Promoting Effects Of Host Oncoprotein Epithelial Growth Factor Receptor In Zika Virus Replication And The Underlying Mechanisms

Background:Epidermal growth factor receptor(EGFR),an oncoprotein mostly located on human cell membranes,is a tyrosine kinase receptor.Once bound by its ligands,EGFR mediates the activation of a variety of cellular signal transduction pathways and plays an important role in regulating cell survival,proliferation,differentiation,migration and apoptosis.Aberrantly excessive activation of the EGFR signaling pathway leads to abnormal cell proliferation,which is closely associated with development and progression of various types of tumors,including non-small cell lung cancer,colon cancer,breast cancer,head and neck cancer,and ovarian cancer.At present,a variety of inhibitors and strategies have been developed for the treatment of malignant tumors by targeting EGFR.Several EGFR inhibitors have now been widely used in the clinic.Zika virus(ZIKV)belongs to the Flaviviridae family of the genus Flavivirus and is a single positive-stranded RNA arbovirus,transmitted mainly through Aedes mosquitoes.The clinical manifestation of ZIKV infection includes headache,fever,conjunctivitis,myalgia,rash and arthralgia,etc.It is noteworthy that ZIKV infection has been associated with neonatal microcephaly,adult Guillain-Barre syndrome,and neurological diseases such as viral encephalitis,meningitis and encephalomyelitis.Due to the lack of full elucidation of the biological characteristics and pathogenic mechanisms of ZIKV,no specific therapeutic drugs or effective preventive vaccines against ZIKV are clinically available at present.It is therefore urgent to further understand the pathogenic mechanisms of ZIKV infection and to identify potential therapeutic targets.Recently,an increasing number of studies have shown that oncoprotein EGFR also plays an important role in viral infection.Viruses can interact with EGFR and modulate the EGFR signaling pathway to facilitate viral entry,replication or escape host immune surveillance.Targeting EGFR or its downstream signaling pathways through specific inhibitors is emerging as an important new strategy to combat viruses.Based on this,this current study mainly explored the effect and mechanism of oncoprotein EGFR and its signaling pathway on ZIKV replication,and provide new therapeutic targets for potential treatment of ZIKV-associated diseases.Objectives:1.To investigate whether EGFR impacts on the degree of ZIKV infection.2.To explore the molecular mechanism of EGFR in altering ZIKV replication3.To determine whether inhibition of EGFR can change infection of ZIKV.Methods:1.To examine the biological function of EGFR in altering ZIKV replication:(1)RNA interference and CRISPR/Cas9 technologies were used to reduce the expression of endogenous EGFR in U251 cells,followed by RT-qPCR and Western blotting analysis to determine whether EGFR expression alters ZIKV replication;(2)Exogenous stable high-expressing EGFR cell lines were established,and then RT-qPCR,Western blotting and immunofluorescence assay were carried out to examine whether EGFR expression promotes ZIKV infection;2.To explore the molecular mechanism via which EGFR affects the degree of ZIKV infection:(1)RT-qPCR assay was used to investigate whether EGFR affects cell binding or entry of ZIKV.(2)Co-immunoprecipitation analysis was performed to determine whether EGFR interacts with ZIKV envelope protein.(3)Western blotting analysis was used to examine the activation of EGFR signaling pathway after ZIKV infection.3.To investigate whether EGFR inhibitor can modulate ZIKV infection:(1)RT-qPCR assay was used to evaluate whether the EGFR inhibitor gefitinib can change cell binding or entry of ZIKV.(2)RT-qPCR assay and Western blotting analysis were conducted to evaluate whether the EGFR inhibitor Gefitinib can affects viral replication.Results:1.Knockdown or knockout of endogenous EGFR in host cells inhibits ZIKV replication.2.Overexpression of ectopic EGFR in host cells is able to promote ZIKV replication.3.ZIKV envelope protein binds EGFR and activates the EGFR signaling pathway to facilitate its cell entry.4.The EGFR inhibitor Gefitinib inhibits ZIKV entry and replication.Conclusion:In summary,this study uncovers that the expression of EGFR promotes ZIKV replication.ZIKV interacts with EGFR through its envelope protein,and activates the EGFR signaling pathway to promote ZIKV cell entry.Importantly,inhibition of EGFR by its inhibitor Gefitinib effectively reduces ZIKV infection.These current findings reveal a previously unrecognized mechanism underlying which EGFR promotes ZIKV replication,and provide a foundation for the potential applicability of EGFR inhibitors in the treatment of diseases caused by ZIKV infection.