UMP-CMP Kinase 2 Inhibits ZIKV Replication And The Underlying Molecular Mechanism

Background and Aims:Zika Virus(ZIKV)is a single-stranded positive RNA virus that can be transmitted through mosquito bite,blood transfusion,vertical transmission and sexual contact.Most ZIKV infected persons are asymptomatic,while pregnant women with ZIKV infection will cause serious neurological diseases and lead to fetal microcephaly or Guillain-Barre syndrome.Up till now,there is still a lack of specific therapeutic drugs and effective vaccines to treat or prevent ZIKV infection.Exploring the interaction between ZIKV and host immune response is essential to develop anti-ZIKV drugs.Interferon(IFN)mediated innate immune response is a vital line of defense against pathogen infection.Previous studies reported that viruses infections stimulated expression of a large number of IFNstimulated genes(ISGs),including CMPK2.CMPK2 has been shown to play an antiviral role in many virus infections,such as in dengue virus(DENV),human immunodeficiency virus(HIV)and human hepatitis E virus(HEV)infections.However,the effect of CMPK2 on the replication of ZIKV and its underlying mechanisms remain to be clarified.Methods:1.A549 cells were infected with ZIKV.CMPK2 mRNA from cells and supernatant was detected by real-time quantitative PCR(RT-qPCR)or CMPK2 protein from cells was detected by Western blot,respectively.2.CMPK2 expression was up-regulated or down-regulated by transfection with plasmid or siRNA into A549 cells prior to ZIKV infection,respectively.48 hours after ZIKV infection,the cellular RNA and protein were collected to detect the replication of ZIKV by RT-qPCR and Western blot.3.Over-expression plasmid or small interfering RNA(siRNA)of RIG-I was transfected into Huh7、Huh7.5.1、A549 cells to up-regulate or down-regulate the expression level of RIG-I,respectively.RIG-I、CMPK2 expression and ZIKV replication were analyzed by real-time PCR and Western blot to explore whether the expression of CMPK2 is dependent on RIG-I.IFNAR deficient U5A cells were used to identify whether the induction of CMPK2 is dependent on IFNAR.4.To investigate whether CMPK2 inhibits ZIKV replication through activating the classical type Ⅰ IFN signaling pathway,we detected the phosphorylation level of STAT1 by Western blot;the activity of interferon stimulating response element(ISRE)by double luciferase reporter gene assay;and the expression levels of downstream ISGs by RTqPCR.Results:1.ZIKV infection stimulated CMPK2 expression at both mRNA and protein levels.2.Overexpression of CMPK2 inhibited the replication of ZIKV.On the contrary,CMPK2 knock-down promoted ZIKV replication.3.Overexpression of RIG-I in RIG-I-deficient Huh7.5.1 cells restored the expression of CMPK2 and inhibited ZIKV replication.While the expresion level of CMPK2 was not affected in IFNAR-deficient U5 A cells following ZIKV infection.4.Overexpression of CMPK2 increased the phosphorylation level of STAT1,ISRE activity and expression levels of other ISGs.Conclusions:ZIKV infection induced CMPK2 expression through activation of the RIG-IMDA5-IFN signaling.CMPK2 overexpression inhibited the replication of ZIKV,while down-regulated CMPK2 promoted ZIKV replication.CMPK2 inbibited ZIKV replication by activating type I IFN-mediated JAK/STAT signaling pathway.Our results clarified the mechanism on how CMPK2 inhibits ZIKV replication and expanded the antiviral scope of CMPK2.