Laboratory for Proteinscience, Proteomics and Epigenetic Signaling (PPES), Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium.
J Immunol Methods. 2011 Sep 30;372(1-2):52-64. doi: 10.1016/j.jim.2011.06.028. Epub 2011 Jul 19.
Expression studies on specific host proteins predominantly use quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based assay to study intracellular expression levels of three important host proteins involved in HIV-1 replication: apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and lens epithelium-derived growth factor (LEDGF/p75). An indirect intracellular staining (ICS) method was optimized using antibodies designed for other applications like enzyme-linked immunosorbent assay (ELISA), confocal imaging, and western blotting. The median fluorescence intensity (MFI) value--a measure for the protein expression level--increased upon higher antibody concentration and longer incubation time, and was reduced following preincubation with recombinant proteins. Staining of stably transfected or knock-down cell lines supported the method's specificity. Moreover, confocal microscopy analysis of peripheral blood mononuclear cells (PBMC), when stained according to the ICS method, confirmed the localization of APOBEC3G and TRIM5α in the cytoplasm, and of LEDGF/p75 in the nucleus. Also, stimulation with mitogen, interferon-alpha, or interferon-beta resulted in detectable, albeit weak, increases in intracellular expression of APOBEC3G and TRIM5α. After optimization, the method was applied to healthy control and HIV-1 infected subjects. For all subjects studied, the memory subset of CD4+ T cells showed significantly higher expression levels of APOBEC3G, TRIM5α, and LEDGF/p75, while the CD16+ subset of monocytes was characterized by higher expression levels of LEDGF/p75. In addition, we observed that therapy-naïve HIV-1 patients tended to have lower expression levels of APOBEC3G and TRIM5α than HIV-1 negative controls. In summary, our data provide proof-of-principle for the detection of specific host factors at the level of a single cell, which may prove useful for our further understanding of their role in virus-host interactions.
表达研究特定的宿主蛋白主要使用定量 PCR 和蛋白质印迹分析。在这项研究中，我们优化了一种基于流式细胞术的测定法，以研究三种参与 HIV-1 复制的重要宿主蛋白的细胞内表达水平：载脂蛋白 B mRNA 编辑酶催化多肽样 3G（APOBEC3G），三肽基 5α（TRIM5α）和晶状体上皮衍生的生长因子（LEDGF/p75）。使用针对酶联免疫吸附测定（ELISA），共聚焦成像和蛋白质印迹等其他应用设计的抗体优化了间接细胞内染色（ICS）方法。抗体浓度越高和孵育时间越长，中荧光强度（MFI）值-一种用于蛋白质表达水平的测量-就会增加，而在用重组蛋白预孵育后则会降低。稳定转染或敲低细胞系的染色支持该方法的特异性。此外，根据 ICS 方法对外周血单核细胞（PBMC）进行的共聚焦显微镜分析证实了 APOBEC3G 和 TRIM5α在细胞质中的定位以及 LEDGF/p75 在核中的定位。此外，有丝分裂原，干扰素-α或干扰素-β的刺激导致 APOBEC3G 和 TRIM5α的细胞内表达可检测到，尽管微弱增加。优化后，该方法应用于健康对照和 HIV-1 感染的受试者。对于所有研究的受试者，CD4+T 细胞的记忆亚群显示出 APOBEC3G，TRIM5α和 LEDGF/p75 的表达水平明显更高，而单核细胞的 CD16+亚群的 LEDGF/p75 表达水平更高。此外，我们观察到未经治疗的 HIV-1 患者的 APOBEC3G 和 TRIM5α表达水平倾向于低于 HIV-1 阴性对照。综上所述，我们的数据为检测单个细胞水平的特定宿主因子提供了原理证明，这可能有助于我们进一步了解它们在病毒-宿主相互作用中的作用。
