The Effect And Mechanism Of MiR-16-5p On Zika Virus Replication

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 NF?B 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;NF?B 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 NF?B,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 NF?B pathway in five aspects:IFNs,ISGs and cytokines expression,ISRE and NF?B 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.