Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation.
Genome-wide shRNA screening identifies host factors involved in early endocytic events for HIV-1-induced CD4 down-regulation.
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DOI：
10.1186/s12977-014-0118-4
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发表时间：
2014-12-13
期刊：
影响因子：
3.3
通讯作者：
Verhasselt B
中科院分区：
文献类型：
--
作者：
Landi A;Vermeire J;Iannucci V;Vanderstraeten H;Naessens E;Bentahir M;Verhasselt B
CD4受体的下调是HIV-1感染的标志之一，它被认为赋予了病毒在体内的选择性复制优势。这一过程主要由三种病毒蛋白介导：Env、VPu和Nef。到目前为止，在HIV-1感染过程中导致感染细胞表面CD4耗尽的机制仍然只有部分特征。在这项研究中，我们试图识别和表征HIV-1诱导的CD4下调中的细胞宿主因素。为了确定参与CD4下调的宿主因素，我们在表达Nef的HeLa CD4+细胞中使用了一个全基因组靶向shRNA慢病毒文库作为CD4下调的诱导剂。我们鉴定了55个基因，主要编码参与网状蛋白介导的内吞作用不同步骤的蛋白质。为了确认和进一步选择命中，我们进行了几轮验证，使用具有不同目标序列的单个shRNA慢病毒载体在HIV-1感染的T细胞中下调基因。通过这一严格的验证设置，我们可以证明，与非靶向shRNA对照相比，DNM3(Dynamin 3)、SNX22(排序连接蛋白22)、ATP6AP1(ATPase、H+转运、溶酶体辅助蛋白1)、HRBL(HIV-Rev结合蛋白样)、IDH3G(异柠檬酸脱氢酶)、HSP90B1(热休克蛋白90 kDaβ成员1)和Eps15(表皮生长因子受体途径底物15)的下调显著增加了HIV感染的SupT1 T细胞中的CD4水平。此外，Eps15、DNM3、IDH3G和ATP6AP1基因敲除显著降低了HIV-1在T细胞中的复制。我们确定了7个基因是HIV-1介导的T细胞中CD4下调的细胞辅助因子。其中七个基因中的四个的缺失也大大减少了T细胞中HIV-1的复制。然而，这些基因除了在HIV介导的CD4下调中发挥作用外，还可能以另一种方式影响HIV-1的复制。我们的发现对HIV-1介导的CD4在质膜和早期内小体水平的下调提供了见解，并确定了四个可能的新的HIV-1复制辅助因素。本文的在线版本(doi：10.1186/s12977-0140118-4)包含补充材料，授权用户可以使用。
Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation. To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells. We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors. The online version of this article (doi:10.1186/s12977-014-0118-4) contains supplementary material, which is available to authorized users.
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