Modulation of Lassa Virus vRNP Activity By Host Cell Factors

项目总结： 阿拉伯拉萨病毒(LASV)每年在西非感染数十万人，导致 拉沙热(LF)病例数量多，与高发病率和死亡率有关。此外，LASV 是一个可信的生物防御威胁。目前还没有FDA许可的疫苗，目前的抗病毒疗法是 限制在标签外使用利巴韦林，这只是部分有效的。LASV在人类健康中的意义 和生物防御准备，加上现有的有限的打击它们的武器库，强调了 对新的抗LASV疗法的需求尚未得到满足。我们假设宿主细胞因子的选择性靶向 病毒核糖核蛋白介导的病毒RNA复制和基因转录所需的过程 (VRNP)，但对宿主细胞的正常新陈代谢和生存是必不可少的，代表了一种新的对抗策略 人类致病ArenaVirus。这种方法将最大限度地减少抗病毒治疗中的常见问题。 抗药性变异体的出现带来的影响。为了实施这一战略，我们建议进行siRNA 全基因组筛选以确定LASV vRNP活性所需的宿主细胞基因。为此，我们有 建立了含有功能性LASV vRNP(LASV/vRNP)的无病毒细胞系，该细胞系可指导LASV vRNP的表达 ZsGreen和Gaussia Luciferase(Gluc)报告基因(RG)来自类似病毒的RNA，又称微型基因组(MG)，以及 结果表明，RG表达(RGE)能准确反映vRNP的活性。我们的具体目标(SA)是： SA 1.进行基于siRNA的全基因组筛选，以确定对P4活性有贡献的宿主细胞因子 LASV vRNP。我们已经使用我们的LASV/vRNP细胞建立了与遗传性HTS兼容的条件，以 确定LASV vRNP活性的修饰物。我们建议对我们的LASV/vRNP细胞进行筛选以对抗达拉康的 On-Target-Plus人类基因组文库用于识别抑制LASV vRNP活性的宿主细胞基因 通过减少Gluc和ZsGreen读数。 SA 2.命中验证。我们将确定不同报告的LASV MG定向表达水平 基因，猫，在被击中的候选细胞中受到RNAi介导的击倒，完全丢失- 通过CRISPR-Cas9的功能缺失，以及药物干预。为了评估命中的特异性，我们将执行MG 与其他RNA病毒的检测。将使用与生物相关的细胞类型进行进一步的HIT验证 在LASV的背景下，包括人上皮细胞和内皮细胞、肝细胞和单核细胞。 从这些研究获得的知识将提供：1)设计针对以下目标的抗病毒战略的基础 针对基本病毒RNA生物合成过程所需的宿主细胞因子，以及2)关于 ArenaVirus与宿主的相互作用将有助于更好地了解ArenaVirus的发病机制。

Project Summary: The arenavirus Lassa (LASV) infects several hundred thousand individuals yearly in West Africa resulting in a high number of Lassa fever (LF) cases that are associated with high morbidity and mortality. In addition, LASV is a credible biodefense threat. There are not FDA-licensed vaccines and current anti-arenaviral therapy is limited to the off-label use of ribavirin that is only partially effective. The significance of LASV in human health and biodefense readiness, together with the limited existing armamentarium to combat them, underscore the unmet need for novel anti-LASV therapeutics. We hypothesize that selective targeting of host cell factors required for virus RNA replication and gene transcription, processes mediated by the virus ribonucleoprotein (vRNP), but dispensable for normal host cell metabolism and survival, represents a novel strategy to combat human pathogenic arenaviruses. This approach would minimize the common problem in antiviral therapy posed by the emergence of drug resistant variants. To implement this strategy, we propose to conduct a siRNA genome-wide screen to identify host cell genes required for the activity of LASV vRNP. For this we have generated a virus-free cell line containing a functional LASV vRNP (LASV/vRNP) that directs expression of the ZsGreen and Gaussia luciferase (Gluc) reporter genes (RG) from a virus-like RNA, aka minigenome (MG), and shown that RG expression (RGE) accurately reflect vRNP activity. Our specific aims (SA) are: SA 1. Conduct siRNA-based genome-wide screen to identify host cell factors that contribute to the activity of LASV vRNP. We have used our LASV/vRNP cells to establish conditions compatible with genetic HTS to identify modifiers of LASV vRNP activity. We propose to screen our LASV/vRNP cells against Dharmacon’s On-Target-Plus human genome library to identify host cell genes that inhibit LASV vRNP activity as reflected by reduction of Gluc and ZsGreen readings. SA 2. Hit validation. We will determine levels of LASV MG-directed expression levels of a different reporter gene, CAT, in cells where hit candidates have been subjected to RNAi-mediated knock down, complete loss- of-function via CRISPR-Cas9, and pharmacological interference. To assess hit specificity we will perform MG assays with other RNA viruses. Further hit validation will be done using cell types that are biologically relevant in the context of LASV including human epithelial and endothelia cells, hepatocytes and monocytes. Knowledge derived from these studies will provide: 1) the foundation for designing antiviral strategies aimed at targeting host cell factors required for essential viral RNA biosynthetic processes, and 2) novel insights about arenavirus-host interactions that will contribute to a better understanding of arenavirus pathogenesis.