HIV Vpr, CRL4.DCAF1 E3 ligase and their targets

项目总结 对HIV的固有免疫力被辅助毒力因子所抵消，后者通常对E3Ub进行重新编程 连接酶通过使受感染细胞被蛋白酶体降解来清除感染细胞中的艾滋病毒限制因子。 事实上，对艾滋病毒辅助蛋白的研究导致了对艾滋病毒主要限制因素的确定， 这反过来又揭示了新的抗病毒机制。HIV-1 VPR辅助蛋白与CRL4DCAF1 E3泛素结合 连接酶，并使用这种酶来对抗特定的复制后DNA修复蛋白，如尿嘧啶DNA 糖基酶(UNG2)、HLTFDNA解旋酶和MUS81结构特异性核酸酶。此外，我们最近 鉴定外切核酸酶1(Exo1)DNA修复蛋白是VPR CRL4DCAF1 E3劫持的新靶点。 据报道，HIV-1 vpr还结合了dna加工蛋白，而dna加工蛋白与一个大的蛋白质复合体有关。 组装在SLX4支架上，连接不同的DNA修复途径。VPR与之交互的事实 并扰乱细胞DNA修复机制的多个方面是耐人寻味的，因为VPR几乎是毫无疑问的 与这一机制的对抗最终应该有利于HIV-1。然而，人们对此知之甚少。 VPR靶向DNA修复蛋白对HIV-1复制的影响及其机制(S) 没有得到彻底的调查。 这个应用集中在DNA修复蛋白是如何被vpr拮抗的以及那些由vpr控制的 SLX4支架对HIV-1复制的影响。我们最近证实，HIV-1 vpr促进HIV-1复制 并将这种作用与VPR通过CRL4DCAF1 E3拮抗DNA修复蛋白的能力联系起来。我们 也证明了一种由SLX4支架控制的未知成分抑制HIV-1 感染。我们假设上述DNA修复蛋白作用于合成 在前病毒整合之前或之后的双链HIV-1 cDNA，从而抑制HIV-1感染，以及 这些影响中的一些可以被HIV-1 VPR抵消。在这里，我们建议描述相互作用的特征 VPR靶向的DNA修复蛋白以及SLX4支架控制的DNA修复蛋白与HIV-1 并阐明它们是如何抑制HIV-1复制的。特别是，我们将描述DNA的行为 修复被VPR拮抗的蛋白质以刺激HIV-1复制(SA1)，识别SLX4 Complex和VPR-CRL4DCAF1 E3靶向HLTF和Exo1作用于(SA2)，并表征DNA修复途径 和抑制HIV-1(SA3)的活性。总体而言，本申请将描述分子机制 通过细胞DNA修复机制介导T淋巴细胞分裂过程中HIV-1复制的抑制 那些被HIV-1VPR用来对抗这种抑制的化合物。

PROJECT SUMMARY Intrinsic immunity to HIV is counteracted by accessory virulence factors, which, typically, reprogram E3 Ub ligases to remove HIV restriction factors from infected cells by targeting them for degradation by proteasome. Indeed, the studies of HIV accessory proteins have led to the identification of major HIV restriction factors, which in turn revealed new anti-viral mechanisms. HIV-1 Vpr accessory protein binds to CRL4DCAF1 E3 ubiquitin ligase and uses this enzyme to antagonize specific postreplication DNA repair proteins, such as uracil DNA glycosylase (UNG2), HLTF DNA helicase and MUS81 structure specific nuclease. Furthermore, we recently identified Exonuclease 1 (Exo1) DNA repair protein as a novel target of the hijacked by Vpr CRL4DCAF1 E3. HIV-1 Vpr was also reported to bind DNA processing proteins, which associate with a large protein complex assembled on SLX4 scaffold that connects to diverse DNA repair pathways. The fact that Vpr interacts with and disrupts multiple aspects of the cellular DNA repair machinery is intriguing, as there is little doubt that Vpr antagonism with this machinery should ultimately benefit HIV-1. Nevertheless, very little is known about how the DNA repair proteins targeted by Vpr impinge on HIV-1 replication and the underlying mechanism(s) have not been thoroughly investigated. This application focuses on how DNA repair proteins antagonized by Vpr and those controlled by the SLX4 scaffold impinge on HIV-1 replication. We recently confirmed that HIV-1 Vpr promotes HIV-1 replication in dividing T cells and linked this effect to Vpr's ability to antagonize DNA repair proteins via CRL4DCAF1 E3. We also demonstrated that an as yet unidentified component controlled by the SLX4 scaffold inhibits HIV-1 infection. We hypothesize that the above DNA repair proteins act on intermediates in the synthesis of the double stranded HIV-1 cDNA prior to, or following provirus integration, thereby inhibiting HIV-1 infection, and that some of these effects are counteracted by HIV-1 Vpr. Here we propose to characterize the interactions between DNA repair proteins targeted by Vpr, as well as those controlled by the SLX4 scaffold, with the HIV-1 cDNA and elucidate how they inhibit HIV-1 replication. In particular, we will characterize the actions of DNA repair proteins that are antagonized by Vpr to stimulate HIV-1 replication (SA1), identify steps that the SLX4 complex and Vpr-CRL4DCAF1 E3-targeted HLTF and Exo1 act on (SA2), and characterize DNA repair pathways and activities that inhibit HIV-1 (SA3). Overall, this application will describe the molecular mechanisms mediating the inhibition of HIV-1 replication in dividing T lymphocytes by cellular DNA repair machinery, and those used by HIV-1 Vpr to antagonize the inhibition.