Identification and Characterization of the Antiviral Response to RSV Infection: A Single Cell Approach

RSV 感染抗病毒反应的鉴定和表征：单细胞方法

• 批准号: 10343844
• 负责人: Sara Sunshine
• 金额: $ 3.42万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2022-10-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10343844
• 关键词: A549; Antiviral Response; Bioinformatics; Biology; Brefeldin A; CRISPR screen; CRISPR-mediated transcriptional activation; Cell Separation; Cells; Complement; Data; Data Set; Epithelial; Epithelial Cells; Foundations; Future; Gene Activation; Gene Expression Profile; Genes; Genetic Transcription; Heterogeneity; Hour; Human; Human Genome; ISG15 gene; Immune response; In Vitro; Individual; Infant Mortality; Infection; Inflammatory Response; Integration Host Factors; Interferons; Knowledge; Label; Life Cycle Stages; Microscopy; Modeling; Molecular; Paracrine Communication; Patients; Phenotype; Play; Poly A; Prevalence; Process; Production; Protein Secretion; Publishing; Research; Respiration; Respiratory Syncytial Virus Infections; Respiratory System; Respiratory syncytial virus; Role; Scientist; Techniques; Testing; Therapeutic; Time; Transcript; Vaccines; Validation; Viral; Viral Load result; Virion; Virus; Virus Diseases; Virus Replication; airway epithelium; antagonist; base; cytokine; experimental study; gene network; genetic signature; immunoregulation; in vivo; inhibitor; insight; interferon antagonist; mutant; novel therapeutic intervention; overexpression; pathogen; prevent; programs; receptor; respiratory; respiratory pathogen; response; single cell sequencing; skills; targeted treatment; therapeutic development; time use; transcriptome

Project Summary/Abstract Respiratory Syncytial Virus (RSV) is a ubiquitous respiratory illness, and the leading viral cause of infant mortality worldwide. Despite its prevalence, there remains no effective vaccine, nor an efficacious RSV-specific therapeutic for infected patients. It is critical that we understand the fundamental biology underlying RSV infection and the subsequent host response to further our understanding of this pathogen, and lay the foundation for novel therapeutic strategies. The temporal dynamics of RSV infection have been characterized in bulk studies in vitro, in vivo, and ex vivo, however, these bulk studies likely underestimate the complexity and heterogeneity of the host respiratory epithelial response to infection. I have performed preliminary experiments to investigate the single cell response of respiratory epithelial cells (A549 cells) to RSV infection and my data revealed viral burden dependent and distinct gene networks activated over a twelve hour course of RSV infection. My data suggests a model in which infection may be considered a dynamic process with a spectrum of transcriptional states indicative of infection state. Indeed, I find expression of interferon stimulated genes (ISGs) is stronger in cells with undetectable RSV transcripts, compared to nearby infected cells. This is indicative of a compromised innate defense state within infected cells, but sensed by the surrounding cells. Given my preliminary studies, I propose a research strategy that seeks to test my hypotheses that the proinflammatory response during infection is largely driven by nearby uninfected cells, and that distinct proinflammatory gene signatures will correlate with different stages of the adjacent viral life cycle. In Aim 1, I will characterize the viral and host single cell temporal transcriptional dynamics of RSV infection in both infected and uninfected bystander cells, and produce single cell trajectories modeling RSV infection. Additionally, I will determine the contribution of paracrine signaling to the proinflammatory response initiated after RSV infection, by blocking protein secretion with Brefeldin A, and investigating the transcriptional response of uninfected bystander cells. In Aim 2, I will complement this study by performing an ISG-wide CRISPRa activation screen to identify host factors that influence both RSV replication and virion production. The completion of this study, which leverages single cell sequencing, bioinformatics, and CRISPR screening techniques, will provide pivotal insights into our understanding of the antiviral response to RSV infection.

项目总结/摘要 呼吸道合胞病毒（RSV）是一种普遍存在的呼吸道疾病，也是导致婴儿死亡的主要病毒原因 国际吧尽管其流行，但仍然没有有效的疫苗，也没有有效的RSV特异性疫苗。 治疗感染的病人。我们必须了解RSV感染的基本生物学机制， 以及随后的宿主反应，以进一步了解这种病原体，并为新的 治疗策略RSV感染的时间动力学已在体外大量研究中表征， 然而，在体内和离体，这些批量研究可能低估了复杂性和异质性， 宿主呼吸道上皮对感染的反应。我已经进行了初步的实验，以调查 呼吸道上皮细胞（A549细胞）对RSV感染的单细胞反应，我的数据显示病毒负荷 在RSV感染的12小时过程中，依赖性和不同的基因网络被激活。我的数据显示 一个模型，其中感染可以被认为是一个动态过程，具有一系列转录状态 指示感染状态。事实上，我发现干扰素刺激基因（ISGs）在细胞中的表达更强 与附近的感染细胞相比，无法检测到RSV转录物。这是一种先天性的 受感染的细胞内的防御状态，但周围的细胞感觉。根据我的初步研究，我建议 这是一项研究策略，旨在验证我的假设，即感染期间的促炎反应主要是 由附近未感染的细胞驱动，并且不同的促炎基因特征将与不同的 相邻病毒生命周期的各个阶段。在目标1中，我将描述病毒和宿主单细胞时间 在感染和未感染的旁观者细胞中RSV感染的转录动力学，并产生单个 模拟RSV感染的细胞轨迹。此外，我将确定旁分泌信号对 RSV感染后通过用布雷菲德菌素A阻断蛋白质分泌而引发的促炎反应，以及 研究未感染的旁观者细胞的转录反应。在目标2中，我将补充这项研究， 进行ISG范围的CRISPRa活化筛选，以鉴定影响RSV复制的宿主因素， 和病毒体的产生。这项研究的完成，利用单细胞测序，生物信息学， CRISPR筛选技术将为我们理解抗病毒反应提供关键见解， RSV感染。

项目成果

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq.

• DOI: 10.1038/s41467-023-41788-4
• 发表时间: 2023-10-06
• 期刊: NATURE COMMUNICATIONS
• 影响因子: 16.6
• 作者: Sunshine, Sara;Puschnik, Andreas S.;Replogle, Joseph M.;Laurie, Matthew T.;Liu, Jamin;Zha, Beth Shoshana;Nunez, James K.;Byrum, Janie R.;Mcmorrow, Aidan H.;Frieman, Matthew B.;Winkler, Juliane;Qiu, Xiaojie;Rosenberg, Oren S.;Leonetti, Manuel D.;Ye, Chun Jimmie;Weissman, Jonathan S.;Derisi, Joseph L.;Hein, Marco Y.
• 通讯作者: Hein, Marco Y.