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(Zika Virus，ZIKV)是一种正在爆炸性传播的黄病毒科新近出现的病毒 穿越南美和中美洲。令人担忧的是，这种病毒通常只会引起轻微的 症状，与报告的婴儿出生病例数量的增加有关 巴西患有小头畸形症，也可能与格林-巴雷综合征的增加有关 综合症。目前还没有治疗或预防寨卡病毒的疗法或许可的疫苗 感染。事实上，对ZIKV的研究还很少，对ZIKV如何形成的基础生物学知之甚少 ZIKV与人类细胞相互作用。这项研究计划的目的是：1)确定关键 这种病毒在感染过程中利用的人为因素和2)描绘先天宿主 细胞对寨卡病毒感染的反应，期望更好地理解 这种病毒与宿主细胞的相互作用可能有助于确定潜在的治疗方法的广泛目标 目标。这项提议将利用两个独立的、免费的前向基因筛查 以前没有应用于黄病毒的药物。它以我们的经验为基础，使用 人类单倍体细胞筛选已鉴定出致病所需的几个人类基因 汉坦病毒。在具体目标1中，插入诱变的单倍体细胞库将 使用ZIKV的致命挑战选择。深度测序将被用来绘制 选择前后人类单倍体文库中的突变插入位点 齐科夫。通过对这两个基因中独立插入的数量进行统计排序 目标1将定义与ZIKV感染有关的重要基因。这些因素的重要性 将通过创建这些基因的表达敲除和敲除来验证这些基因 用ZIKV复测传染性。AIM 2将识别当被激活时可以 使用改进的CRISPR/CAS9系统限制ZIKV感染。为了实现这一目标，我们将 利用RNA引导的切割缺陷Cas9蛋白和融合的sgRNAs的DNA结合 到强大的转录激活剂。包含70,000个sgRNA的文库，这些sgRNA针对的是 人类的每一个基因都将被转导到细胞中。细胞内sgRNA的Illumina测序 致命的ZIKV挑战前后的种群将被用于识别sgRNAs(和 相应的基因)，这些基因富含在抵抗或限制感染的细胞中。生物信息学 将使用工具来定义限制ZIKV感染的途径或细胞过程。 这些目标共同作用于启动一项研究计划，该计划将产生重要的基础科学 关于这种新病毒的数据。