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、黄热病和 寨卡病毒在热带和亚热带地区。登革热病毒由四种血清型（DENV 1 -4）组成， 全球每年约有3.9亿感染患者和约2.5万人死亡。没有有效的药物， 针对DENV感染的许可疫苗;然而，有一项全球倡议来消灭这些蚊子 群体通过人工转染来自Dorosophia的Wolbachia，wMel或wMelPop菌株到Ae. 蚊子这些细胞内细菌已被证明可以减少蚊子的数量，缩短蚊子的寿命。 蚊子的寿命，使可能减少登革病毒的传播。此外，据透露， wMel和wMelPop对Ae具有较强的抗病毒保护作用。agypti。相反，关于沃尔巴克氏体对 AE.白纹伊蚊仍然有限。我们集中研究了DENV 2在wMelPop感染的小鼠中复制的机制。 C6/36细胞系（Ae. albopictus）。在将DENV 2感染至幼稚C6/36细胞或wMelPop-C6/36细胞后，细胞免疫学检测显示， RNAseq分析显示，wMelPop抑制DENV 2诱导的细胞基因表达。 在上清液中，病毒拷贝数不因存在或不存在以下物质而显著改变： wMelPop，尽管LLC/MK2细胞中的空斑试验用从wMelPop-C6/36细胞回收的病毒 显著减少。用于监测病毒复制的免疫荧光染色显示， wMelPop-C6/36细胞在未处理的C6/36细胞中复制良好，但在BHK-21细胞中复制延迟。序列 RNAseq结果显示，wMelPop在E（Glu 202 Gly）、NS 2A、NS 2B、NS 2C、NS （Leu201Phe）、NS2B（Thr94Ile、Thr101Ile、Ile114Thr）、NS4B（Tyr99Cys）和NS5Pol（Leu716Val）。 这些突变中有5个发生在NS 2A、NS 2B和NS 4 B的跨膜结构域（TMD）处或附近，所有突变均发生在NS 2A、NS 2B和NS 4 B中。 其中的蛋白质是在内质网（ER）中形成复制区室所必需的。观察到 该突变体病毒通过在幼稚C6/36细胞中复制保持所有这些突变，尽管在BHK-21细胞中， 将这些突变恢复为WT序列，除了在NS 5处和在NS 3处的进一步获得性突变（在NS 3中的Val 97 Ile）。 蛋白酶和解旋酶结构域中的Ile 212 Val）。这些结果表明，必须有一个结构或功能 蚊子和哺乳动物细胞之间ER的差异以及wMelPop诱导的突变功能差 在哺乳动物细胞中，需要在NS 3处进一步突变。在目标1中，我们将扩大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 感染性克隆具有个别突变，并检查TMD-NS蛋白，E，NS 3 在NS 5在目的2中，我们将研究突变的TMD-NS蛋白是否表达并定位于ER， 形成复制区室并与NS 3相互作用。在目标3中，我们将确定负责的变异蚊子 一种影响DENV 2复制并导致导致复制不全的突变的细胞因子 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.