Analysis of flavivirus infection on the cellular lipidome - implications for virus particle production and replication.

黄病毒是节肢动物传播的RNA病毒，在世界范围内引起显著的人类发病率和死亡率。在过去的50年中，已经出现了许多黄病毒，包括登革热病毒（DENV）、日本脑炎病毒（JEV）、西尼罗河病毒（WNV）和最近的寨卡病毒（ZIKV），以引起现在严重的全球公共卫生问题的疾病。热带和亚热带地区的国家，如泰国，承受着这些疾病的最大经济和社会代价，这些疾病由于蚊子媒介的存在而成为地方病。据估计，仅DENV感染每年就给泰国造成约2.9亿美元的直接和间接成本。然而，用于预防蚊媒黄病毒病的完全保护性和安全的疫苗仅适用于黄热病病毒（YFV）和JEV。因此，需要开发改进的黄病毒疫苗和抗病毒疗法。更好地了解黄病毒与其宿主之间的相互作用将有助于改进控制措施。先前的研究已经确定黄病毒调节细胞脂质代谢以促进病毒复制和组装，并且血清脂质的变化与疾病的严重程度相关。然而，关于感染期间脂质代谢的总体变化如何具体影响i）病毒颗粒的脂质组成及其感染性ii）从感染细胞分泌的脂质类型和iii）疾病过程，知之甚少。关于不同的黄病毒如何改变宿主和病毒脂质组以及是否涉及常见脂质也知之甚少。英国和泰国的研究人员已经使用高通量质谱法（MS）进行了初步研究，以研究DENV感染如何i）调节细胞内和分泌的蛋白质谱ii）调节细胞脂质组和iii）影响DENV颗粒的组成。这些研究已经揭示，与未感染的细胞相比，参与脂质代谢和/或脂蛋白的蛋白质在DENV感染期间被差异调节和分泌，并且DENV-2感染的细胞的脂质组学谱被改变。在这项调查中，我们建议使用先进的高通量液相色谱-MS来分析如何脂质代谢在黄病毒感染过程中受到干扰，并明确定义的脂质是黄病毒复制和颗粒组装的关键。具体来说，我们将检查感染性黄病毒颗粒和病毒样颗粒（VLP）的脂质含量和用于黄病毒复制感染过程中的细胞膜。我们还将确定黄病毒感染是否会导致细胞分泌的脂质发生变化。这些研究将使用泰国关注的三种黄病毒进行; DENV，JEV和ZIKV，以确定黄病毒是否依赖于脂质代谢的常见变化进行复制和颗粒组装。这些脂质在感染中的重要性将通过RNA敲低和/或药物治疗改变其合成来证实。我们还将确定病毒颗粒的脂质组成是否影响宿主对病毒进入的反应和病毒颗粒的免疫原性。从长远来看，这些知识将被用作i）操纵细胞脂质合成以增加VLP的分泌以产生改进的疫苗的基础，ii）鉴定黄病毒复制所必需的脂质生物合成途径和新的抗病毒疗法的潜在靶点，以及iii）增加我们对病毒发病机理的了解。该项目将建立在英国和泰国合作伙伴之间的现有合作基础上，并开发泰国合作伙伴使用高通量方法分析病毒颗粒以及病毒感染如何重塑细胞脂质组的能力，不仅针对黄病毒，还针对其他新兴的虫媒病毒，这些病毒没有治疗方法。

Flaviviruses are arthropod-borne RNA viruses that cause significant human morbidity and mortality worldwide. Over the last 50 years, a number of flaviviruses including; dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and recently Zika virus (ZIKV) have emerged to cause diseases that are now serious global public health concerns. Countries in tropical and sub-tropical regions, such as Thailand, bear the greatest economic and societal cost of these diseases, which are endemic due to the presence of the mosquito vectors. It is estimated that DENV infections alone cost Thailand some US$290 million per year in direct and indirect costs. However, fully protective and safe vaccines for prevention of mosquito-borne flavivirus diseases are only available for yellow fever virus (YFV) and JEV. As such, there is a need to develop improved flavivirus vaccines and anti-viral therapies. A greater understanding of the interaction between flaviviruses and their hosts will facilitate improved control measures. Previous studies have established that flaviviruses modulate cellular lipid metabolism to promote virus replication and assembly and that changes in serum lipids are associated with disease severity. However, there is little known concerning how the overall changes to lipid metabolism during infection specifically affect i) the lipid composition of the virus particle and its infectivity ii) the types of lipids that are secreted from infected cells and iii) disease processes. Little is also known concerning how different flaviviruses alter the host and virus lipidome and whether common lipids are involved.The UK and Thai investigators have already conducted preliminary studies using high-throughput mass spectrometry (MS) to investigate how DENV infection i) modulates intracellular and secreted protein profiles ii) modulates the cellular lipidome and iii) influences the composition of DENV particles. The studies have revealed that proteins involved in lipid metabolism and/or lipoproteins are differentially regulated and secreted during DENV infection and that the lipidomic profile of DENV-2 infected cells is altered, compared to uninfected cells. In this investigation we propose to use advanced high-throughput liquid chromatography-MS to analyse how lipid metabolism is perturbed during flavivirus infection and specifically define the lipids that are key for flavivirus replication and particle assembly. Specifically, we will examine the lipid content of infectious flavivirus particles and virus-like-particles (VLPs) and the cellular membranes used for flavivirus replication during infection. We will also determine whether flavivirus infection results in changes in the lipids secreted from cells. The studies will be done using three flaviviruses of concern in Thailand; DENV, JEV and ZIKV, to determine if flaviviruses rely on common changes to lipid metabolism for their replication and particle assembly. The importance of these lipids in infection will be confirmed by altering their synthesis by RNA knockdown and/or drug treatment. We will also determine whether the lipid composition of virus particles affects the host response to viral entry and the immunogenicity of virus particles.In the longer term, this knowledge will be used as a basis to i) manipulate cellular lipid synthesis to increase the secretion of VLPs to produce improved vaccines ii) identify lipid biosynthetic pathways essential for flavivirus replication and potentially targets for novel anti-viral therapies and iii) increase our knowledge of viral pathogenesis. The project will build on existing collaborations between the UK and Thai partners and develop the capability of the Thai partners to use high-throughput approaches to analyse virus particles and how viral infection remodels the cellular lipidome, not only for flaviviruses, but also for other emerging arboviruses for which no therapies exist.