Research On The Role Of TRPV4 In The Process Of Influenza And Zika Virus Infection

In recent years,viral infectious diseases have brought great challenges to social public health security,such as influenza caused by influenza virus,Zika virus disease caused by Zika virus,and acute respiratory distress syndrome caused by SARS-Co V-2.Therefore,strengthening the monitoring,prevention and control of viral infectious diseases has attracted great attention to the international community.At present,the prevention and treatment of infectious diseases is mainly based on vaccination and antiviral treatment.However,the vaccine only targets known circulating strains and has a lag time due to long development cycle.Most of the existing antiviral drugs target viral proteins.Although significant progress has been made in the clinical treatment of viral infectious diseases,there are some defects such as easy to develop viral resistance and unable to completely eliminate the virus.Since viruses need host cells to complete self-replication,host factors play important roles in virus proliferation,which provides favorable conditions for finding effective antiviral drug targets.As an important signaling factor in cells,calcium ions(Ca²⁺)play an important role in viral invasion,viral genome replication,virion maturation,and progeny viral release.By hijacking host calcium channels,virus disturbs intracellular Ca²⁺homeostasis and provides favorable conditions for virus self-replication.Transient receptor potential vanillate isoform 4（TRPV4）is a Ca²⁺permeable nonselective cationic channel protein on the plasma membrane of cells.TRPV4mediates Ca²⁺influx and promotes viral replication when cells are exposed to Zika virus,suggesting that TRPV4 plays an important role in the process of viral infection.This study aims to reveal the important role of TRPV4-regulated Ca²⁺signaling in Zika virus（ZIKV）and influenza virus（IV）infection and pathogenesis.First,by measuring the intracellular calcium concentration,it was found that Zika virus or influenza virus infection can lead to an increase in intracellular calcium concentration.Compared with the control group,the content of Ca²⁺in BHK cells increased by about 1.26 times after 24hours of Zika virus infection,while the content of Ca²⁺in A549 cells increased by about 4.04 times after 24 hours of infection with influenza virus H5N6 strain.Western blot experiments showed that Zika virus or influenza virus infection upregulated TRPV4 protein expression levels.In addition,indirect immunofluorescence results showed that increasing intracellular Ca²⁺concentration did not significantly promote Zika virus or influenza virus infection in host cells,while decreasing intracellular Ca²⁺concentration significantly inhibited Zika virus or influenza virus infection.Secondly,after Zika virus or influenza virus infection,the activator（GSK1016790A）and inhibitor（HC-067047）of TRPV4 channel protein were used to treat the cells,respectively,and it was found that the activation of TRPV4 channel did not promote the infection of cells by the virus,however,inhibition of TRPV4 could significantly reduce the viral titer in infected cells and significantly inhibit the infection of cells by Zika virus or influenza virus.Subsequently,gene editing experiments showed that overexpression of TRPV4 could not promote the infection of Zika virus or influenza virus to cells,while silencing TRPV4 could significantly reduce the infection of virus to cells.Furthermore,using mice as model animals,the potential biological function of TRPV4 in the pathogenesis of Zika virus or influenza virus was evaluated.In Zika-infected A129 mice,TRPV4inhibitor（HC-067047）treatment alleviated the sharp weight reduction of virus-infected mice,improved the survival rate of infected mice by 25%,significantly reduced the m RNA expression of viral gene NS5 in infected mouse tissues（brain,liver,spleen,kidney,testis and epididymis）,and improved histopathological damage caused by viral infection.In addition,in influenza-infected BALB/c mice,TRPV4 inhibitor（HC-067047）treatment also alleviated the sharp weight reduction of virus-infected mice,improved the survival rate of infected mice by 20%,significantly reduced the virus titer in the lung tissue of infected mice,and improved the pathological damage of lung tissue caused by virus infection.Similarly,in TRPV4 knockout mice infected with Zika virus or influenza virus,knockout TRPV4 ameliorated tissue pathological damage caused by viral infection compared to controls.Notably,when influenza-infected TRPV4 knockout mice were infected,TRPV4 knockout improved the survival rate of influenza-infected mice compared to the control group（protection rate was 20%）.Combined with in vitro experiments,we reveal the important role of TRPV4 in regulating Zika virus or influenza virus infection and pathogenesis.Finally,we also analyze the potential regulatory mechanism of TRPV4 in the process of influencing influenza virus infection.Western blot analysis showed that influenza virus infection increased the phosphorylation levels of IκBαand NF-κB/p65 proteins,leading to the activation of the NF-κB signaling pathway.In addition,real-time quantitative PCR（RT-q PCR）results showed that influenza virus infection increased the m RNA expression levels of cytokines（TNF-α,IL-1β,IL-6）and interferons（IFN-α,IFN-β）.Importantly,inhibition of TRPV4 restored the NF-κB signaling pathway induced by influenza virus infection to a resting state and significantly reduced cytokine expression after influenza virus infection.This suggests that TRPV4 has a potential regulatory role in related diseases caused by abnormal cytokine expression.Taken together,this study reveals the potential role of TRPV4 channels in Zika and influenza virus infection and pathogenesis.In addition,we have revealed the biological function of TRPV4in regulating the NF-κB signaling pathway induced by viral infection and elucidated the important role of this protein in regulating the expression levels of cytokines.This not only provides a new insight into the pathogenesis of Zika virus and influenza virus infection,but also lays an important foundation for the development of antiviral drugs.