Mechanism of wMelPop-induced DENV2 transmembrane domain mutations in NS2A, NS2B and NS4B

wMelPop 诱导 NS2A、NS2B 和 NS4B 中 DENV2 跨膜结构域突变的机制

• 批准号: 10302753
• 负责人: Tadahisa Teramoto
• 金额: $ 23.4万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302753
• 关键词: Aedes; Affect; Amino Acids; Antiviral Agents; Area; Arthropod Vectors; Attenuated; Bacteria; Biogenesis; Blood; Cell Line; Cells; Cessation of life; Competence; Culicidae; Data; Defensins; Dengue; Dengue Infection; Dengue Virus; Endoplasmic Reticulum; Exoribonucleases; Female; Flavivirus; Frequencies; Gene Expression; Genome; Habitats; Hospitalization; Human; Individual; Induced Mutation; Infection; Inherited; Integral Membrane Protein; Lead; Lipid Bilayers; Longevity; Mammalian Cell; Medicine; Membrane; Membrane Proteins; Molecular Chaperones; Monitor; Mutate; Mutation; Nonstructural Protein; Pathway interactions; Patients; Peptide Hydrolases; Plaque Assay; Population; Positioning Attribute; Principal Investigator; Process; Proteins; RNA; Serotyping; Small Interfering RNA; Stains; Structure; Tertiary Protein Structure; Transcript; Transmembrane Domain; Vaccines; Viral; Viral Genome; Viral Proteins; Virus; Virus Replication; Wolbachia; Yellow Fever; Zika Virus; antimicrobial peptide; base; cecropin; egg; feeding; fitness; helicase; lipid metabolism; male; mutant; offspring; programs; protein aggregation; protein complex; protein distribution; protein folding; severe dengue; small molecule inhibitor; sperm cell; transcriptome; transcriptome sequencing; transmission process

Aedes(Ae.) aegypti and Ae. albopictus are two major mosquito species transmitting dengue, Yellow fever and Zika viruses in tropical and subtropical regions. Dengue viruses consist of four serotypes (DENV1-4) that cause ~390 million infected patients and ~25,000 deaths globally each year. There is neither an effective medicine nor licensed vaccine against DENV infection; however there is a global initiative to eradicate these mosquito populations by artificially transinfecting Dorosophia-originated Wolbachia, wMel or wMelPop strain to both Ae. mosquitoes. These intracellular bacteria have been shown to reduce the mosquito population and shorten the mosquito lifespans, enabling possible reduction of DENV transmission. Furthermore, it has been revealed that wMel or wMelPop induce strong antiviral protection to Ae. agypti. In contrast, the data on Wolbachia’s effect on Ae. albopictus is still limited. We have focused on the mechanism of DENV2 replication in wMelPop-transinfected C6/36 cell line (Ae. albopictus). After infecting DENV2 to naïve C6/36 cells or wMelPop-C6/36 cells, cellular transcripts by RNAseq analysis revealed that wMelPop suppressed DENV2-induced cellular gene expressions. In the supernatants, the virus copy numbers were not significantly altered by the presence or absence of wMelPop, although the plaque assay in LLC/MK2 cells with the recovered viruses from wMelPop-C6/36 cells were significantly reduced. Immunofluorescent staining to monitor virus replication showed that the virus from wMelPop-C6/36 cells replicated well in naïve C6/36 cells, but delayed replication in BHK-21 cells. Sequence results by RNAseq revealed that wMelPop caused seven amino acid alterations at E (Glu202Gly), NS2A (Leu201Phe), NS2B (Thr94Ile, Thr101Ile, Ile114Thr), NS4B (Tyr99Cys), and NS5Pol (Leu716Val) in DENV2. Five of these mutations occurred at or close to transmembrane domains (TMDs) in NS2A, NS2B and NS4B, all of which are necessary to form replication compartment in the endoplasmic reticulum (ER). It was observed that this mutant virus kept all these mutations through replication in naïve C6/36 cells, although in BHK-21 cell, it was reverting these mutations to WT sequence except at NS5 and further acquired mutations at NS3 (Val97Ile in protease and Ile212Val in helicase domains). These results suggest that there must be a structural or functional difference of ER between mosquito and mammalian cells and that wMelPop-induced mutations function poorly in mammalian cells and require further mutations at NS3. In Aim 1, we will expand the effect of wMelPop in replication efficiency and mutation induction to all DENV serotypes as well as Zika virus. We will create the infectious clones having the individual mutations and examine the correlations among TMD-NS proteins, E, NS3 and NS5. In Aim 2, we will investigate if the mutated TMD-NS proteins are expressed and localized at ER to form replication compartment and interact with NS3. In Aim 3, we will identify the responsible altered mosquito cellular factor that is affecting DENV2 replication and causing the mutations that lead to replication incompetence in mammalian cells.

埃及伊蚊（Ae.）和伊蚊。白纹伊蚊是传播登革热、黄热病和黄热病的两种主要蚊子 热带和亚热带地区的寨卡病毒。登革热病毒由四种血清型 (DENV1-4) 组成，可引起 全球每年约有 3.9 亿感染患者和约 25,000 人死亡。既没有有效的药物，也没有 获得许可的预防登革热病毒感染的疫苗；然而，有一项全球倡议来消灭这些蚊子 通过将源自 Dorosophia 的 Wolbachia、wMel 或 wMelPop 菌株人工转染至两种 Ae 来控制种群。 蚊子。这些细胞内细菌已被证明可以减少蚊子数量并缩短蚊子寿命 蚊子的寿命，从而可能减少登革热病毒的传播。此外，据透露， wMel 或 wMelPop 可诱导针对 Ae 的强抗病毒保护。阿吉普蒂人。相比之下，有关沃尔巴克氏体对 艾。白纹伊蚊数量仍然有限。我们重点研究了 wMelPop 转染的 DENV2 复制机制 C6/36 细胞系（白纹伊蚊）。将 DENV2 感染幼稚 C6/36 细胞或 wMelPop-C6/36 细胞后，细胞 RNAseq 分析的转录结果显示，wMelPop 抑制 DENV2 诱导的细胞基因表达。 在上清液中，病毒拷贝数没有因存在或不存在而显着改变。 wMelPop，尽管使用从 wMelPop-C6/36 细胞中回收的病毒对 LLC/MK2 细胞进行噬菌斑测定 均显着减少。监测病毒复制的免疫荧光染色表明，该病毒来自 wMelPop-C6/36 细胞在初始 C6/36 细胞中复制良好，但在 BHK-21 细胞中复制延迟。顺序 RNAseq 结果显示 wMelPop 引起 E (Glu202Gly)、NS2A 处的 7 个氨基酸改变 DENV2 中的 (Leu201Phe)、NS2B (Thr94Ile、Thr101Ile、Ile114Thr)、NS4B (Tyr99Cys) 和 NS5Pol (Leu716Val)。 其中 5 个突变发生在 NS2A、NS2B 和 NS4B 的跨膜结构域 (TMD) 处或附近，全部 其中对于在内质网（ER）中形成复制室是必需的。据观察， 这种突变病毒通过在幼稚 C6/36 细胞中复制而保留了所有这些突变，尽管在 BHK-21 细胞中，它是 将这些突变恢复为 WT 序列，除了 NS5 处以及 NS3 处进一步获得的突变（Val97Ile 解旋酶结构域中的蛋白酶和 Ile212Val）。这些结果表明一定存在结构或功能 蚊子和哺乳动物细胞之间 ER 的差异以及 wMelPop 诱导的突变功能不佳 在哺乳动物细胞中，需要 NS3 进一步突变。在目标 1 中，我们将扩展 wMelPop 的效果 所有 DENV 血清型以及寨卡病毒的复制效率和突变诱导。我们将创建 具有个体突变的感染性克隆并检查 TMD-NS 蛋白、E、NS3 之间的相关性 和 NS5。在目标 2 中，我们将研究突变的 TMD-NS 蛋白是否表达并定位于 ER 以 形成复制室并与 NS3 相互作用。在目标 3 中，我们将确定负责的变异蚊子 影响 DENV2 复制并导致导致复制无能的突变的细胞因子 在哺乳动物细胞中。