JoVE Journal > Immunology and Infection
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Immunology and Infection

通过转版免疫沉淀和质谱测定,在HIV-1复制过程中核子因子的识别

Published: June 26, 2019
doi:
10.3791/59329
, , , , , ,
1Molecular and Cellular Biology Department,Beckman Research Institute at the City of Hope, 2Irell & Manella Graduate School of Biological Sciences, 3Department of Molecular Immunology,Beckman Research Institute and the City of Hope

Summary

在这里,我们描述了在质谱中存在HIV-1复制的Rev免疫沉淀。所述方法可用于识别艾滋病毒-1感染周期中涉及的核素因子,并适用于其他疾病模型,用于对研究不足的途径进行表征。

Abstract

HIV-1感染周期需要病毒蛋白与宿主因子相互作用,以促进病毒复制、包装和释放。感染周期进一步要求形成具有HIV-1 RNA的病毒/宿主蛋白复合物,以调节拼接和使核细胞质迁移。HIV-1 Rev蛋白通过具有非电子cis作用靶点-Rev响应元件(RRE)的多面化实现HIV-1 mRNA的核出口。富精素图案(ARM)的COOH-总站内存在核定位信号(NoLS),允许在核素中积累Rev/RRE复合物。除了调解与mRNA无关的核出口和拼接外,还推测核因子通过各种其他功能支持HIV-1的传染周期。在存在HIV-1复制的质谱中,我们描述了一种野生型(WT)Rev的免疫沉淀方法,与Rev核苷酸突变(删除和单点Rev-NoLS突变)相比。与核细胞质迁移相关的核核素因子(核细胞因子B23和核素C23)以及细胞拼接因子,在Rev-NoLS突变存在时失去与Rev的相互作用。各种其他核因子,如snoRNA C/D盒58,被识别失去与Rev突变的相互作用,但他们在HIV-1复制周期的功能仍然未知。这里介绍的结果表明,这种方法用于识别维持HIV-1感染周期的病毒/宿主核素因子。该方法中使用的概念适用于需要描述研究不足的通路的其他病毒和疾病模型。

Introduction

核细胞被假定为病毒复制所需的各种细胞宿主和病毒因子的相互作用场。核细胞是一个复杂的结构,分为三个不同的隔间:纤维室,密集的纤维室和粒状隔间。HIV-1 Rev 蛋白专门在粒状隔间内进行本地化;但是,此本地化模式的原因未知。在 NoLS 序列中存在单点突变(Rev 突变 4、5 和 6)的情况下,Rev 保持核苷酸模式,并且以前曾证明可以挽救 HIV-1HXB2复制,但与 WT Rev 1 相比,效率降低.所有单点突变都无法维持HIV-1NL4-3感染周期。在NoLS序列中存在多个单点突变(Rev-NoLS突变2和9)时,已观察到Rev分散在整个细胞核和细胞质,并且未能挽救HIV-1HXB2复制1。此蛋白质组学研究的目标是破译与 Rev 介导的 HIV-1 感染途径有关的核细胞因子和非核细胞因子。Rev 免疫沉淀条件通过与核蛋白 B23 磷蛋白的相互作用进行优化,该蛋白先前已被证明在存在核突变时与 Rev 失去交互作用。

过去,对细胞因子进行了广泛的研究;然而,这是在没有病毒发病机制的情况下完成的。在这项研究中,通过HIV-1复制过程中的Rev相互作用,一种蛋白质是核磷蛋白B23,也称为核对霉素(NPM)、核糖核酸或NO38(两栖动物2、3)、4.B23表示为三种同种体(NPM1、NPM2和NPM3)-核素/核质核伴生家族的所有成员5、6。NPM1分子伴菜功能在核小体的适当组装,在染色质高阶结构7,8参与的蛋白质/核酸复合物的形成,以及防止聚集和通过N端核心域(残留物1-120)错误折叠目标蛋白9。NPM1功能通过核细胞核和细胞质10、11、内转录间隔序列中前核糖体RNA的处理,扩展到核糖体成因12,13,并逮捕核蛋白聚合期间核细胞组装14,15。NPM1与抑制凋亡16和肿瘤抑制剂ARF 17、18和p5319的致稳定有关,揭示了其作为致癌因子和肿瘤抑制器的双重作用。NPM1参与基因组稳定性、中位复制和转录的细胞活动。NPM1在细胞周期相间的核糖核酸中,在线虫期间沿染色体边缘,在线虫发生结束时在核细胞体(PNB)中发现。NPM2和NPM3的研究不如NPM1,在恶性肿瘤20期间,NPM1的表达水平发生了改变。

NPM1被记录在各种核/核细胞质穿梭中,通过内部NES和NLS9,21,并以前曾报告推动HIV-1 Tat和Rev蛋白的核进口。在B23结合域-β-乳酸酶融合蛋白的存在下,Tat在细胞质内错位,失去转活活性;这表明Tat对B232有很强的亲和力。另一项研究在缺乏含有RRE的mRNA的情况下建立了Rev/B23稳定复合物。在RRE mRNA的存在下,Rev与B23分离,优选与HIV RRE结合,导致B232的位移。在亚核水平上,Tat转能和B23HIV mRNA的转用交换过程在哪里发生,目前不得而知。两种蛋白质都假定通过B23相互作用同时进入核素。预期其他宿主细胞蛋白参与HIV核蛋白通路。本蛋白质组学调查中描述的方法将有助于阐明在HIV-1发病机制过程中涉及的核细胞因子与宿主细胞因子的相互作用。

蛋白质组学研究是通过表达Rev NoLS单点突变(M4、M5和M6)和HIV-1HXB2生产的多种精氨酸替代(M2和M9)开始的。在此模型中,一个HeLa细胞系可稳稳地表达Rev缺陷HIV-1HXB2(HLfB)转染WT Rev和Rev核糖核细胞突变,这些突变在3’末端含有标志标记。WT Rev 的存在将允许病毒复制发生在 HLfB 培养,与不挽救 Rev 缺乏 (M2 和 M9) 的 Rev-NoLS 突变相比,或允许病毒复制发生,但效率不如 WT Rev (M4、M5 和 M6)1。细胞莱沙在病毒增殖后48小时后在Rev表达的情况下被收集,并在免疫沉淀后使用针对Rev/B23相互作用优化的莱沙缓冲液进行免疫沉淀。介绍了使用不同盐浓度的莱沙缓冲液优化,并在银色或库马西染色SDS-PAGE凝胶中比较和分析HIV-1 Rev的蛋白质洗脱方法。第一种蛋白质组学方法涉及通过串联质谱直接分析来自表达的WT Rev、M2、M6和M9的洗脱样品。第二种方法将WT Rev、M4、M5和M6的改性与第一种方法进行比较。与WT Rev相比,对Rev-NoLS突变的肽亲和力进行了分析,并显示了蛋白质识别概率。这些方法揭示了在HIV-1复制期间参与HIV-1 mRNA传输和与Rev拼接的潜在因素(核核和非核苷酸)。总体而言,所述细胞溶解、IP和洗脱条件适用于感兴趣的病毒蛋白,用于了解激活和调节感染途径的宿主细胞因子。这也适用于研究各种疾病模型的持久性所需的细胞宿主因素。在此蛋白质组学模型中,HIV-1 Rev IP 针对 B23 相互作用进行了优化,以阐明与核细胞质穿梭活动和 HIV-1 mRNA 结合有关的核苷酸因子。此外,可以开发与HLfB细胞系类似的细胞系,以稳稳地表达缺乏关键蛋白质的传染病模型,以研究感兴趣的感染途径。

Protocol

1. 细胞培养 在Dulbeco的改性Eagle介质(DMEM)中保持HLfB,辅以10%胎儿牛血清(FBS)、2mM L-谷氨酰胺和1mM丙酸钠,在组织培养处理的100毫米板中。在加湿的培养箱中保持细胞培养在37°C,提供5%CO2。通过汇入细胞密度为1 x 106细胞/mL。 丢弃细胞培养基。用 10 mL 的 1x 磷酸盐缓冲盐水 (PBS) 轻轻冲洗细胞。在不破坏细胞层的情况下取出并丢弃 1x PBS。 向细胞中加入2 mL的1x胰蛋白酶…

Representative Results

与WT Rev.WT Rev-3’标志和pcDNA-标志相比,Rev-NoLS单点和多点精氨酸突变与各种亚细胞定位模式相对应,在与细胞宿主因子相互作用的能力方面进行了检查在HLfB文化中表达向量。蛋白质复合物从总细胞赖沙处理,并沾染银染色试剂。Rev-NoLS-3’标志在三种不同的液化缓冲条件下可检测到(约18 kDa),其中含有图1中不同浓度的NaCl(137 mM、200 mM和300 mM)。B23在含低盐浓度(137 mM,通道2⁄4)的莱?…

Discussion

对在HIV-1存在的情况下比较Rev-NoLS突变和WT Rev的质谱分析,以了解病毒复制周期中涉及的核因子。这将确定病毒感染所需的核苷酸成分。核卵石B23对Rev-NoLS具有很高的亲和力,在Rev3的核粒定位和Rev绑定HIV mRNA 22的核细胞质迁移中具有功能。B23与Rev-NoLS突变的亲和力,其中含有单个或多个精氨酸替代物,在病毒产生过程中通过Rev-3’flag的免疫沉淀进行评估(?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢芭芭拉·费尔伯博士和乔治·帕夫拉基斯博士为国家卫生研究院(NIH)艾滋病研究和参考试剂项目、国家过敏和传染病研究所提供的HLfB粘附性培养疾病(NIAID),NIH。作者还承认NIH、格兰特AI042552和AI029329提供的财务来源。

Materials

Acetic acid Fisher Chemical A38S-212
Acetonitrile Fisher Chemical A955-500
Acrylamide:Bisacrylamide BioRad 1610158
Ammonium bicarbonate Fisher Chemical A643-500
Ammonium persulfate Sigma-Aldrich 7727-54-0
ANTI-Flag M2 affinity gel Sigma-Aldrich A2220
anti-Flag M2 mouse monoclonal IgG Sigma-Aldrich F3165
BioMax MS film Carestream 8294985
Bio-Rad Protein Assay Dye Reagent Concentrate, 450 mL Bio-Rad 5000006
B23 mouse monoclonal IgG Santa Cruz Biotechnologies sc-47725
Bromophenol blue Sigma-Aldrich B0126
Carnation non-fat powdered milk Nestle N/A
Cell scraper ThermoFisher Scientific 179693PK
C18IonKey nanoTile column Waters 186003763
Corning 100-mm TC-treated culture dishes Fisher Scientific 08-772-22
Dithiothreitol Thermo Scientific J1539714
1 x DPBS Corning 21-030-CVRS
ECL Estern blotting substrate Pierce 32106
Ethanol, 200 proof Fisher Chemical A409-4
FBS Gibco 16000044
Formic Acid Fisher Chemical A117-50
GelCode blue stain reagent ThermoFisher 24590
Glycerol Fisher Chemical 56-81-5
goat-anti-mouse IgG-HRP Santa Cruz Biotechnologies sc-2005
Iodoacetamide ACROS Organics 122270050
KimWipe delicate task wiper Kimberly Clark Professional 34120
L-glutamine Gibco 25030081
Methanol Fisher Chemical 67-56-1
NanoAcuity UPLC Waters N/A
Pierce Silver Stain Kit Thermo Scientific 24600df
15-mL Polypropylene conical tube Falcon 352097
Prestained Protein Ladder, 10 to 180 kDa Thermo Scientific 26616
Protease inhibitor cocktail Roche 4693132001
Purified BSA New England Biolabs B9001
PVDF  Western blotting membrane Roche 3010040001
Sodium Pyruvate Gibco 11360070
10 x TBS Fisher Bioreagents BP2471500
TEMED BioRad 1610880edu
Triton X-100 detergent solution BioRad 1610407
Trizaic source Waters N/A
trypsin-EDTA Corning 25-051-CIS
Tween 20 BioRad 1706531
Synapt G2 mass spectrometer Waters N/A
Whatman filter paper Tisch Scientific 10427813
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Tags

HIV-1Nucleolar FactorsRev ImmunoprecipitationMass SpectrometryViral FactorsHost FactorsInfectious CycleProtein CharacterizationHeLa CellsPlasmid TransfectionCell LysisProtease InhibitorImmunoprecipitation

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Arizala, J. A. C., Chomchan, P., Li, H., Moore, R., Ge, H., Ouellet, D. L., Rossi, J. J. Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry. J. Vis. Exp. (148), e59329, doi:10.3791/59329 (2019).
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