Deep mutational scanning of the Zika virus NS5 protein

寨卡病毒 NS5 蛋白的深度突变扫描

基本信息

批准号：
10171769
负责人：
Matthew J Evans
金额：
$ 21.81万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
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/10171769
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 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

项目摘要

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.

黄病毒代表了严重的全球健康挑战。这些病毒（包括寨卡病毒（ZIKV））的研究对于帮助防止流行病的传播至关重要，理想情况下将导致疗法的可用性。对病毒复制机制的更深入的理解将有助于抗病毒发育。黄病毒NS5蛋白编码多种关键的病毒活性。其中包括酶促甲基转移酶和聚合酶活性，这是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必需的NS5活性，大概是在传染性病毒组装或释放的水平上。最后，NS5还是一个主要的干扰素拮抗剂，面对潜在的宿主先天免疫反应，允许感染。除了甲基转移酶和聚合酶酶促活性的基本元素（例如底物结合和活性位点残基）外，还几乎没有什么了解的序列决定因素，这些决定因素有助于这类活动。我们假设ZIKV NS5编码可以在遗传上分离的许多重叠函数。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个氨基变体组成的库。为此，我们将使用我们产生的当代ZIKV菌株的新型感染性克隆。我们将筛选该库的能力，以进行RNA复制并通过感染细胞传播，并研究仅一天后出现病毒（仅允许复制而不是扩散，发生）和几天（这将允许在细胞之间传播病毒）。每种活性的突变耐受性将由深度测序（在两个时间点被感染细胞中的病毒RNA）确定，并将其与初始质粒文库进行比较。我们还将在用干扰素处理的细胞中传递该文库，以选择保留抑制STAT2的能力的突变体，STAT2是先天免疫反应的介体。该活动将通过深度测序类似地定义。测序方法将使用强大的下一代测序技术同时测定所有可能的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。该项目的结果将提供对Flavivirus宿主细胞相互作用和复制机制的深入见解，这些机制可能有助于开发ZIKV的治疗努力，并可能进一步应用于与其他未来病毒疫情作斗争。