Investigation of the HIV-1 matrix interactome during virus replication.
Investigation of the HIV-1 matrix interactome during virus replication.
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DOI：
10.1002/prca.201400189
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发表时间：
2016-02
期刊：
影响因子：
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通讯作者：
Belshan M
中科院分区：
文献类型：
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作者：
Li Y;Frederick KM;Haverland NA;Ciborowski P;Belshan M
像所有病毒一样，人类免疫缺陷病毒1型（HIV-1）需要宿主细胞因子进行生产性复制。这些因素的鉴定可能会导致新的细胞为基础的抑制剂的发展。将Strep标签插入HIV-1 gag基因的Matrix区的C末端。所得病毒具有复制能力并用于感染Jurkat T细胞。用Strep-Tactin琼脂糖亲和纯化基质复合物。使用SWATH质谱法进行蛋白质定量，将数据进行log-2转换，并使用Bonferroni校正的学生t检验来确定统计学显著性。通过免疫印迹验证了几种候选蛋白，并通过siRNA敲低测定研究了它们在病毒感染中的作用。共发现17种蛋白质在感染与未感染和未标记的对照样品之间存在统计学差异。通过免疫印迹法证实Ku 70、Ku 80和YB-1与基质相互作用。敲除两种候选物EZRIN和YB-1，增强了体外HIV感染。Strep标签允许在病毒复制的情况下捕获病毒蛋白复合物。几个先前描述的因素被确定，并发现至少有两个候选蛋白质在HIV-1感染中发挥作用。这些数据进一步增加了我们对HIV-宿主细胞相互作用的理解。
Like all viruses, Human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. A Strep-tag was inserted into the C-terminus of the Matrix region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. Matrix complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using SWATH mass spectrometry, data was log-2 transformed, and student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. Ku70, Ku80 and YB-1 were confirmed to interact with Matrix by immunoblot. Knockdown of two candidates, EZRIN and YB-1, enhanced HIV infection in vitro. The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV-host cell interactions.