Deep mutational scanning of the Zika virus NS5 protein

寨卡病毒 NS5 蛋白的深度突变扫描

• 批准号: 10057677
• 负责人: Matthew J Evans
• 金额: $ 26.45万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057677
• 关键词: Active Sites; Affect; Amino Acids; Animal Model; Antiviral Agents; Antiviral Therapy; Attenuated; Binding Sites; Biological Assay; Biological Models; Cell Communication; Cell Culture Techniques; Cells; Codon Nucleotides; Complement; Complementary DNA; Data; Development; Disease Outbreaks; Elements; Epidemic; Flavivirus; Flavivirus Infections; Future; Genetic; Genome; Goals; Harvest; Immune Evasion; Impairment; Individual; Infection; Innate Immune Response; Interferons; Libraries; Life Cycle Stages; Link; Maps; Mediator of activation protein; Methyltransferase; Modeling; Molecular Virology; Mutagenesis; Mutation; Nonstructural Protein; Pathogenesis; Phase; Phenotype; Plasmids; Play; Polymerase; Proteins; Public Health; Puerto Rican; RNA replication; Research; Role; STAT2 gene; Signal Transduction; Site; Technology; Therapeutic Intervention; Time; Transfection; Tropism; Vaccines; Variant; Viral; Viral Genome; Viral Proteins; Virus; Virus Assembly; Virus Diseases; Virus Replication; Work; Zika Virus; cell type; combat; deep sequencing; env Gene Products; global health; human pathogen; in vivo; innovation; insight; mutant; mutation screening; neutralizing antibody; new therapeutic target; next generation sequencing; novel; prevent; protein structure; response; reverse genetics; secondary infection; therapeutic development; tool; viral RNA; virology

Flaviviruses represent serious global health challenges. Research on these viruses, including Zika virus (ZIKV), is crucial to help prevent the spread of epidemics and will ideally result in the availability of therapies. Antiviral development would be aided by a deeper understanding of the mechanisms of viral replication. The flavivirus NS5 protein encodes multiple critical proviral activities. These include the enzymatic methyltransferase and polymerase activities, which are required for viral genome amplification inside infected cells. We have also identified an NS5 activity that is required for the spread of virus between cells, presumably at the level of infectious virus assembly or release. Finally, NS5 is also a major interferon antagonist that permits infection in the face of potential host innate immune responses. Apart from the basic elements of the methyltransferase and polymerase enzymatic activities, such as substrate binding and active site residues, little to nothing is known about the sequence determinants that contribute to this vast array of activities. We hypothesize that ZIKV NS5 encodes numerous overlapping functions that can be genetically separated. To identify these determinants, we will generate libraries comprised of all possible single amino variants in the NS5 protein. To do so, we will use a novel infectious clone of a contemporary ZIKV strain that we generated. We will screen this library for the capacity to undergo RNA replication and spread by infecting cells and looking at which viruses are present after only one day (to allow only replication, not spread, to occur) and several days (which will allow spread of viruses between cells). The mutational tolerance for each activity will be determined by deep sequencing, the viral RNAs in infected cells at the two time points and comparing them to the initial plasmid library. We will also passage this library in cells treated with interferon to select mutants that retain the capacity to inhibit STAT2, a mediator of innate immune response. This activity will be similarly defined by deep sequencing. The sequencing approach will use powerful next-generation sequencing technologies to assay all possible NS5 single amino acid mutations simultaneously and is a highly scalable strategy to study ZIKV evolutionary potential. The results of this project will provide in-depth insights into flavivirus host cell interactions and replication mechanisms that may aid in the development of therapeutic efforts for ZIKV and could perhaps be further applied in combating other future virus outbreaks.

黄病毒代表着严重的全球健康挑战。对包括寨卡病毒 (ZIKV) 在内的这些病毒的研究对于帮助预防流行病的传播至关重要，并且理想情况下将有助于提供治疗方法。对病毒复制机制的更深入了解将有助于抗病毒药物的开发。黄病毒 NS5 蛋白编码多种关键的前病毒活性。其中包括酶促甲基转移酶和聚合酶活性，这是受感染细胞内病毒基因组扩增所必需的。我们还确定了病毒在细胞之间传播所需的 NS5 活性，大概是在感染性病毒组装或释放的水平上。最后，NS5 也是一种主要的干扰素拮抗剂，在潜在宿主先天免疫反应的情况下允许感染。除了甲基转移酶和聚合酶酶活性的基本要素（例如底物结合和活性位点残基）之外，人们对促成如此大量活性的序列决定因素知之甚少。我们假设 ZIKV NS5 编码许多可以通过基因分离的重叠功能。为了识别这些决定因素，我们将生成由 NS5 蛋白中所有可能的单氨基变体组成的文库。为此，我们将使用我们生成的当代 ZIKV 毒株的新型感染性克隆。我们将筛选该文库进行 RNA 复制和传播的能力，方法是感染细胞，并在一天（仅允许复制而不是传播）和几天（这将允许病毒传播）后查看哪些病毒存在细胞之间）。每种活性的突变耐受性将通过深度测序、两个时间点感染细胞中的病毒 RNA 并将其与初始质粒库进行比较来确定。我们还将在用干扰素处理的细胞中传代该文库，以选择保留抑制先天免疫反应介质 STAT2 能力的突变体。该活动将通过深度测序进行类似的定义。该测序方法将使用强大的下一代测序技术来同时检测所有可能的 NS5 单氨基酸突变，并且是研究 ZIKV 进化潜力的高度可扩展的策略。该项目的结果将深入了解黄病毒宿主细胞相互作用和复制机制，这可能有助于开发 ZIKV 治疗方法，并可能进一步应用于对抗未来其他病毒的爆发。