The chaperone protein p32 stabilizes HIV-1 Tat and strengthens the p-TEFb/RNAPII/TAR complex promoting HIV transcription elongation.
The chaperone protein p32 stabilizes HIV-1 Tat and strengthens the p-TEFb/RNAPII/TAR complex promoting HIV transcription elongation.
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DOI：
10.1073/pnas.2217476120
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发表时间：
2023-01-03
影响因子：
11.1
通讯作者：
Valente, Susana T.
中科院分区：
文献类型：
--
作者：
Li, Chuan;Mori, Luisa P.;Lyu, Shuang;Bronson, Ronald;Getzler, Adam J.;Pipkin, Matthew E.;Valente, Susana T.
此处所列结果突出显示：1）鉴定调节HIV转录的宿主因子的染色质亲和纯化策略; 2）鉴定一些尚不清楚的调节HIV转录的宿主因子，即支架蛋白p32; 3）p32增强HIV反式激活因子达特蛋白的半衰期并增强其与反式激活应答元件（TAR）p-TEFb的结合，3）达特抑制剂dCA除了阻断Tat-TAR相互作用外，还破坏了达特与p32的结合，这部分解释了它的作用; 4）破坏p32与达特的相互作用可用于针对HIV的治疗干预。HIV基因表达受HIV转录激活因子、达特、宿主转录因子和染色质重塑复合物的组合活性调节。为了鉴定调节HIV转录的宿主因子，我们使用特异性单向导RNA和内切核酸酶缺陷型Cas9对整合的HIV启动子进行染色质亲和纯化，然后进行质谱分析。支架蛋白p32，也称为ASF/SF 2剪接因子相关蛋白，被鉴定为活跃转录HIV启动子中存在但沉默启动子中不存在的最富集因子之一。染色质免疫沉淀分析证实了活性HIV启动子上的p32的存在及其由达特增强的募集。HIV使用达特有效地将正转录延伸因子B（p-TEF B）（CDK 9/CCNT 1）募集到TAR（从HIV转录物的前59 bp形成的RNA二级结构），以增强RNAPII转录延伸。p32的RNA干扰显著降低了原代CD 4 +T细胞和HIV慢性感染细胞中的HIV转录，与HIV剪接或p32抗剪接活性无关。相反，p32的过表达特异性地增加Tat依赖的HIV转录。发现p32与达特的碱性结构域直接相互作用，增强了达特的稳定性和半衰期。相反，p32通过N-和C-末端结构域与达特结合。可能由于其支架特性，p32还促进了达特与TAR、p-TEFb和RNAPII的结合，从而增强了Tat依赖性HIV转录。总之，我们确定p32作为宿主因子，其与达特蛋白相互作用并稳定该蛋白，促进依赖于塔特的转录调控，并且可以探索用于HIV靶向转录抑制。
Results presented here highlight: 1) a chromatin affinity purification strategy to identify host factors regulating HIV transcription; 2) identification of a few yet unknown host factors regulating HIV transcription, namely the scaffold protein p32; 3) p32 enhances the half-life of the HIV transactivator Tat protein and strengthens its association with trans-activation response element (TAR), p-TEFb, and RNAPII to promote polymerase transcriptional elongation and HIV amplification; 3) the Tat inhibitor dCA, besides blocking Tat–TAR interaction, was found to disrupt Tat binding to p32 partly explaining its potency; 4) disrupting p32 interaction with Tat may be explored for therapeutic interventions against HIV. HIV gene expression is modulated by the combinatorial activity of the HIV transcriptional activator, Tat, host transcription factors, and chromatin remodeling complexes. To identify host factors regulating HIV transcription, we used specific single-guide RNAs and endonuclease-deficient Cas9 to perform chromatin affinity purification of the integrated HIV promoter followed by mass spectrometry. The scaffold protein, p32, also called ASF/SF2 splicing factor-associated protein, was identified among the top enriched factors present in actively transcribing HIV promoters but absent in silenced ones. Chromatin immunoprecipitation analysis confirmed the presence of p32 on active HIV promoters and its enhanced recruitment by Tat. HIV uses Tat to efficiently recruit positive transcription elongation factor b (p-TEFb) (CDK9/CCNT1) to TAR, an RNA secondary structure that forms from the first 59 bp of HIV transcripts, to enhance RNAPII transcriptional elongation. The RNA interference of p32 significantly reduced HIV transcription in primary CD4+T cells and in HIV chronically infected cells, independently of either HIV splicing or p32 anti-splicing activity. Conversely, overexpression of p32 specifically increased Tat-dependent HIV transcription. p32 was found to directly interact with Tat’s basic domain enhancing Tat stability and half-life. Conversely, p32 associates with Tat via N- and C-terminal domains. Likely due its scaffold properties, p32 also promoted Tat association with TAR, p-TEFb, and RNAPII enhancing Tat-dependent HIV transcription. In sum, we identified p32 as a host factor that interacts with and stabilizes Tat protein, promotes Tat-dependent transcriptional regulation, and may be explored for HIV-targeted transcriptional inhibition.
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