摘要

背景研究：

寨卡病毒（Zika Virus）是一种蚊媒传播病毒，感染后可能导致严重的神经系统症状，如格林巴利综合征；围产期感染寨卡病毒可以导致胎儿小头畸形。由于目前仍缺乏有效的疫苗和特异性的抗寨卡病毒药物，了解寨卡病毒与宿主之间的相互作用有利于寻找新的抗寨卡病毒策略。MicroRNA（miRNA）是一类长约20nt的短链RNA，可以结合至信使RNA（message RNA，mRNA）的3’端非编码区，阻断mRNA的翻译或者介导mRNA的降解，在宿主和病原体相互作用中发挥着重要作用，是抗病毒基因治疗的重要研究方向之一。有研究表明，寨卡病毒感染神经细胞和星形胶质细胞会引起一系列miRNA表达水平的变化，但具体作用及机制尚不清楚。MiR-16-5p在以往的研究中常被作为感染性肺炎、皮肤绘图症和类风湿性关节炎等疾病的标志物，也有研究发现miR-16-5p可以抑制肠病毒71（EV71）复制，促进病毒引起的细胞凋亡。我们的前期研究也显示，CYLD（cylindromatosis）是miR-16-5p的潜在作用靶点之一。CYLD是一种去泛素化酶，不仅可以通过解聚视黄酸诱导基因蛋白I（retinoic acid-inducible gene-I，RIG-I）上K63的泛素链从而抑制其活性，调控干扰素相关信号通路；还能够抑制核因子κB（nuclear factor κB，NFκB）通路介导的各种炎性因子的产生。这些研究结果都提示miR-16-5p与感染及免疫密切相关，但miR-16-5p对寨卡病毒复制是否有影响及其具体作用机制是否与CYLD存在关联，目前都尚不清楚。

探究miR-16-5p在寨卡病毒感染中的作用，并明确其作用靶点及影响寨卡病毒复制的分子机制。

研究内容及方法：

（1）寨卡病毒感染A549细胞或293T细胞， 收集细胞内核酸，检测miR-16-5p的表达水平变化。

（2）在A549细胞中转染miR-16-5p后，感染寨卡病毒，荧光定量PCR（quantitative Real-time PCR，qRT-PCR）和western blot检测寨卡病毒核酸与蛋白水平，评估寨卡病毒复制情况；通过qRT-PCR检测I型干扰素、干扰素刺激基因及炎性因子表达水平；通过双荧光素酶报告基因实验检测干扰素刺激应答元件（interferon sensitive response element，ISRE）及NFκB活性，评估I型干扰素信号通路NFκB信号通路的活化情况。在A549细胞与HeLa细胞中转染miR-16-5p，通过流式细胞术检测细胞凋亡情况。

（3）通过网站（www.targetscan.com）预测miR-16-5p可能作用的靶点，结合其对干扰素信号通路及NFκB信号通路的影响进一步筛选出靶点CYLD，并通过以下方法进行了靶点确认：1）在A549细胞中转染miR-16-5p，通过western blot 检测CYLD的蛋白表达水平；2）分别转染已经构建了靶序列结合位点的p-miR-CYLD-WT与其突变p-miR-CYLD-MUT，使用双荧光素酶报告基因检测确定其靶向的结合序列。

（6）寨卡病毒感染A549细胞，转染siCYLD或CYLD表达质粒抑制或增加CYLD表达，通过qRT-PCR或western blot检测细胞内寨卡病毒的复制水平，通过qRT-PCR检测细胞内先天免疫相关基因表达水平，通过双荧光素酶报告基因实验检测NFκB及ISRE的活性。在HeLa细胞中转染siCYLD，通过流式细胞术检测细胞凋亡。

（7）在A549细胞中转染寨卡病毒非结构蛋白质粒（NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5），通过qRT-PCR检测细胞内miR-16-5p与CYLD的表达水平。

研究结果：

（1）寨卡病毒能够感染A549细胞和293T细胞，且显著抑制miR-16-5p的表达。

（2）高表达miR-16-5p抑制寨卡病毒复制，促进Ⅰ型干扰素产生，增强ISRE活性和NFκB活性，促进干扰素刺激基因和炎性细胞因子表达，促进细胞凋亡。

（3）高表达miR-16-5p抑制了p-miR-CYLD-WT的荧光表达，降低了CYLD的表达水平。

（4）抑制CYLD表达可以抑制寨卡病毒复制，促进I型干扰素、干扰素刺激基因和炎性基因表达，增加ISRE活性和NFκB活性，并促进HeLa细胞凋亡。过表达CYLD可以促进寨卡病毒复制，抑制I型干扰素、干扰素刺激基因和炎性基因表达，并抑制ISRE活性和NFκB活性。

（5）寨卡病毒NS1蛋白下调miR-16-5p的表达，增强CYLD的表达。

研究结论：

研究结果表明，一方面，MiR-16-5p通过靶向CYLD，降低CYLD的表达水平，促进I型干扰素介导的抗病毒作用和NFκB信号通路有关的炎症反应，从而抑制寨卡病毒复制；另一方面，寨卡病毒NS1蛋白可以抑制宿主miR-16-5p表达，有利于病毒的复制。这些结果提示miR-16-5p在寨卡病毒与宿主相互作用中发挥着重要作用，为探索寨卡病毒感染治疗策略提供了新思路。

关键词：寨卡病毒；miR-16-5p；干扰素；干扰素刺激基因；CYLD；NFκB；

Abstract

Background: Zika virus (Zika Virus) is a mosquito-borne virus. ZIKV infection may result in serious neurological symptoms, including Guillain-Barre syndrome, while perinatal ZIKV infection is responsible for fetal microcephaly. Unfortunately, neither effective vaccine nor specific anti-ZIKV drug is available. Therefore, understanding the interaction between ZIKV and the host will be helpful to develop new anti-ZIKV strategies. MicroRNA is short-stranded RNA with a length of about 20 nt, which can bind to the 3'UTR region of mRNA, blocking translation or mediating the degradation of mRNA. Although the molecular mechanism remained to be elucidated. It has been reported that ZIKV-infected nerve cells and astrocytes experienced dysregulation of miRNA expression, indicating that miRNA may play important roles in ZIKV infection. MiR-16-5p, the marker of some infectious or immune diseases such as infectious pneumonia, Dermatology and rheumatoid arthritis, was shown to inhibit the virus replication and promote cell apoptosis in enterovirus 71 (EV71) infection. Moreover, our previous study identified CYLD (cylindromatosis) as a potential target of miR-16-5p. CYLD, a deubiquitinating enzyme that depolymerize the ubiquitin chain from K63, can modulate the interferon(IFN) signaling pathway by deactivating RIG-I. It can also inhibit NFκB pathway, suppressing the expression of inflammatory cytokines. These results indicate that miR-16-5p is closely associated with infection and immunity. However, whether and how miR-16-5p is involved in ZIKV replication is still unknown.

Aims: In this study, we aim to investigate the effect of miR-16-5p on ZIKV infection, identify the target of miR-16-5p and explore the underlying mechanism.

Methods:

(1) A549 cells and 293T cells were infected with ZIKV and miR-16-5p expression were measured by qRT-PCR.

(2) To explore the effect of miR-16-5p on ZIKV replication and innate immune response, A549 cells and 293T cells were transfected with miR-16-5p mimic followed by ZIKV infection. The expression of ZIKV NS5 mRNA, IFNs, typical ISGs and inflammatory factors were tested using qRT-PCR. ZIKV NS5 protein was measured by western blot; NFκB and ISRE activity were measured by luciferase assay. To investigate the effect of miR-16-5p on apoptosis, A549 cells and HeLa cells were transfected with miR-16-5p and analyzed by flow cytometry.

(3) To validate the target of miR-16-5p, 1) the luciferase expression was measured following co-transfection of miR-16-5p and p-miR-CYLD-WT or p-miR-CYLD-MUT in A549 cells, and 2) the CYLD expression were measured by western blot in A549 cells transfected with miR-16-5p.

(4) To confirm the effect of CYLD on ZIKV replication, CYLD expression was inhibited or unregulated by RNAi or CYLD plasmid transfection, respectively. ZIKV replication, expression of IFNs, ISGs, pro-inflammatory cytokines, activity of ISRE and NFκB, cell apoptosis were evaluated by qRT-PCR, western blot, luciferase assay and/or flow cytometry.

(5) To investigate the effect of ZIKV on miR-16-5p, A549 cells were transfected with ZIKV non-structure protein plasmid, and the expression of miR-16-5p and CYLD was analyzed by qRT-PCR.

Results:

(1) ZIKV infection and replication suppressed miR-16-5p expression in A549 cells and 293T cells.

(2) MiR-16-5p overexpression inhibited ZIKV replication, enhanced the expression of IFNs, ISGs and pro-inflammatory cytokines, and promoted cell apoptosis.

(3) Overexpression of miR-16-5p downregulated p-miR-CYLD-WT luciferase expression and CYLD protein level.

(4) Silence or overexpress CYLD can modulate the IFN signal pathway and NFκB pathway in five aspects: IFNs, ISGs and cytokines expression, ISRE and NFκB activity. Silence CYLD promotes HeLa cells apoptosis.

(5) ZIKV NS1 suppressed miR-16-5p expression and upregulated CYLD expression.

Conclusion: On the one hand, miR-16-5p targeted CYLD to inhibit ZIKV replication through promoting host innate immune response, anti-viral inflammation and cell apoptosis; On the other hand, ZIKV NS1 downregulated miR-16-5p expression to benefit ZIKV replication. Our results indicated that miR-16-5p is closely associated with the interaction between ZIKV and host, providing new insights into the role of miR-16-5p in viral infection.

Key words: Zika virus; miR-16-5p; Interferon; ISGs; CYLD; NFκB;