Choma Maja K, Lumb Jennifer, Kozik Patrycja, Robinson Margaret S
University of Cambridge, Cambridge Institute for Medical Research, Cambridge, CB2 0XY, United Kingdom.
PLoS One. 2015 Oct 14;10(10):e0140404. doi: 10.1371/journal.pone.0140404. eCollection 2015.
The HIV-1-encoded protein, Nef, plays a key role in the development of AIDS. One of Nef's functions is to keep MHC class I off the surface of infected cells, a process that requires the host proteins clathrin and AP-1. To identify other proteins involved in this pathway, we carried out a genome-wide siRNA library screen on HeLa cells co-expressing HLA-A2 and an inducible form of Nef. Out of 21,121 siRNA pools, 100 were selected for further analysis, based on their ability to either inhibit or enhance downregulation of MHC-I by Nef. When cells were treated with the same siRNA pools as those used in the screen, 79% produced a similar phenotype. However, when the cells were treated with different siRNA reagents targeting the same genes, only 16% produced a similar phenotype. This indicates that most of the hits found in the original screen are likely to have been off-target, an important concern that is often not taken into account in siRNA screening studies. Nevertheless, we identified novel host factors involved in Nef-induced downregulation of MHC-I, including four genes, MIIP, CAMSAP3, SLC6A3, and KCTD19, where multiple reagents produced a strong inhibitory effect on Nef activity. Other hits slightly below our very high stringency cutoff point may also deserve further study. Thus, our dataset is a valuable resource for scientists investigating the pathogenesis of HIV.
人类免疫缺陷病毒1型（HIV-1）编码的蛋白Nef在艾滋病发展过程中起关键作用。Nef的功能之一是使MHC I类分子不在被感染细胞表面表达，这一过程需要宿主蛋白网格蛋白和AP-1。为了鉴定参与该途径的其他蛋白，我们对共表达HLA-A2和可诱导形式Nef的HeLa细胞进行了全基因组siRNA文库筛选。在21,121个siRNA池当中，基于它们抑制或增强Nef介导的MHC-I下调的能力，选择了100个进行进一步分析。当用与筛选中相同的siRNA池处理细胞时，79%产生了相似的表型。然而，当用靶向相同基因的不同siRNA试剂处理细胞时，只有16%产生了相似的表型。这表明在最初筛选中发现的大多数命中结果可能是脱靶效应，而这一重要问题在siRNA筛选研究中常常未被考虑。尽管如此，我们鉴定出了参与Nef诱导的MHC-I下调的新宿主因子，包括四个基因，即MIIP、CAMSAP-3、SLC6A3和KCTD19，多种试剂对这些基因产生了对Nef活性的强烈抑制作用。其他略低于我们非常严格的筛选标准的命中结果可能也值得进一步研究。因此，我们的数据集是科学家研究HIV发病机制的宝贵资源。
