Mechanism Of EGFR Regulating Hantaan Virus Replication

Hemorrhagic Fever with Renal Syndrome(HFRS)is an acute infectious disease caused by hantavirus infection.HFRS is a global epidemic that threatens social and public health,as well as human health and life.China is the region most threatened by HFRS,accounting for 90% of all HFRS cases worldwide.It is imperative to address the urgent issue of preventing and treating HFRS.According to the geographical distribution of hantavirus natural hosts,Hantaan virus(HTNV)is the principal pathogen causing HFRS in China,but its infection mechanism is unknown.Currently,treatment of HFRS is supportive,and there are no FDA-approved specific antiviral medicines available.Therefore,it is necessary to enhance our comprehension of the mechanism of HTNV infection further.Epidermal Growth Factor Receptor(EGFR)is a receptor tyrosine kinase(RTK)that is widely expressed in epidermal cells throughout the body.Multiple signaling pathways that are triggered by ligand binding are involved in regulating cell proliferation,growth and migration.It has been reported that EGFR is not only closely related to the development of cancer,but also widely involved in the infection of several viruses.Viruses including HCV,ZIKV,HBV,TGEV,HCMV,HSV,IAV exploit EGFR-mediated endocytosis and EGFR-regulated signaling pathways to facilitate their invasion,replication and modulate host defense responses.However,there are few studies concerning the role of EGFR in HTNV infection.The author previously found that EGFR can influence the formation of HTNV offspring viruses,but the mechanism is unknown.This study provided a theoretical foundation for the screening of anti-HTNV specific medicines through uncovering the mechanism of EGFR in HTNV infection,which has important clinical significance.The role of EGFR in the production of HTNV progeny viruses can be divided into two aspects.On the one hand,EGFR controls the invasion of HTNV;on the other hand,EGFR directly impacts HTNV replication in cells.To further validate the influence of EGFR on the formation of HTNV progeny viruses,HTNV was utilized to infect cell lines that stably overexpress,knock down,and knock out EGFR.Overexpression of EGFR significantly promoted the expression of HTNV nucleocapsid protein(NP),replication of S segment,and generation of progeny viruses,according to Western blot,RT q PCR,Plaque assay,respectively.However,knockdown or knockout endogenous EGFR could inhibit the expression of HTNV NP protein,nucleic acid replication,and offspring virus production.The results suggest that EGFR not only influences the formation of HTNV progeny virus,but also governs HTNV nucleocapsid protein expression in cells and nucleic acid replication.Subsequently,we utilized classic virus binding and entry experiments to detect the amount of virus that binds to cell surfaces and internalizes into cells at 4℃ and37℃,respectively.The results showed that overexpression or knockdown of EGFR had no effect on HTNV binding to host cells,but EGFR-overexpressing increased the internalization of HTNV;knocking down EGFR results in a decrease in internalized viruses,indicating that EGFR may be involved in HTNV invasion.Finally,the author observed an increase in HTNV replication levels in stable overexpressing EGFR intracellular domain cell,implying that the EGFR intracellular region is involved in HTNV viral replication.The virus minigene replicon system is a powerful tool for detecting the effect of target genes on viral transcription and replication.The Hantavirus minigene reporting system was utilized by the author to detect whether EGFR is involved in the transcription and replication of the HTNV genome.The findings revealed that EGFR greatly boosted HTNV genome transcription and replication,and that the kinase domain in the intracellular region of EGFR is a crucial domain that promotes viral genome transcription and replication.EGFR K745 is a critical amino acid for EGFR kinase function.The author discovered that overexpression of the EGFR Cyto K745 A mutant can reduce the influence of the EGFR intracellular region on HTNV replication.Erlotinib is an inhibitor that specifically targets the EGFR kinase domain.Adding different concentrations of Erlotinib to A549 cells for HTNV infection showed a dose-dependent inhibition of HTNV replication by Erlotinib,indicating that EGFR kinase activity plays an important role in its function.In order to further investigate the mechanism by which EGFR enhances HTNV replication,the author examined the interaction between HTNV structural proteins and EGFR.EGFR intracellular domain interacted with HTNV NP during transient transfection and virus infection,according to co-immunoprecipitation experiment.The colocalization of EGFR and HTNV NP in cells was also confirmed by immunofluorescence.The truncated mutants of EGFR and HTNV NP were used for co-immunoprecipitation and GST pulldown experiments.It was found that the kinase domain of EGFR was the key domain for interaction with HTNV NP,and the interaction between HTNV NP and EGFR was not dependent on its RNA functional domain.Based on the above results,it is indicated that EGFR affects HTNV replication through its interaction with HTNV NP.In summary,it was discovered that EGFR has a positive regulatory effect on HTNV replication.In addition to participating in HTNV invasion,EGFR mainly relies on its kinase activity to promote the transcription and replication of the HTNV genome,and it was revealed the interaction between HTNV NP and EGFR.These findings reveal the mechanism of Hantavirus infection,and provide new targets for the treatment of HFRS.