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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.

黄病毒是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，并将理想地导致治疗的可用性。对病毒复制机制的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。这些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需的。我们还鉴定了病毒在细胞间传播所需的NS5活性，推测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，其在面对潜在的宿主先天免疫应答时允许感染。除了甲基转移酶和聚合酶酶活性的基本元素，如底物结合和活性位点残基，几乎一无所知的序列决定因素，有助于这一系列的活动。我们假设ZIKV NS5编码许多可以在遗传上分离的重叠功能。为了鉴定这些决定簇，我们将产生由NS5蛋白中所有可能的单氨基变体组成的文库。为此，我们将使用我们产生的当代ZIKV毒株的新型感染性克隆。我们将通过感染细胞来筛选该文库进行RNA复制和传播的能力，并在仅一天（仅允许复制，而不是传播发生）和几天（允许病毒在细胞之间传播）后观察哪些病毒存在。将通过深度测序、两个时间点感染细胞中的病毒RNA并将其与初始质粒文库进行比较来确定每种活性的突变耐受性。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STAT2（先天免疫应答的介体）的能力的突变体。该活动将通过深度测序进行类似定义。测序方法将使用强大的下一代测序技术来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策略。该项目的结果将为黄病毒宿主细胞相互作用和复制机制提供深入的见解，这可能有助于ZIKV治疗工作的发展，并可能进一步应用于对抗其他未来的病毒爆发。