Modulation of Lassa Virus vRNP Activity By Host Cell Factors

宿主细胞因子对拉沙病毒 vRNP 活性的调节

基本信息

批准号：
9321544
负责人：
Juan C. de la Torre
金额：
$ 31.25万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-02-15 至 2019-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9321544
关键词：
Africa Antiviral Agents Antiviral Therapy Applications Grants Area Arenaviridae Arenavirus Biological Biological Assay CRISPR/Cas technology Cell Density Cell Line Cell Survival Cells Cellular Metabolic Process Cellular biology Collaborations Complex Containment Drug resistance Epithelial Fatality rate Foundations Gene Expression Generations Genes Genetic Genetic Transcription Genomic Library Health Hepatocyte Human Human Genome Individual Knowledge Label Lassa Fever Lassa virus Luciferases Lymphocytic choriomeningitis virus Measures Mediating Molecular Biology Monitor Morbidity - disease rate Nucleoproteins Old World Arenaviruses Pathogenesis Pathogenicity Pharmacology Phenotype Polymerase Process Proteins RNA RNA Interference RNA Viruses RNA interference screen RNA replication Readiness Reading Recording of previous events Reporter Reporter Genes Ribavirin Ribonucleoproteins Small Interfering RNA Specificity Technology Therapeutic Toxic effect Transcription Process Transfection Travel Vaccines Validation Variant Viral Genome Viral Hemorrhagic Fevers Virus Virus Replication base biodefense cell type clinically significant combat design functional genomics genome-wide analysis insight knock-down loss of function monocyte mortality neglect novel novel strategies pathogen stem transmission process viral RNA

项目摘要

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.

项目概要：沙粒病毒拉沙病毒 (LASV) 每年在西非感染数十万人，导致大量拉沙热 (LF) 病例与高发病率和死亡率相关。是一种可信的生物防御威胁，目前尚无 FDA 许可的疫苗，目前的抗沙粒病毒疗法是有效的。仅限于利巴韦林的标签外使用，其仅部分有效 LASV 对人类健康的重要性。和生物防御准备情况，以及现有的有限的武器装备，突显了我们一直在努力解决选择性靶向宿主细胞因子的问题。病毒 RNA 复制和基因转录所需的病毒核糖核蛋白介导的过程（vRNP），但对于正常宿主细胞代谢和生存来说是可有可无的，代表了一种对抗病毒的新策略这种方法可以最大限度地减少抗病毒治疗中的常见问题。为了实施这一策略，我们建议进行 siRNA 治疗。全基因组筛选以确定 LASV vRNP 活性所需的宿主细胞基因。产生了含有功能性 LASV vRNP (LASV/vRNP) 的无病毒细胞系，该细胞系指导来自病毒样 RNA（又名小基因组 (MG)）的 ZsGreen 和高斯荧光素酶 (Gluc) 报告基因 (RG)，以及表明 RG 表达 (RGE) 准确反映 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，以确定有助于活性的宿主细胞因子 LASV vRNP。我们使用 LASV/vRNP 细胞建立了与遗传 HTS 兼容的条件。识别 LASV vRNP 活性的修饰因子我们建议针对 Dharmacon 筛选我们的 LASV/vRNP 细胞。 On-Target-Plus 人类基因组文库可识别抑制 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基因的 LASV MG 定向表达水平。基因，CAT，在细胞中，其中命中候选者已受到RNAi介导的敲低，完全丧失- 为了评估命中特异性，我们将进行 MG。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。从这些研究中获得的知识将提供：1）设计抗病毒策略的基础针对重要病毒 RNA 生物合成过程所需的宿主细胞因子，以及 2）关于沙粒病毒与宿主的相互作用将有助于更好地了解沙粒病毒发病机制。