小分子抑制剂处理等方式进行了各种转录分析，以评估BRCA1在HIV-1转录中的作用。我们通过体外结合试验检测了Tat与BRCA1的结合情况，并通过染色质免疫沉淀检测了BRCA1与长末端重复序列（LTR）的结合情况。
发现BRCA1对病毒转录很重要，因为缺乏BRCA1的细胞显示出HIV-1 Tat依赖性转录严重降低，功能获得或丧失研究导致转录增强或降低。此外，检测到Tat与BRCA1 aa504 - 802形成复合物。对BRCA1磷酸化效应激酶ATR和ATM的小分子抑制降低了Tat依赖性转录，而Chk2抑制剂则无作用。此外，在病毒启动子处发现了BRCA1，用姜黄素和ATM抑制剂处理降低了BRCA1与LTR的结合。重要的是，这些发现已在高度相关的HIV感染模型中得到验证，表明BRCA1磷酸化影响Tat依赖性转录。
BRCA1在HIV-1启动子处的存在突出了这种多面蛋白在HIV-1感染中的新功能。BRCA1途径或使BRCA1磷酸化的酶可能潜在地用作联合抗逆转录病毒疗法基于宿主的补充治疗，因为有多种强效ATM抑制剂正在作为化疗药物进行研发。
