| Viral infections can cause serious diseases that endanger human health.They often cause global concern,worry and even panic.For example,an outbreak of Zika virus(ZIKV)has occurred in the Americas,and its infection has caused microcephaly and Guillain-Barre syndrome.Dengue virus(DENV)affects approximately 390 million people each year,resulting in dengue fever,dengue haemorrhagic fever,and Dengue Shock Syndrome.Hepatitis C virus(HCV)infection causes chronic hepatitis,cirrhosis,and hepatocarcinoma,which affects approximately 170 million people worldwide.As absolute intracellular parasites,viruses must enter host cells to initiate replication,spread,and maintenance of viral infection.Therefore,the interference of virus into the host cell pathway will prevent viral infection,and it is an efficient and excellent antiviral strategy.Most viruses use cell endocytosis machinery to enter the host cell.The virus releases the viral nucleic acid into the cell by membrane fusion or penetration at the plasma membrane,endosome,endoplasmic reticulum,and nuclear membrane.This fusion or penetration process is usually associated with pH,and regulates the pH of cells and organelles is a major function of the ion channel.Obviously,ion channels should play a very important role in the process of virus entering the host cell.However,the relationship between host ion channels and viral infections is rarely explored.Ion channels are membrane channel proteins that exchange ion between inside and outside of the cytoplasmic membrane or organelle membrane,and play an important role in maintaining various physiological functions of cells.Kv1.3 potassium channels are widely distributed on the surface of plasma membrane and organelle membrane of excitatory and non-excitatory cells,and mediate K+ outflow of cell plasma membrane,K+ influx of organelle membrane,maintain resting membrane potential and regulate membrane repolarization.Kv1.3 potassium channels are crirical to regulate cell proliferation,apoptosis,and cell volume.The abnormal expression of potassium channel Kv1.3 closely related to insulin resistance,neurodegenerative diseases,autoimmune diseases,and cancer,thus Kv1.3 potassium channel is considerated as a target for the treatment and diagnosis of various diseases.However,the relationship between the Kv1.3 potassium channel and viral infection has solely been studied.First,this study analyzed the gene expression and function of defensin BmKDfsin3 from scorpion Mesobuthus martensii.Using the scorpion infection model,it revealed that the scorpion defensin BmKDfsin3 has a constitutive transcriptional expression pattern.In the HCV Huh7.5.1 cell infection system,BmKDfsin3 was able to inhibit HCV replication in a concentration-dependent manner,and its IC50 for inhibiting HCV replication was 2.34 μM.At the same time,BmKDfsin3 also has a selective inhibition of Kv1.3 potassium channel activity,and the IC50 for inhibiting Kv1.3 potassium channel activity is 23.4 nM.Our laboratory found that the IC50 values of the Kv1.3 potassium channel-selective scorpion toxin peptide inhibitors BmKTX-D33H and ADWX-1 blocking Kv1.3 potassium channel activity were 15.4 pM and 1.89 pM respectively,and they were found to be effective on HCV replication in this study,its IC50 for inhibition of HCV replication was 1.22 μM and 24.20 nM,respectively.Studies suggest that Kv1.3 potassium channel-selective scorpion toxin peptide inhibitors may be universal in affecting HCV replication,and that their inhibition of HCV replication function is positively correlated with the Kv1.3 channel activity inhibitory function.These experimental results show that the Kv1.3 potassium channel which is detected by the molecular probe of defensin BmKDfsin3 may affect HCV replication.Second,the expression and activity of Kvl.3 potassium channel in HCV-infected human hepatocyte cell line Huh7.5.1 and liver tissue were studied.RT-PCR,Western blot and immunohistochemistry revealed that Kvl.3 potassium channels were expressed in both human liver cell line Huh7.5.1 and liver tissue at mRNA and protein levels.Using the whole cell patch clamp technique,Kv1.3 potassium channel current can also be detected on the surface of Huh7.5.1 cell membrane.The study also found that HCV replication upregulates Kv1.3 potassium channel mRNA and protein levels in the Huh7.5.1 cell line in an interferon-independent manner,and immunohistochemical analysis showed that Kvl.3 protein expression was also elevated in liver tissues of HCV-infected patients.At the same time,in the Kv1.3-stable,a Huh7.5.1 cell line that is stably over-expressing the Kv1.3 potassium channel,HCV replication not only up-regulates the mRNA and protein expression of the Kv1.3 potassium channel,but also enhances the cell membrane surface Kv1.3 potassium channel current.These experimental results have revealed that HCV infection can up-regulate the expression of Kv1.3 potassium channels and increase the Kvl.3 potassium channel current of the host cell membrane,suggesting that the host cell Kv1.3 potassium channel is closely related to HCV replication.Then,the effect of Kv1.3 potassium channel on HCV replication was studied.The Kv1.3 potassium channel was over-expressed in Huh7.5.1 cells by molecular cloning,cell transfection,and confirmed by qRT-PCR,Western blotting.The experimental results showed that overexpression of Kv1.3 potassium channels in host cells inhibited HCV replication,down-regulated overexpression of Kv1.3 potassium channel can restore its inhibitory effect on HCV replication and Kv1.3 potassium channel protein knockout promotes HCV replication.The results of these in vitro cell experiments show that the host cell Kv1.3 potassium channel can effectively inhibit HCV replication.The"phase of function" experiments have shown that the Kv1.3 potassium channel inhibits the entry of HCV into the host cell:it does not affect the integrity and infectivity of HCV virus particles and does not affect the adsorption phase of HCV into the host cell but the post-adsorption phase of HCV entry into the cell process.Electron transmission microscopy observed that the Kvl.3 potassium channel blocked HCV particles in the vesicles.These studies indicate that the host cell Kv1.3 potassium channel inhibits HCV entry into host cells and is closely related to the viral envelope and endosome membrane fusion phases.Next,this study clarified the mechanism by which Kv1.3 potassium channel inhibits HCV entry into host cells.In Huh7.5.1 cells,Kv1.3 potassium channels are located on acidic organelles such as early endosomes,late endosomes,lysosomes,and could be colocalized with HCV in the process of entry.Overexpression of the Kv1.3 potassium channel results in an increase in the pH of the acidic organelles.The cell-cell membrane fusion study found that Kv1.3 potassium channel can inhibit the HCV E1E2 envelope protein-mediated membrane fusion process.And similar to the acidification inhibitors BAF and Vacuolin-1,overexpression of Kv1.3 potassium channels can amplify acidic organelles.These experimental results indicate that the Kvl.3 potassium channel of the host cell affects the fusion of the HCV envelope with the endosome membrane by inhibiting the acidification of the endosome,thereby preventing the release of RNA genome of HCV into the cytoplasm and eventually limiting the entry of HCV into the host cell.Finally,the Kv1.3 potassium channel was studied to limit the broad spectrum of virus entry into host cells.The Flaviviridae envelope viruses DENV and ZIKV have a similar cell entry process to HCV and both enter the host cell via a membrane fusion process mediated by endosome acidification.The results of qRT-PCR,Western blot.plaque assay,transmission electron microscopy,and membrane fusion showed that the Kv1.3 potassium channel of the host cell can also affect the fusion of the viral envelope with the endosome membrane of the host cell,thus effectively inhibiting the DENV and ZIKV copies.Unlike HCV,DENV,and ZIKV,the membrane fusion process of SeV,the envelope virus of the Paramyxoviridae,into the cell occurs at neutral pH conditions and on the cytoplasmic membrane surface.Viral RNA detection experiments showed that overexpression of Kvl.3 potassium channels did not affect SeV replication.Thus,the Kvl.3 potassium channel inhibits the replication of a class of viruses that enter the host cell via membrane fusion processes mediated by endosomes acidification,and is a broad-spectrum antiviral protein that restricts virus entry into host cells.In summary,this study first used defensin BmKDfsin3 from scorpion Meobuthus martensii as a molecular probe,and found that the Kvl.3 potassium channel of the host cell probably affects HCV replication.Then,it was revealed that the Kvl.3 potassium channel of the host cell has the function of inhibiting the membrane fusion of HCV into the cell,and the anti-HCV mechanism of the host cell Kvl.3 potassium channel inhibiting the fusion process of the membrane by affecting the acidification of the endosome is proved.The Kv1.3 potassium channel inhibits the replication of a class of viruses that enter the host cell via membrane fusion process mediated by endosomes,and is a broad-spectrum antiviral protein that restricts virus entry into host cells.In this study,we first discovered the new function of the Kv1.3 potassium channel in inhibiting the entry of virus into the host cell at the membrane fusion stage,and provided new strategies and approaches for the prevention and treatment of infections such as HCV,DENV and ZIKV and other viruse,and also provides knowledge for the host cell ion channel as broad-spectrum antiviral targets. |
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