Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins
Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins
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DOI：
10.1128/jvi.06796-11
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发表时间：
2012-02-01
影响因子：
5.4
通讯作者：
Santos, Nuno C.
中科院分区：
文献类型：
--
作者：
Carvalho, Filomena A.;Carneiro, Fabiana A.;Santos, Nuno C.
登革热病毒（DENY）影响数百万人，每年造成2万多人死亡。目前尚无针对由DENV感染引起的疾病的有效治疗方法，部分原因是缺乏对病毒生命周期基本方面的了解，包括病毒组分与细胞区室之间相互作用的分子基础。在这里，我们表征了DEW衣壳（C）蛋白和肝脂滴（LD）之间的相互作用的性质，这是最近被证明是病毒复制周期所必需的。Zeta电位分析显示LD的表面负电荷，平均表面电荷为-19 mV。用C蛋白滴定LD导致表面电荷增加，其在+13.7 mV处达到平台，表明病毒蛋白-LD相互作用将蛋白阳离子表面暴露于水性环境。原子力显微镜（AFM）为基础的力谱测量进行了使用C蛋白功能化的AFM针尖。发现C蛋白-LD相互作用是强的，具有33.6pN的单一（非）结合力。这种结合依赖于细胞内高浓度的钾离子，但不依赖于钠离子。在DEW感染的细胞中，Na+/K+-ATP酶的抑制导致C蛋白从LD中解离，并且感染性病毒的产生被抑制50倍，但不抑制RNA复制，这表明钾依赖性相互作用的生物学相关性。有限的蛋白水解的LD表面受损的C蛋白-LD的相互作用，并在特定的抗体的存在下的力测量表明，围脂蛋白3（TIP 47）是主要的DEW C蛋白配体的LD的表面上。
Dengue virus (DENY) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DEW capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of -19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C proteinfunctionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na+/K+-ATPase in DEW-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DEW C protein ligand on the surface of LDs.