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

伊蚊(Aedes(Ae.)Aegypti和Ae.白纹伊蚊是传播登革热的两种主要蚊种，黄热病和白纹伊蚊 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2.5万人死亡。既没有有效的药物，也没有有效的药物 针对DENV感染的许可疫苗；然而，有一项全球倡议来根除这些蚊子 通过人工将桃树起源的沃尔巴克氏菌、wMel或wMelPop菌株转移到两个Ae. 蚊子。这些胞内细菌已被证明可以减少蚊子的数量，并缩短 蚊子寿命延长，可能减少DENV传播。此外，据透露， WMel或wMelPop对Ae有较强的抗病毒保护作用。阿吉普提。相比之下，沃尔巴克氏菌对人类健康的影响 天哪。白纹伊蚊的数量仍然有限。我们重点研究了DENV2在wMelPop感染中的复制机制 C6/36细胞系(Ae.白纹伊蚊)。在DENV2感染幼稚C6/36细胞或wMelPop-C6/36细胞后， RNAseq分析显示，wMelPop抑制了DENV2诱导的细胞基因表达。 在上清液中，病毒拷贝数不会因存在或不存在而显著改变 WMelPop，尽管从wMelPop-C6/36细胞中回收的病毒在LLC/MK2细胞中的空斑分析 显著减少了。免疫荧光染色监测病毒复制情况显示，病毒来自 WMelPop-C6/36细胞在幼稚的C6/36细胞中复制良好，但在BHK-21细胞中复制延迟。数列 RNAseq结果显示，wMelPop在E(Glu202Gly)、NS2A上引起7个氨基酸变化 (Leu201Phe)、NS2B(Thr94Ile、Thr101Ile、Ile114Thr)、NS4B(Tyr99Cys)和NS5Pol(Leu716Val)。 其中5个突变发生在NS2A、NS2B和NS4B的跨膜区(TMD)或接近跨膜区(TMD)，均为 它们是在内质网(ER)中形成复制室所必需的。据观察， 这种突变病毒通过在幼稚的C6/36细胞中复制来保持所有这些突变，尽管在BHK-21细胞中 将这些突变恢复到除NS5以外的WT序列和NS3的进一步获得性突变(Val97Ile in 解旋酶结构域的蛋白酶和Ile212Val)。这些结果表明，一定存在结构或功能上的 蚊子和哺乳动物细胞内质网的差异及wMelPop诱导的突变作用不佳 在哺乳动物细胞中，并需要在NS3进一步突变。在目标1中，我们将扩展wMelPop在 对所有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及其与TMD-NS蛋白、E、NS3的相关性 和NS5。在目标2中，我们将研究突变的TMD-NS蛋白是否在内质网表达和定位到 形成复制间隔，并与NS3相互作用。在目标3中，我们将识别责任改变的蚊子 影响DENV2复制并导致导致复制无能的突变的细胞因素 在哺乳动物细胞中。

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.