Promoting Effects Of Host Exostosin Glycosyltransferase 2 In Zika Virus Replication And The Underlying Mechanisms

Background:Exostosin glycosyltransferase 2(EXT2)is a member of the exostosin family,which is closely related to the synthesis of heparan sulfate proteoglycans.Previous studies have found that the exostosin family serves as tumor suppressor,and the mutation inactivation of EXT2 can trigger the formation of a type of benign tumor known as Hereditary multiple exostoses(HMO).Zika virus(ZIKV)is a single positive-stranded RNA virus of the genus Flavivirus,and transmitted mainly through mosquitoes.The main clinical manifestations of patients infected with Zika virus include headache,fever,muscle pain,arthralgia,conjunctivitis,myelitis,Guillain-Barre syndrome and viral meningitis,etc.Pregnant women infected with ZIKV can cause congenital Zika virus syndrome,including neonatal microcephaly,which greatly damages the health of newborns.Due to the lack of fully understood the biological characteristics and pathogenesis of ZIKV,thus far there is no specific therapeutic drugs or vaccines for ZIKV.It is therefore urgent to further explore the pathogenic mechanism of ZIKV and to identify potential drug targets.Recently,studies have shown that EXT1,homologous to EXT2,can inhibit porcine reproductive and respiratory syndrome virus infection by degrading its NSP3 and NSP5 proteins through the proteasome pathway.In addition,there are few studies linking EXT2 to viral infections.Our previous analysis of the public genes dataset based on the CRISPR/Case9 system for whole genome knockout screening and found that EXT2 was an essential factor for the replication of flavivirus.Based on this,this study mainly explores the effect and mechanism of EXT2 on ZIKV replication,aiming to provide a new drug target for potential treatment of ZIKV-associated diseases.Objectives:1.To investigate whether EXT2 impacts on the degree of ZIKV replication.2.To explore the molecular mechanism of EXT2 on ZIKV replication.Methods:1.To examine the biological function of EXT2 in on ZIKV replication:(1)RNA interference was used to reduce the expression of endogenous EXT2 in A549 and U251 cells,followed by RT-qPCR and Western blotting analysis to examine whether EXT2 expression alters ZIKV replication.(2)Exogenous overexpressing EXT2 cell lines were established,and then RT-qPCR,Western blotting and immunofluorescence assay were used to determine whether EXT2 expression promotes ZIKV replication.2.To explore the molecular mechanism by which EXT2 affects the degree of ZIKV replication:(1)The effects of EXT2 on interferon and interferon-stimulating genes were verified by ELISA and RT-qPCR.(2)The effects of EXT2 on interferon signaling pathway were detected by luciferase reporter assays.(3)The interaction between EXT2 and key molecules of interferon signaling pathway was verified by co-immunoprecipitation assays.Results:1.Knockdown of endogenous EXT2 in host cells inhibits ZIKV replication.2.Overexpression of EXT2 in host cells is able to promote ZIKV replication.3.EXT2 can inhibit the production of type Ⅰ interferon and the expression of interferon-stimulating genes.4.EXT2 can competitively inhibit the interaction between IKK and IRF3,TBK1 and IRF3,thereby repressing the activation of IRF3.Conclusion:In summary,this study found that EXT2 can promote ZIKV replication by inhibiting the production of type Ⅰ interferon via competitively blocking the interaction between IKK and IRF3,TBK1 and IRF3.The study provides potential new drug targets for the treatment of Zika virus disease.