宿主ARF4和ARF5基因敲除小鼠的构建及其在寨卡病毒感染中的作用

Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice

摘要:

本研究旨在构建宿主ADP-核糖基化因子4 (ADP-ribosylation factor 4, ARF4)和ADP-核糖基化因子5 (ADP-ribosylation factor 5, ARF5)基因敲除小鼠，明确ARF4和ARF5基因对寨卡病毒感染的作用。首先利用CRISPR-Cas9技术，获得ARF4和ARF5单基因敲除小鼠(ARF4KO和ARF5KO)，并通过杂交繁育获得双基因敲除小鼠(ARF4KO/ARF5KO)，通过PCR法鉴定小鼠基因型，定量RT-PCR法检测目标基因的敲除效果。之后，用寨卡病毒感染基因敲除小鼠，采集第2、4、 6天小鼠血液和各组织样本，提取核酸后用RT-qPCR法检测病毒载量，用HE染色观察组织病理变化。结果显示，用ARF4和ARF5特异引物，分别在ARF4KO^–/+、ARF5KO^–/–以及ARF4KO^–/+/ARF5KO^–/–小鼠扩增到与目标基因大小一致的条带。RT-qPCR检测结果显示，ARF4KO^–/+小鼠各组织中ARF4 mRNA水平显著降低，其敲除效率在37.8%-50.0%之间，ARF5表达水平无变化。ARF5KO^–/–小鼠组织中ARF5 mRNA完全敲除，ARF4无变化。ARF4KO^–/+/ARF5KO^–/–小鼠在肺、肾和睾丸中ARF4 mRNA水平显著降低，ARF5完全敲除。最后，用寨卡病毒分别感染基因敲除小鼠和野生型小鼠。结果显示，与野生型小鼠相比，ARF4KO^–/+小鼠在各时间点血清中病毒载量均显著下降，但ARF5KO^–/–小鼠与ARF4KO^–/+/ARF5KO^–/–小鼠无明显变化。同时，与野生型小鼠相比，ARF4KO^–/+小鼠各组织病毒载量没有显著降低，但其大脑和睾丸的病理性改变减轻。本研究利用CRISPR-Cas9技术成功构建了ARF4和ARF5基因敲除小鼠，并证实ARF4是寨卡病毒感染必需的宿主蛋白，为后续深入研究ARF4和ARF5在寨卡病毒感染致病中的作用机制以及抗病毒药物研发奠定了基础。

Abstract:

The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, ARF5 and ARF4 genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice. Fertilized eggs were transplanted to obtain ARF4 or ARF5 knockout (ARF4KO or ARF5KO) mice, and ARF4/5 double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice (ARF4KO/ARF5KO). Then, the mouse genotypes were identified by PCR to identify the positive knockout mice, and RT-qPCR was employed to examine the knockout efficiency. The mice were then infected with ZIKV and the blood and tissue samples were collected after 2, 4, and 6 days. RT-qPCR was then employed to determine the virus load, and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue. The results showed that expected PCR bands were detected from ARF4KO^–/+, ARF5KO^–/–, and ARF4KO^–/+/ARF5KO^–/– mice, respectively. The results of mRNA transcription measurement indicated the significant knockdown of ARF4 by 37.8%–50.0% but not ARF5 in ARF4KO^–/+ compared with the wild-type mice. Meanwhile, complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO^–/– mice. Additionally, completed knockout of ARF5 and down-regulated mRNA level of ARF4 in the lung, kidney, and testis were detected in ARF4KO^–/+/ARF5KO^–/–mice in comparison with the wild-type mice. The virus load in the serum decreased in ARF4KO^–/+ mice, while it showed no significant change in ARF5KO^–/– or ARF4KO^–/+/ARF5KO^–/– mice compared with that in the wild type. Meanwhile, ARF4KO^–/+ mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice. We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study. ARF4 rather than ARF5 is essential for ZIKV infection in vivo. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.