Modulation of Lassa Virus vRNP Activity By Host Cell Factors

项目摘要： 拉沙沙粒病毒（LASV）每年在西非感染数十万人， 拉沙热（LF）病例数量多，发病率和死亡率高。此外，LASV 是一个可信的生物防御威胁没有FDA许可的疫苗，目前的抗沙粒病毒治疗是 仅限于仅部分有效的利巴韦林的标签外使用。LASV在人类健康中的意义 和生物防御准备，加上有限的现有装备，以打击他们，强调 对新型抗LASV治疗剂的未满足需求。我们假设选择性靶向宿主细胞因子 病毒RNA复制和基因转录所需，由病毒核糖核蛋白介导的过程 （vRNP），但对正常的宿主细胞代谢和存活无效，代表了一种新的对抗 人类致病性沙粒病毒。这种方法将最大限度地减少抗病毒治疗中的常见问题 这是由抗药性变异体的出现引起的。为了实施这一策略，我们建议进行siRNA 全基因组筛选以鉴定LASV vRNP活性所需的宿主细胞基因。为此， 产生了含有功能性LASV vRNP（LASV/vRNP）的无病毒细胞系，所述功能性LASV vRNP指导 来自病毒样RNA的ZsGreen和Gaussia荧光素酶（Gluc）报告基因（RG），也称为微型基因组（MG），以及 表明RG表达（RGE）准确地反映了vRNP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，以鉴定有助于 LASV vRNP。我们已经使用我们的LASV/vRNP细胞来建立与遗传HTS相容的条件， 鉴定LASV vRNP活性修饰剂。我们建议筛选我们的LASV/vRNP细胞对Dharmacon的 On-Target-Plus人类基因组文库，以鉴定抑制LASV vRNP活性的宿主细胞基因， 通过降低Gluc和ZsGreen读数。 SA 2.命中验证。我们将测定不同报告基因的LASV MG定向表达水平 基因，CAT，在细胞中，命中候选人已经受到RNAi介导的敲低，完全丢失- 通过CRISPR-Cas9的功能和药理学干扰。为了评估命中特异性，我们将进行MG 与其他RNA病毒进行比较。将使用生物学相关的细胞类型进行进一步的命中验证 在LASV的情况下，包括人上皮和内皮细胞、肝细胞和单核细胞。 从这些研究中获得的知识将提供：1）设计抗病毒策略的基础， 靶向病毒RNA生物合成过程所需的宿主细胞因子，以及2）关于 沙粒病毒-宿主相互作用，这将有助于更好地了解沙粒病毒的发病机制。

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.