Discovering host factors impacting ZIKV infection via forward genetic screens

通过正向遗传筛选发现影响 ZIKV 感染的宿主因素

• 批准号: 9265644
• 负责人: Paul Bates
• 金额: $ 23.54万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9265644
• 关键词: Affect; Americas; Andes Virus; Barbados; Bioinformatics; Biology; Brazil; CRISPR/Cas technology; Cell physiology; Cells; Central America; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Colombia; Country; Coupled; Critical Pathways; DNA Binding; Data; Dengue; Dengue Virus; Dependovirus; Development; Ebola virus; Engineering; Enterovirus 68; Exhibits; Family; Family member; Flavivirus; French Polynesia; Funding; Funding Mechanisms; Gene Activation; Genes; Genetic; Genetic Screening; Goals; Guide RNA; Guillain-Barré Syndrome; Haiti; Hantavirus; Haploid Cells; Haploidy; Human; Human Genome; Immune response; Immunologic Factors; Infection; Insertional Mutagenesis; Integration Host Factors; Knowledge; Lassa virus; Libraries; Link; Location; Maps; Methods; Mexico; Microcephaly; Pathogenicity; Pathway interactions; Pharmacology; Population; Process; Protein Isoforms; Proteins; Recording of previous events; Reporting; Research; Research Proposals; Rift Valley fever virus; Site; South America; Symptoms; System; Technology; Therapeutic; Transcription Coactivator; Treatment Efficacy; Vaccines; Venezuela; Viral; Virus; Virus Diseases; Virus Replication; West Nile virus; Zika Virus; base; deep sequencing; design; expectation; experience; experimental study; gain of function; genome-wide; knock-down; knockout gene; loss of function; member; negative affect; novel; pathogen; prevent; programs; response; screening; therapeutic target; tool; transmission process; whole genome

Zika virus (ZIKV) is an emergent viruses of the family Flaviviridae that is spreading explosively through South & Central America. Of concern, the virus, which usually causes only mild symptoms, has been linked to a reported increase in the number of cases of babies born in Brazil with microcephaly and may also be associated with an increase in Guillain-Barré syndrome. Currently there are no therapeutics or licensed vaccines to treat or prevent ZIKV infection. Indeed ZIKV is understudied and very little is known about the basic biology of how ZIKV interacts with human cells. The aim of this research proposal is to 1) identify key human factors exploited by this virus during infection and to 2) delineate innate host cellular responses to Zika infection with the expectation that a better understanding of how this virus interacts with host cells may aid in the broad goal of identifying potential therapeutic targets. This proposal will utilize two independent, complimentary forward genetic screens that have not previously been applied to flaviviruses. It builds on our experience using a human haploid cell screen that has identified several human genes required by pathogenic hantaviruses. In specific aim 1 a library of insertionally-mutagenized haploid cells will be selected using lethal challenge by ZIKV. Deep sequencing will be used to map the locations of mutagenic insertion sites within the human haploid library prior to and following selection with ZIKV. By statistically ranking the number of independent insertions into genes within these two populations, aim 1 will define genes important for ZIKV infection. The importance of these genes will be validated by creating expression knockdowns and knockouts of these genes to retest infectivity with ZIKV. Aim 2 will identify cellular genes that when activated can restrict ZIKV infection using a modified CRISPR/Cas9 system. To accomplish this goal we will use RNA-guided DNA binding of a cleavage-defective Cas9 protein and sgRNAs that are fused to strong transcriptional activators. A library of >70,000 sgRNAs that target every isoform of every human gene will be transduced into cells. Illumina sequencing of the sgRNAs in the cell population before and after lethal ZIKV challenge will be used to identify sgRNAs (and the corresponding genes) that are enriched in cells that resist or restrict infection. Bioinformatic tools will be employed to define pathways or cellular processes restricting ZIKV infection. Together, these aims serve to initiate a research program that will yield important basic scientific data on this emergent virus.

寨卡病毒（ZIKV）是黄病毒科的一种新兴病毒，正在爆炸性传播 穿越南美洲和中美洲。令人担忧的是，这种病毒通常只引起轻微的 症状，已被链接到一个报告中的婴儿出生的病例增加， 巴西患有小头畸形，也可能与格林巴利综合征的增加有关 综合征目前还没有治疗或预防ZIKV的治疗剂或许可的疫苗 感染事实上，ZIKV是研究不足，很少有人知道的基本生物学如何 ZIKV与人类细胞相互作用。本研究的目的是：（1）确定关键 在感染过程中被这种病毒利用的人的因素和2）描述先天宿主 细胞对寨卡病毒感染的反应，期望更好地了解如何 这种病毒与宿主细胞相互作用可能有助于确定潜在的治疗药物的广泛目标。 目标的该提案将利用两个独立的、互补的正向遗传筛选 这是以前没有应用于黄病毒的。它建立在我们使用 人类单倍体细胞筛选已经鉴定了致病性所需的几个人类基因， 汉他病毒在具体的目的1中，将插入诱变的单倍体细胞的文库 使用ZIKV的致死性攻击进行选择。深度测序将用于绘制 在用以下方法选择之前和之后，在人单倍体文库内的诱变插入位点 ZIKV。通过对这两个基因中独立插入的数量进行统计排名， 在群体中，目的1将定义对ZIKV感染重要的基因。这些的重要性 基因将通过产生这些基因的表达敲低和敲除来验证， 用ZIKV重新测试感染性。目标2将识别细胞基因，当激活时， 使用改良的CRISPR/Cas9系统限制ZIKV感染。为了实现这一目标，我们将 使用RNA引导的切割缺陷型Cas9蛋白和融合的sgRNA的DNA结合 强大的转录激活因子。一个包含> 70，000个sgRNA的文库，其靶向每种sgRNA亚型， 所有人类基因都会被导入细胞细胞中sgRNA的Illumina测序 在致死ZIKV攻击之前和之后的群体将用于鉴定sgRNA（并且在致死ZIKV攻击之前和之后的群体将用于鉴定sgRNA）。 相应的基因）富集在抵抗或限制感染的细胞中。生物信息 将使用工具来定义限制ZIKV感染的途径或细胞过程。 总之，这些目标有助于启动一项研究计划，将产生重要的基础科学成果。 关于这个新出现的病毒的数据