Role Of Hsa?Circ?0001613 In The Replication Of Zika Virus And Its Underlying Mechanism

Background:Zika virus(Zika virus,ZIKV)is a single-stranded RNA virus that belongs to the Flavivirus family with high neurotrophic and teratogenic effects.As a mosquito-borne virus,ZIKV can also be transmitted by blood transfusion.ZIKV infection can cause Guillain-Barre in adults or give birth to babies with microcephaly if women infected during pregnancy,which attracts global attention.Up till now,neither an effective vaccine nor specific antiviral treatment is available.Therefore,it is essential to investigate the pathogenesis of Zika virus infection.Circular RNAs(circRNAs),with a special covalently closed structure,are formed by back-splicing of pre-mRNA and involved in various gene regulations.Studies have shown that circRNAs play an important role in the regulation of host antiviral immunity and in virus replication.However,the role of circRNAs in ZIKV infection and the underlying mechanism has not been reported.Type ? interferons(mainly IFN-?/?),as key cytokines of the host anti-viral innate immune system,play an important role in limiting virus infections.Through binding to the IFN surface receptors(IFNARs),IFN-?/? activates the JAK/STAT signaling pathway and stimulates the activity of the nuclear interferon-stimulated response element ISRE,leading to the production of several hundred interferon-stimulated genes(ISGs)to inhibit virus replication.In our previous study,we found IFN-?/? and several ISGs can regulate ZIKV replication.However,whether circRNAs regulate ZIKV through IFN pathway is unclear.Aims:In this study,we sought to identify the circRNAs profiles in A549 cells and ZIKV infected A549 cells.We will select one circRNAs(hsa_circ₀001613)significantly regulated by ZIKV infection and explore its role in ZIKV replication and preliminarily mechanism of activating the JAK/STAT signaling pathway mediated by type I interferon.Content:In this study,two parts of experiments were designed to explore the role of circRNAs in Zika virus replication and the underlying mechanisms.In the first part,circRNA expression profiles of Zika virus-infected adenocarcinomic human alveolar basal epithelial cells(A549).We chose hsa_circ₀001613 to dissect its role in ZIKV replication and to explore the underlying mechanism in JAK/STAT signal transduction pathway mediated by type-I IFN in the second part.Methods:(1)ZIKV were infected in A549 cells.Total RNAs and proteins were extracted and ZIKV replication was analyzed both at mRNA and protein levels by quantitative real-time PCR(qRT-PCR)and western blotting(WB),respectively.(2)Total RNAs in A549 cells with or without Zika virus infection were extracted by Trizol method and were used for circRNA RNA-seq analysis.Differentially expressed circRNA profiling following Zika virus infection was obtained by bioinformatics analysis.Selected circRNAs were validated using qRT-PCR,agarose Gel electrophoresis and RNase R digestion experiments.(3)The expression of hsa_circ₀001613 in different cell lines and after three RNA viruses(ZIKV,DENV and HCV)infections were detected by qRT-PCR.Subcellular location of hsa_circ₀001613 was also analyzed.hsa_circ₀001613 was knocked down by siRNA in A549 cells before infected with ZIKV,ZIKV replication was monitored both at mRNA and proteins levels by qRT-PCR and western blotting(for ZIKV NS1),respectively.Dual-luciferase reporter assay was employed to analyze the activity of Interferon-sensitive response element(ISRE).And finally,expression levels of IFN-?/? and interferon-stimulated genes(ISGs)were determined by qRT-PCR.Results:We confirmed that Zika virus can infect and replicated effectively in A549 cells.We identified 11 up-regulated and 34 down-regulated circRNAs following ZIKV infection by circRNA RNA-seq and bioinformatics analysis.In addition,we validated three differentially expressed circRNAs,which are consistent with the RNA-seq results.We confirmed that hsa_circ₀001613 was expressed in A549 cells,293T cells and Huh 7.5.1 cells though the expression level various in different cells following infection with different RNA viruses(ZIKV,DENV and HCV).The expression level of hsa_circ₀001613 decreased significantly in A549 cells following ZIKV or DENV-2 infection.By contrast,its expression level was significantly up-regulated after HCV JFH1 infection in Huh7.5.1 cells.Subcellular location analysis revealed that hsa_circ₀001613 was mainly located in the cytoplasm.Knockdown of hsa_circ₀001613 inhibited ZIKV replication,while significantly upregulated IFN-?/? production and enhanced ISRE activity leading to the increased expression of downstream interferon-stimulated genes(ISGs).ConclusionWe identified 46 significantly different expressed circRNAs after ZIKV infected A549 cells by circRNA RNA-seq,of which 11 were significantly up-regulated and 34 were significantly down-regulated.ZIKV infection significantly suppressed hsa_circ₀001613 expression in A4549 cells.Preliminary study indicated that hsa_circ₀001613 knockdown inhibited ZIKV replication possibly through activating type-? IFN signaling pathway as showed by increased ISGs expression and ISRE activity.