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，正在呈爆炸性传播 穿过南美洲和中美洲。值得关注的是，这种病毒通常只会引起轻微的症状 症状，据报道与出生的婴儿病例数增加有关 巴西患有小头畸形，也可能与格林-巴利病的增加有关 综合症。目前尚无治疗或预防 ZIKV 的疗法或获得许可的疫苗 感染。事实上，ZIKV 的研究还不够充分，人们对 ZIKV 的基本生物学知之甚少。 ZIKV 与人体细胞相互作用。本研究计划的目的是 1) 确定关键 该病毒在感染过程中利用人为因素并2）描绘先天宿主 细胞对寨卡感染的反应，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治疗方法的广泛目标 目标。该提案将利用两个独立的、互补的正向遗传筛选 以前尚未应用于黄病毒。它建立在我们使用 人类单倍体细胞筛选，已鉴定出致病性所需的几个人类基因 汉坦病毒。在具体目标 1 中，将建立插入诱变单倍体细胞文库 通过 ZIKV 的致命挑战进行选择。深度测序将用于绘制位置图 在选择之前和之后，人类单倍体文库中的诱变插入位点 寨卡病毒。通过对这两个基因中独立插入的数量进行统计排名 对于人群，目标 1 将定义对 ZIKV 感染重要的基因。这些的重要性 将通过创建这些基因的表达敲低和敲除来验证基因 用 ZIKV 重新测试感染性。目标 2 将识别细胞基因，这些基因在被激活时可以 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 DNA 结合切割缺陷型 Cas9 蛋白和融合的 sgRNA 强转录激活剂。包含 >70,000 个 sgRNA 的文库，针对每种亚型 每个人类基因都会被转导到细胞中。细胞内 sgRNA 的 Illumina 测序 致命 ZIKV 攻击之前和之后的人群将用于识别 sgRNA（以及 相应的基因），这些基因在抵抗或限制感染的细胞中富集。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。 这些目标共同启动了一项研究计划，该计划将产生重要的基础科学成果 有关这种新出现的病毒的数据。