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，在世界范围内引起严重的人类发病率和死亡率。在过去的50年里，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）和最近出现的寨卡病毒（ZIKV）引起的疾病现已成为严重的全球公共卫生问题。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费用。然而，预防蚊媒黄病毒病的完全保护和安全疫苗仅适用于黄热病病毒（YFV）和JEV。因此，有必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。先前的研究已经证实，黄病毒通过调节细胞脂质代谢来促进病毒的复制和组装，并且血清脂质的变化与疾病的严重程度有关。然而，关于感染期间脂质代谢的总体变化如何特异性影响i)病毒颗粒的脂质组成及其传染性ii)受感染细胞分泌的脂质类型以及iii)疾病过程，目前知之甚少。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普通脂质也知之甚少。英国和泰国的研究人员已经使用高通量质谱（MS）进行了初步研究，以调查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iii)影响DENV颗粒的组成。这些研究表明，在DENV感染期间，参与脂质代谢和/或脂蛋白的蛋白质受到不同的调节和分泌，并且与未感染的细胞相比，DENV-2感染细胞的脂质组学谱发生了改变。在这项研究中，我们建议使用先进的高通量液相色谱-质谱分析在黄病毒感染过程中脂质代谢是如何被扰乱的，并明确定义黄病毒复制和颗粒组装的关键脂质。具体来说，我们将检查感染性黄病毒颗粒和病毒样颗粒（VLPs）的脂质含量以及在感染期间用于黄病毒复制的细胞膜。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令人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共同变化来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敲除和/或药物治疗来改变它们的合成来证实。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，这些知识将被用作以下方面的基础：(1)操纵细胞脂质合成以增加VLPs的分泌以生产改进的疫苗；(2)确定黄病毒复制所必需的脂质生物合成途径和新型抗病毒治疗的潜在靶点；(3)增加我们对病毒发病机制的了解。该项目将建立在英国和泰国合作伙伴之间现有合作的基础上，并发展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，还针对其他没有治疗方法的新出现的虫媒病毒。

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.