Li Bo, Wang Di, Xie Xiaoxue, Chen Xiaoli, Liang Guorui, Xing Dan, Zhao Teng, Wu Jiahong, Zhou Xinyu, Li Chunxiao
School of Public Health, The Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, Guiyang 550025, China.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.
Viruses. 2024 Mar 28;16(4):525. doi: 10.3390/v16040525.
E-20-monooxygenase (E20MO) is an enzymatic product of the shade (shd) locus (cytochrome p450, E20MO). Initially discovered in Drosophila, E20MO facilitates the conversion of ecdysone (E) into 20-hydroxyecdysone (20E) and is crucial for oogenesis. Prior research has implicated 20E in growth, development, and insecticide resistance. However, little attention has been given to the association between the E20MO gene and DENV2 infection. The transcriptome of Ae. aegypti cells (Aag2 cells) infected with DENV2 revealed the presence of the E20MO gene. The subsequent quantification of E20MO gene expression levels in Aag2 cells post-DENV infection was carried out. A CRISPR/Cas9 system was utilized to create an E20MO gene knockout cell line (KO), which was then subjected to DENV infection. Analyses of DENV2 copies in KO and wild-type (WT) cells were conducted at different days post-infection (dpi). Plasmids containing E20MO were constructed and transfected into KO cells, with pre- and post-transfection viral copy comparisons. Gene expression levels of E20MO increased after DENV infection. Subsequently, a successful generation of an E20MO gene knockout cell line and the verification of code-shifting mutations at both DNA and RNA levels were achieved. Furthermore, significantly elevated DENV2 RNA copies were observed in the mid-infection phase for the KO cell line. Viral RNA copies were lower in cells transfected with plasmids containing E20MO, compared to KO cells. Through knockout and plasmid complementation experiments in Aag2 cells, the role of E20MO in controlling DENV2 replication was demonstrated. These findings contribute to our understanding of the intricate biological interactions between mosquitoes and arboviruses.
E - 20 - 单加氧酶（E20MO）是阴影（shd）基因座的一种酶促产物（细胞色素P450，E20MO）。E20MO最初在果蝇中被发现，它促进蜕皮激素（E）转化为20 - 羟基蜕皮激素（20E），对卵子发生至关重要。先前的研究表明20E与生长、发育和抗杀虫剂有关。然而，E20MO基因与登革热病毒2型（DENV2）感染之间的关联却很少受到关注。感染DENV2的埃及伊蚊细胞（Aag2细胞）的转录组显示存在E20MO基因。随后对DENV感染后Aag2细胞中E20MO基因表达水平进行了定量分析。利用CRISPR/Cas9系统创建了一个E20MO基因敲除细胞系（KO），然后对其进行DENV感染。在感染后不同天数（dpi）对KO细胞和野生型（WT）细胞中的DENV2拷贝数进行了分析。构建了含有E20MO的质粒并转染到KO细胞中，对转染前后的病毒拷贝数进行了比较。DENV感染后E20MO的基因表达水平升高。随后，成功构建了E20MO基因敲除细胞系，并在DNA和RNA水平上验证了移码突变。此外，在KO细胞系的感染中期观察到DENV2 RNA拷贝数显著升高。与KO细胞相比，转染含有E20MO质粒的细胞中病毒RNA拷贝数较低。通过在Aag2细胞中进行敲除和质粒互补实验，证明了E20MO在控制DENV2复制中的作用。这些发现有助于我们理解蚊子与虫媒病毒之间复杂的生物相互作用。
