人单核细胞系中HIV-1前病毒转录调节新型研究模型的建立

Establishment of a model system for studying the transcriptional regulation mechanism of HIV-1 in latently infected human monocyte cell lines

摘要:

【目的】抗反转录病毒疗法(ART)能够有效控制人免疫缺陷病毒(Human immunodeficiency virus-1，HIV-1)的复制，但是不能将其完全清除。至2012年底，全球仍有3 500万HIV-1感染者，同年约160万人死于艾滋病(Acquired immune deficiency syndrome，AIDS)及其相关疾病。HIV-1感染难以根治的主要原因之一是机体内HIV-1潜伏储存库(Reservoir)的存在。HIV-1潜伏储存库主要由CD4+ T细胞和单核巨噬细胞构成，与CD4+ T细胞相比，目前研究者对单核巨噬系细胞中HIV-1病毒复制机制尚不明了，且缺乏适宜的研究体系。因此，为探讨单核细胞活化或分化信号对HIV-1复制的影响，我们建立了旨在研究HIV-1前病毒转录调控机制的人单核巨噬细胞系模型。【方法】构建env区域移码突变和nef区域携带EGFP或NanoLuc报告基因的HIV-1 NLnGFP-Kp或NLnNanoLuc-Kp重组病毒，分别感染2种人单核细胞系THP-1或U937细胞。通过有限稀释法制备单克隆细胞系，利用流式细胞术或NanoLuc荧光素酶活性分析检测报告基因的表达。筛选EGFP或NanoLuc阴性表达的细胞克隆，经激活剂佛波酯(Phorbol-12-myristate-13-acetate，PMA)刺激后鉴定潜伏感染的细胞克隆。【结果】研究中鉴定了4个HIV-1潜伏感染的细胞克隆。其中2个是表达EGFP的THP-1克隆，2个是以NanoLuc为报告基因的U937克隆。这些克隆在PMA刺激处理后皆有报告基因的表达，而在恒态条件下未检测到报告基因的表达。【结论】成功建立了4个HIV-1潜伏感染的人单核细胞系克隆，该模型有助于理解单核巨噬系细胞的HIV-1病毒复制机制，可能成为进一步研究HIV-1前病毒转录调控机制的有力工具。

关键词:人免疫缺陷病毒，前病毒，转录调控，单核巨噬细胞，细胞模型，再活化

Abstract:

[Objective] With the use of anti-retroviral therapy (ART) for HIV-infected individuals, a greater control of viral replication is now possible. In 2012, however, more than 35 million people were living with HIV-1 and more than 1.6 million people died of AIDS or HIV-1 related diseases worldwide. One of the main reasons for difficulties in completely eradicating HIV-1 in vivo is the reservoir problem. Although the CD4+ T-cells and monocytes or macrophages are thought to be the reservoir of HIV-1 in vivo, the viral replication mechanism in monocytes or macrophages is yet to be studied, as compared with that of CD4+ T-cells, because of the lack of an appropriate system. Thus, to evaluate the effects of the activating or differentiating signals in monocytes on the replication of HIV-1, we attempted to establish the HIV-1 latently infected human monocyte cell lines. [Methods] We used two recombinant HIV-1 viruses, NLnGFP-Kp and NLnNanoLuc-Kp, which have a frame-shift mutation in the env region and have EGFP or NanoLuc gene, respectively, as a marker in the nef region. Two human monocyte linage cell lines, THP-1 and U937 cells, were each infected with one of the two viruses. For cloning of the infected cells, the limiting dilution method was used and the expression levels of these marker genes were determined by flow cytometry or luciferase assay. From the cell clones not expressing either marker gene, we selected latently infected cell clones after stimulating them with phorbol-12-myristate-13-acetate (PMA). [Results] We obtained four cell clones that were considered to be latently infected with HIV-1. Two of them from THP-1 had EGFP as a marker and two of them from U937 had NanoLuc as a marker. All these cell clones expressed their marker genes when stimulated with PMA but in the steady state condition of their cultures at an undetectable level. [Conclusion] We established four cell clones latently infected HIV-1 from human monocyte lineage cell lines. These cells can be a useful tool for a better understanding of the transcriptional regulation mechanisms in the HIV-1 replication.

Key words:human immunodeficiency virus (HIV), provirus, transcriptional regulation, monocyte-macrophage, cell model system, reactivation