EASL2015:有针对性的RNAi筛选使用高通量传染性模型系统揭示了

StephenW

RS-2979
Viral hepatitis
Hepatitis B & D - Experimental

A TARGETED RNAI SCREEN USING A HIGH-THROUGHPUT INFECTIOUS MODEL SYSTEM UNCOVERS GLYPICAN GPC5 AS A HOST FACTOR FOR HEPATITIS B AND D VIRUS ENTRY
Eloi R. Verrier* 1, 2, Charlotte Bach1, 2, Laura Heydmann1, 2, Amelie Weiss3, Mickael Renaud3, Perrine Fritsch3, François Habersetzer4, Georges  Abou-Jaoudé5, David Durantel6, 7, Catherine Schuster1, 2, Laurent Brino3, Camille Sureau5, Mirjam B. Zeisel1, 2, Thomas F. Baumert1, 2, 4
1Institut de Recherche sur les Maladies Virales et Hépatiques, Inserm U1110, 2Université de Strasbourg, Strasbourg, 3Plateforme de Criblage Haut-débit, IGBMC, Illkirch, 4Pôle Hépato-digestif, Institut Hospitalo-universitaire, Strasbourg, 5Laboratoire de Virologie Moléculaire, INTS, Paris, 6Centre de Recherche en Cancérologie de Lyon, Inserm U1052, 7Université de Lyon, Lyon, France


Corresponding author’s email: [email protected]


Background and Aims:
Chronic hepatitis B virus (HBV) infection and its co-infection with hepatitis D virus (HDV) are leading causes of liver disease and cancer world-wide. Viral entry is the first step of infection, plays a key role in spread and control of infection and has been shown to be a viable target for the development of curative therapeutic strategies. HBV and HDV viruses infect exclusively hepatocytes and share the same envelope proteins and entry pathway. Heparan sulfate proteoglycans (HSPGs) have been shown to mediate HBV and HDV attachment at the hepatocyte cell surface before interacting with sodium taurocholate cotransporting polypeptide (NTCP). However, the detailed mechanisms of entry and its host cell-dependency factors are still poorly understood.  Using a targeted RNAi screen we aimed to investigate the role of HSPG core proteins in HBV/HDV entry.
Methods:
We established a high-throughput HDV infection model using Huh7 cells overexpressing NTCP and susceptible to HDV infection. Unlike previous approaches, this system does not require dimethylsulfoxide or polyethylene glycol to facilitate entry, thus providing a model closely mimicking natural infection. Using a small library of siRNAs targeting the core members of the HSPG family, we performed a targeted screen to evaluate the role of individual HSPG core proteins in HDV entry.
Results:
While the silencing of the expression of most HSPG core genes did not result in strong modulation of infection, silencing of Glypican GPC5 expression induced a marked and significant decrease of HDV infection as shown by immunofluorescence of HDV infected Huh7-NTCP+ cells and RT-PCR of HDV RNA. Using HepG2 cells overexpressing NTCP and individual siRNAs, we demonstrate that GPC5 silencing is a host cell-dependency factor for HBV infection. Silencing of GPC5 in HepG2-NTCP cells resulted in a marked decrease of both HBV-positive cells and HBV pregenomic RNA, confirming the functional role of this host cell surface protein for both HDV and HBV infection.
Conclusions:
Collectively, this targeted RNAi screen in a high-throughput infectious model system uncovers GPC5 as an entry factor for hepatitis B and D viruses. These results advance our understanding of cell entry of HBV and HDV and open new avenues for therapies aiming at HBV cure. Since glypicans have been shown to play a role in the control of cell division and growth regulation, virus-GPC5 interactions may also play a role for pathogenesis of virus-induced liver disease.


Disclosure of Interest: None Declared

StephenW

RS-2979

病毒性肝炎

乙肝＆D - 实验





有针对性的RNAi筛选使用高通量传染性模型系统揭示了磷脂酰肌醇聚糖GPC5作为东道主因子肝炎B和D病毒进入

埃洛伊R.维利亚* 1，2，夏洛特BACH1，2，劳拉Heydmann1，2，天使爱美丽Weiss3，迈克尔Renaud3，珀赖恩Fritsch3，弗朗索瓦Habersetzer4，乔治·阿布 - Jaoudé5，大卫Durantel6，7，凯瑟琳Schuster1，2，洛朗Brino3，卡米尔Sureau5，米莉亚B. Zeisel1，2，托马斯F. Baumert1，2,4

1Institut德RECHERCHE河畔莱弊病Virales等Hépatiques，INSERM U1110，2Université斯特拉斯堡，斯特拉斯堡，3Plateforme去Criblage的Haut-借记卡，IGBMC，伊尔基希，4POLE肝，饭后，研究所Hospitalo-区大学，斯特拉斯堡，5Laboratoire德VirologieMoléculaire，INTS，巴黎，6Centre德RECHERCHE连接Cancérologie里昂，INSERM U1052，7Université里昂，里昂，法国



通讯作者的邮箱：[email protected]



背景和目的：慢性乙型肝炎病毒（HBV）感染及其共同感染肝炎病毒（HDV）的肝脏疾病和癌症世界性的主要原因。病毒进入是病毒感染的第一个步骤中，起着关键作用在传播和感染的控制，并已被证明是用于治疗性治疗策略的开发一种可行的目标。 HBV和HDV病毒感染肝细胞专用和共享相同的包膜蛋白和入门途径。硫酸乙酰肝素蛋白多糖（聚糖）已被证明与牛磺胆酸钠cotransporting多肽（NTCP）相互作用之前进行调解的HBV和HDV附着在肝细胞表面上。但是，入口的详细机制和其宿主​​细胞的依赖因素仍然知之甚少。通过有针对性的RNAi筛选我们的目的是调查HSPG核心蛋白在HBV / HDV进入角色。

方法：建立使用的Huh7细胞过度NTCP和易受HDV感染高通量HDV感染模型。不同于以往的方法中，该系统不需要二甲基亚砜或聚乙二醇，以方便条目，从而提供了一个模型密切模仿自然感染。使用针对HSPG家族的核心成员siRNA的一个小型图书馆，我们进行了有针对性的屏幕，以评估各个HSPG核心蛋白的HDV进入角色。

结果：虽然大多数HSPG核心基因的表达的沉默没有导致强烈感染调制，诱发如图HDV的免疫荧光HDV感染的标记，并显著降低磷脂酰肌醇聚糖GPC5表达沉默感染的Huh7-NTCP +细胞和RT- PCR HDV RNA的。使用HepG2细胞过表达NTCP和个人的siRNA，我们证明了GPC5沉默是HBV感染宿主细胞的依赖因素。 GPC5在HepG2-NTCP细胞的沉默导致两者的HBV阳性细胞和HBV前基因组RNA的显着减少，证实此宿主细胞表面蛋白为HDV和HBV感染的功能性作用。

结论：总的来说，在高通量传染性模型系统本靶向RNAi筛选揭示GPC5如肝炎B和D病毒的条目的因素。这些结果推进我们的HBV和HDV和开辟新的途径进入细胞的疗法针对乙肝治疗的认识。由于glypicans已被证明在细胞分裂和生长调节控制作用，病毒GPC5相互作用也可以起到一定的作用为病毒性肝病的发病机制。



股权变动：无申报