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单病毒示踪技术揭示艾滋病毒有效侵染巨噬细胞过程
at :2017/4/13 10:43:34    

艾滋病毒有效入侵细胞释放遗传物质是病毒生命周期的重要环节,但其具体机制仍不清楚。近期,中国科学院武汉病毒研究所崔宗强研究员与中国科学院生物物理研究所张先恩研究员、美国乔治梅森大学吴云涛教授合作,实时动态揭示了单个HIV-1病毒以内吞方式入侵人原代巨噬细胞,病毒囊膜与细胞初级内体(early endosome)发生膜融合,在初级内体释放病毒核心实现有效侵染的过程。

 

 

研究人员首先建立了细胞辅助的病毒包装荧光量子点技术。在HIV-1病毒组装过程中,病毒Vpr蛋白被生物素化并与链霉亲和素修饰的量子点(SA-QDs)特异结合,包装进入成熟的病毒粒子中,从而获得病毒锥形核心内包装量子点的艾滋病毒,该病毒可用于单颗粒病毒长时间实时示踪。结合病毒囊膜以及细胞组分等多重荧光标记,实时动态观察到单个HIV-1病毒内吞入侵人原代巨噬细胞过程,并且病毒囊膜与初级内体膜发生融合,释放病毒核心实现病毒的有效感染。一系列药物抑制实验证实了该过程对病毒建立有效感染的重要作用。此外,研究人员也发现细胞微丝结构对HIV-1入侵人原代巨噬细胞建立有效感染具有重要作用。

 

该研究实时动态解析了单个艾滋病毒入侵人原代巨噬细胞过程,揭示了病毒核心释放这一瞬时动态分子事件的过程和机制,对深入理解艾滋病毒在重要天然宿主巨噬细胞中的感染机制具有重要意义,也将为开发新的抗病毒药物提供思路。

 

这一研究成果已在ACS Nano期刊在线发表(DOI:10.1021/acsnano.7b00275),李芹和李炜为论文共同第一作者。该研究得到了国家重大科学研究计划、国家自然科学基金、中国科学院重点项目、中国科学院青年创新促进会等基金项目的资助。华中科技大学参与了该项研究。

 

 量子点标记示踪艾滋病毒有效侵染巨噬细胞途径

 

Abstract: Macrophages are one of the major targets of human immunodeficiency virus (HIV-1), but the viral entry pathway remains poorly understood in these cells. Noninvasive virus labeling and single-virus tracking are effective tools for studying virus entry. Here, we constructed a quantum dot (QD)-encapsulated infectious HIV-1 particle to track viral entry at a single-particle level in live human primary macrophages. QDs were encapsulated in HIV-1 virions by incorporating viral accessory protein Vpr-conjugated QDs during virus assembly. With the HIV-1 particles encapsulating QDs, we monitored the early phase of viral infection in real time and observed that, during infection, HIV-1 was endocytosed in a clathrin-mediated manner; the particles were translocated into Rab5A-positive endosomes, and the core was released into the cytoplasm by viral envelope-mediated endosomal fusion. Drug inhibition assays verified that endosome fusion contributes to HIV-1 productive infection in primary macrophages. Additionally, we observed that a dynamic actin cytoskeleton is critical for HIV-1 entry and intracellular migration in primary macrophages. HIV-1 dynamics and infection could be blocked by multiple different actin inhibitors. Our study revealed a productive entry pathway in macrophages that requires both endosomal function and actin dynamics, which may assist in the development of inhibitors to block the HIV entry in macrophages.
 

本文来自“中科院武汉病毒研究所”
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