Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System
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Author:
Date:
2018-04-20
[Abstract] The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations ...
[摘要] 乙型肝炎病毒(HBV)是一种重要的全球人类病原体,并且是肝炎,肝硬化和肝癌的主要原因。 HBV衣壳由单个病毒蛋白的多个拷贝组成,衣壳或核心蛋白(HBc)在病毒生命周期中起着多重作用,并且最近已经成为开发抗HBV病毒疗法的主要靶标。尽管已经开发了几种用于研究HBV衣壳组装的系统,包括异源过表达系统如细菌和昆虫细胞,使用纯化蛋白质和哺乳动物细胞培养系统进行体外组装,但对非生理浓度HBc和盐以及难以操纵装配的宿主调节物在生理相关条件下的衣壳装配的详细研究方面存在主要限制。我们最近开发了基于兔网织红细胞裂解物(RRL)的哺乳动物无细胞系统,其中HBc以生理浓度表达并在近生理条件下组装成衣壳。该系统已经揭示了HBc装配要求,这是以前装配系统所不能预料的。此外,该系统中的衣壳组装受可容易操作的内源宿主因子调控。在这里,我们提供了这种无细胞衣壳装配系统的详细协议,包括如何操纵调节装配的宿主因子的说明。
【背景】乙型肝炎病毒(HBV)是一种重要的全球人类病原体,其长期感染全世界数以亿计的人并且代表病毒性肝炎,肝硬化和肝癌的主要原因(Seeger等人, 2013; Trepo et。,2014)。 HBV通过逆转录RNA中间体(所谓的前基因组RNA(pgRNA))在核衣壳内(NC)复制其基因组DNA(一种宽松的环状部分双链DNA(RC ...
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In vivo Live Imaging of Calcium Waves and Other Cellular Processes during Fertilization in Caenorhabditis elegans
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Author:
Date:
2017-04-05
[Abstract] Fertilization calcium waves are a conserved trigger for animal development; however, genetic analysis of these waves has been limited due to the difficulty of imaging in vivo fertilization. Here we describe a protocol to image calcium dynamics during in vivo fertilization in the genetic animal model Caenorhabditis elegans. This protocol consists of germline microinjection of a chemical calcium indicator, worm immobilization, live imaging, and image processing that quantifies the calcium fluorescence in the oocyte region moving in the field-of-view during ovulation. This imaging protocol can also be used to image other cellular processes during in vivo fertilization in C. elegans, such as membrane fusion and cytoskeletal dynamics.
[摘要] 受精钙波是动物发育的保守触发因素; 然而,这些波的遗传分析由于在体内成像难以受精而受到限制。 在这里,我们描述了在遗传动物模型秀丽隐杆线虫中的体内受精期间成像钙动力学的方案。 该方案包括化学钙指示剂的种系显微注射,蠕虫固定,活体成像和图像处理,其量化在排卵期间在视野内移动的卵母细胞区域中的钙荧光。 该成像方案也可以用于在体内受精期间对其他细胞过程进行成像。 线虫,如膜融合和细胞骨架动力学。
受精钙波在卵活化中起关键作用,并通过使用体外施肥系统在各种生物体中进行了分析。 线虫秀丽隐杆线虫由于其半透明的身体,适合于在体内成像受精。 受精钙成像在C. 塞缪尔等人报道了使用化学钙指示剂的线虫。 (2001)。 我们在这里描述了通过应用高速旋转盘共焦显微镜和图像处理方法从成像方法修改的方案,其在排卵期间分割在视场中移动的受精卵母细胞区域。 该协议能够精确定量描述钙波的时间动力学和波形图的遗传分析。
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