Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice
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.
万方数据
维普
