Department of Molecular Biology and Microbiology, Case Western Reserve School of Medicine, Cleveland, Ohio, USA.
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
mBio. 2018 Oct 23;9(5):e01732-18. doi: 10.1128/mBio.01732-18.
Viral accessory proteins hijack host cell E3 ubiquitin ligases to antagonize innate/intrinsic defenses and thereby provide a more permissive environment for virus replication. Human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr reprograms CRL4 E3 to antagonize select postreplication DNA repair enzymes, but the significance and role of these Vpr interactions are poorly understood. To gain additional insights, we performed a focused screen for substrates of CRL4 E3 reprogrammed by HIV-1 Vpr among known postreplication DNA repair proteins and identified exonuclease 1 (Exo1) as a novel direct HIV-1 Vpr target. We show that HIV-1 Vpr recruits Exo1 to the CRL4 E3 complex for ubiquitination and subsequent proteasome-dependent degradation and that Exo1 levels are depleted in HIV-1-infected cells in a Vpr-dependent manner. We also show that Exo1 inhibits HIV-1 replication in T cells. Notably, the antagonism of Exo1 is a conserved function of main group HIV-1 and its ancestor Vpr proteins in the simian immunodeficiency virus from chimpanzee (SIVcpz) lineage, further underscoring the relevance of our findings. Overall, our studies (i) reveal that HIV-1 Vpr extensively remodels the cellular postreplication DNA repair machinery by impinging on multiple repair pathways, (ii) support a model in which Vpr promotes HIV-1 replication by antagonizing select DNA repair enzymes, and (iii) highlight the importance of a new class of restrictions placed on HIV-1 replication in T cells by the cellular DNA repair machinery. HIV-1 polymerase reverse transcribes the viral RNA genome into imperfectly double-stranded proviral DNA, containing gaps and flaps, for integration into the host cell chromosome. HIV-1 reverse transcripts share characteristics with cellular DNA replication intermediates and are thought to be converted into fully double-stranded DNA by cellular postreplication DNA repair enzymes. Therefore, the finding that the HIV-1 accessory protein Vpr antagonizes select postreplication DNA repair enzymes that can process HIV-1 reverse transcripts has been surprising. Here, we show that one such Vpr-antagonized enzyme, exonuclease 1, inhibits HIV-1 replication in T cells. We identify exonuclease 1 as a member of a new class of HIV-1 restriction factors in T cells and propose that certain modes of DNA "repair" inhibit HIV-1 infection.
病毒辅助蛋白劫持宿主细胞 E3 泛素连接酶，以拮抗先天/内在防御，从而为病毒复制提供更宽松的环境。人类免疫缺陷病毒 1 型（HIV-1）辅助蛋白 Vpr 重新编程 CRL4 E3 以拮抗选择的复制后 DNA 修复酶，但这些 Vpr 相互作用的意义和作用知之甚少。为了获得更多的见解，我们在已知的复制后 DNA 修复蛋白中进行了针对 HIV-1 Vpr 重新编程的 CRL4 E3 底物的重点筛选，并鉴定出外切核酸酶 1（Exo1）为 HIV-1 Vpr 的新的直接靶标。我们表明，HIV-1 Vpr 将 Exo1 募集到 CRL4 E3 复合物中进行泛素化和随后的蛋白酶体依赖性降解，并且 HIV-1 感染细胞中的 Exo1 水平以 Vpr 依赖的方式耗尽。我们还表明，Exo1 抑制 T 细胞中的 HIV-1 复制。值得注意的是，Exo1 的拮抗作用是主要组 HIV-1 和其祖先 Vpr 蛋白在来自黑猩猩的猿猴免疫缺陷病毒（SIVcpz）谱系中的保守功能，进一步强调了我们研究结果的相关性。总体而言，我们的研究（i）揭示了 HIV-1 Vpr 通过影响多种修复途径，广泛重塑细胞复制后 DNA 修复机制，（ii）支持了 Vpr 通过拮抗选择的 DNA 修复酶促进 HIV-1 复制的模型，以及（iii）突出了细胞 DNA 修复机制对 T 细胞中 HIV-1 复制的新的一类限制的重要性。HIV-1 聚合酶将病毒 RNA 基因组逆转录成含有缺口和瓣的不完全双链前病毒 DNA，用于整合到宿主细胞染色体中。HIV-1 逆转录产物与细胞 DNA 复制中间体具有共同特征，并且被认为通过细胞复制后 DNA 修复酶转化为完全双链 DNA。因此，HIV-1 辅助蛋白 Vpr 拮抗可处理 HIV-1 逆转录产物的选择复制后 DNA 修复酶的发现令人惊讶。在这里，我们表明，Vpr 拮抗的一种酶，外切核酸酶 1，抑制 T 细胞中的 HIV-1 复制。我们将外切核酸酶 1 鉴定为 T 细胞中 HIV-1 限制因子的新类别之一，并提出某些 DNA“修复”模式抑制 HIV-1 感染。
