| Zika virus is a newly emerging mosquito borne pathogen,belonging to Flaviviridae of Flaviviridae.Zika virus infection can cause serious neurological complications,including microcephaly of newborn,congenital malformation,fetal death and Guillain Barre syndrome in adults.Since the first large-scale outbreak of Zika virus in Yapu islands in 2007,it has spread rapidly to various regions of the world,causing millions of people to be infected and seriously threatening human health.These serious outbreaks forced the World Health Organization(WHO)to list Zika virus as a global emergency public health event in 2016.Although the research on the pathogenesis and immune escape mechanism of Zika virus is in progress,there are still many unsolved problems in the mechanism of host restriction and immune escape.At present,there is no effective treatment or vaccine for zikv in clinical practice.Therefore,it is particularly important to clearly define the host factors of Zika virus and the molecular mechanism of their role in the development of safe and effective therapeutic strategies and preventive drugs and vaccines.Innate immune response is the host's first line of defense against viral infections.Type ? interferon signaling pathway plays an important role in defending against viral infections.Once cells sense pathogen invasion,a series of signal transduction pathways are triggered,finally,hundreds of IFN stimulating genes(ISGS)with antiviral and immunoregulatory functions were induced to express,so as to play an antiviral role.Studies have found that non-structural proteins of flavivirus can antagonize the IFN-I signaling pathway,so that the virus escapes the host's immune response.Therefore,based on the antiviral effect of IFN-I signaling pathway and the antagonistic effect of non-structural proteins of flavivirus on IFN-I signaling pathway,in this project,we mainly study the influence of non structural protein of Zika virus on the gene expression of type I interferon signaling pathway.In this study,293T cells were transfected with Zika virus non-structural protein NS1,NS3 or NS4A expression plasmids,and collected cell protein samples and total cell RNA 48h after transfections.Western blot was used to verify Zika virus non-structural protein NS1.NS3 and NS4A were overexpressed in 293T cells.At the same time,the IFN-?response signal pathway PCR array chip was used to detect the total RNA samples of the three groups of cells.The obtained experimental data was analyzed online on the PCR Array data analysis website on QIAGEN's official website,and the genes that were significantly up-regulated or down-regulated were analyzed.The results of the gene chip were verified by fluorescent quantitative PCR.At the same time,A549 cells were infected by Zika virus,and the expression of these genes was checked by qPCR to verify whether they were consistent with the verification results of gene chip.KEGG website and literature were used to explore the effects of these genes involved in the signal pathway or the gene that play an important role on Zika virus replication.As result,Western blot proved that Zika virus non-structural proteins NS1,NS3,NS4A were overexpressed in 293T cells after transfection with ZIKV NS1,NS3 or NS4A expression plasmids.Through IFN-? response signal pathway PCR array chip to detect detection and q-PCR verification,a total of 7 type ? interferon signaling pathways induced genes significant changes in gene expressio,including IRF7,ISG20,CCL2,JAK2,DDX58,MYD88,TNFSF10.The expression of ZIKV NS1 mediated the gene expression changes of IRF7,ISG20,JAK2 and tnfsf10,in which the gene expression of IRF7,ISG20 up-regulated,and the gene expression of TNFSF10 and JAK2 down-regulated.The expression of ZIKV NS3 gene mediates the change of DDX58 and IRF7 gene expression,in which the expression of IRF7 gene up-regulated and DDX58 gene down-regulated.The expression of ZIKV NS4A mediated the changes of gene expression of DDX58,CCL2,TNFSF10 and MYD88,in which the expression of CCL2 gene up-regulated,while that of DDX58,tnfsf10 and MYD88 gene down-regulated.At the same time,the expression of ISG20,IRF7 and CCL2 genes in A549 cells infected with Zika virus was generally increased,which was basically consistent with the results of microarray.The expression of MYD88 gene was generally decreased,which was basically consistent with the results of microarray.However,the expression of DDX58 and TNFSF10 gene was generally increased,which was contrary to the results of microarray verification.The expression of JAK2 gene was up-regulate before 8 hours,and then decreased.In conclusion,the expression of ZIKV NS 1 up-regulated IRF7 and ISG20 genes,and down-regulated TNFSF10 and JAK2 genes;the expression of ZIKV NS3 up-regulated IRF7 gene expression and down-regulated DDX58 gene expression;the expression of ZIKV NS4A expression up-regulated CCL2 gene expression and down-regulated DDX58,TNFSF10 and MYD88 gene expression... |
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