Mechanism Of Protein 4.1R In Zika Virus Infection

BackgroundZika virus（ZIKV）belongs to the positive-strand RNA virus family of the flavivirus genus.The virus is a typical mosquito-borne flavivirus that can also be transmitted from mother to child,sexually transmitted and blood.ZIKV is expressed in the form of polyprotein and contains 3 structural proteins（C,pr M/M,E）and 7 non-structural proteins（NS1-5）.Adults infected with ZIKV are usually asymptomatic,but can also cause mild fever,headache,joint pain,conjunctivitis,etc;ZIKV infection in pregnant women can cause severe fetal microcephaly,neurodevelopmental defects and even death.Exploring the mechanism of action between ZIKV and host cells can provide new directions and ideas for the prevention and treatment of Zika virus.Membrane skeletal protein 4.1R is a member of the 4.1 superfamily and was originally discovered in human mature red blood cells.Protein 4.1R can promote the binding of spectrin-actin and help the connection of the cell membrane skeleton to the membrane,it can interact with a variety of transmembrane proteins,and it plays an extremely important role in the maintenance of normal cell morphology,conglutination,signal transduction and other physiological functions.Preliminary laboratory studies have shown that protein 4.1R exerts a negative regulatory effect on CD4⁺T lymphocyte activation and proliferation by inhibiting CD4⁺T cell receptor TCR-mediated signal transduction;4.1R-deficient mouse CD4⁺T cell over-activation and over-proliferation,and increase the production of IL-2 and IFNγ.Some literature studies have found that the cytoskeletal protein plays a key role in the process of viral infection and replication.SPTBN1,a member of spectrin family,plays an importent role in the subsequent steps of virus entry into cells.SPTBN1 can firmly bind to HIV-1 gag p55 and promote HIV-1 infection of macrophages;Actin is the membrane skeleton protein necessary for DENV-Ⅱand DENV-Ⅳto infect host cells.It can interact with the DENV envelope protein to participate in the entry of DENV into host cells.However,the role and mechanism of protein 4.1R in virus infection and replication have not been reported.This study used wild-type and protein 4.1R-deficient mouse embryonic fibroblasts to investigate whether protein 4.1R affects DENV/ZIKV-infected MEF cells.The results fully suggest that protein 4.1R is involved in the entry process of ZIKV-infected host cells.Protein 4.1R may become a new therapeutic target,which is expected to promote the progress of ZIKV treatment.Methods1.Using PCR amplification and Western Blot technology,the expression of protein 4.1R in 4.1R^+/+MEF and 4.1R^-/-MEF cells was identified from the gene and protein levels.2.Construction of eukaryotic expression recombinant plasmids p EGFP-4.1R（135k Da）,p EGFP-4.1R（80 k Da）and p CMV3-E-HA.3.Real-time quantitative PCR was used to detect the effect of protein 4.1R on DENV infection and replication.4.Using DENV-ⅡLuc⁺replicon and detecting the activity of luciferase reporter gene to determine the effect of protein 4.1R on DENV replication.5.Real-time quantitative PCR and Western Blot technology were used to detect the effect of protein 4.1R on ZIKV infection at the RNA level and protein level.6.The plaque method was used to detect the infectivity of progeny virus in ZIKV7.Flow cytometry was used to detect the expression of 4.1R^+/+MEF and 4.1R^-/-MEF cell surface receptors AXL and Tyro3.8.Real-time fluorescence quantitative PCR technology was used to detect the virus binding and entry of ZIKV infected 4.1R^+/+MEF and 4.1R^-/-MEF cells.9.Electron microscopy was used to detect the virus entry and replication of ZIKV10.Co-immunoprecipitation method was used to detect the interaction between protein 4.1R and ZIKV E protein.Results1.PCR amplification results showed that the 4.1R^+/+MEF cell c DNA amplified two bands of about 2500 bp and 1700 bp,while absent in 4.1R^-/-MEF cells;Western blot results showed that 4.1R^+/+MEF cells expressed two subtypes of 4.1R protein with sizes of 135 k Da and 80 k Da,while absent in 4.1R^-/-MEF cells.2.Molecular cloning technology was used to successfully construct eukaryotic expression recombinant plasmids p EGFP-4.1R（80 k Da）,p EGFP-4.1R（135 k Da）and p CMV3-E-HA.3.Using real-time quantitative PCR to detect the effect of protein 4.1R on DENV infection and replication,the results showed that compared with the positive control of Vero cells,the viral load of 4.1R^+/+MEF cells and 4.1R^-/-MEF cells was very low and there was a significant difference（P<0.01）,but there was no significant difference in viral load between 4.1R^+/+MEF cells and 4.1R^-/-MEF cells.4.After DENV-ⅡLuc⁺was infected with Vero,4.1R^+/+MEF and 4.1R^-/-MEF cells respectively and cultured for 48 h,the effect of protein 4.1R on DENV replication was determined by detecting the activity of luciferase reporter gene in the cells.The luciferase test results showed that compared with Vero,the activity level of the luciferase reporter gene in 4.1R^+/+MEF cells and 4.1R^-/-MEF cells was very low and there was an extremely significant difference（P<0.0001）;while there is no significant difference in activity between 4.1R^+/+MEF cells and 4.1R^-/-MEF cells.5.Real-time quantitative PCR and Western Blot technology were used to detect the effect of protein 4.1R on ZIKV infection and replication.The results showed that ZIKV could infect Vero and 4.1R^+/+MEF cells,while the level of viral RNA in the supernatant and cells collected after 48 hours of infection of 4.1R^-/-MEF cells was much lower than that of Vero and 4.1R^+/+MEF cells,the difference was significant and statistically significant;After 48 hours of infection with ZIKV,the positive control Vero cells and 4.1R^+/+MEF cells showed bands about 53 k Da,indicating that 4.1R^+/+MEF cells and Vero cells can express ZIKV E protein after infection;while absent in 4.1R^-/-MEF cells.To sum up the experimental results,the loss of protein 4.1R can affect ZIKV-infected MEF cells.6.The plaque method was used to detect the infectivity of progeny virus in ZIKV infected 4.1R^+/+MEF and 4.1R^-/-MEF cells.The results showed that the supernatant of Vero cells and 4.1R^+/+MEF cells infected could appear plaque after 5 d of culture after re-infection with Vero,but the supernatant after infection with 4.1R^-/-MEF cells could not show phagocytosis.The spots indicate that the progeny virus produced after4.1R^+/+MEF cell infection is infectious,while the progeny virus produced after 4.1R^-/-MEF cell infection is not infectious.7.The expression of receptors AXL and Tyro3 on 4.1R^+/+MEF and 4.1R^-/-MEF cells was detected by flow cytometry.The results showed that the receptors AXL and Tyro3 were expressed on 4.1R^+/+MEF and 4.1R^-/-MEF cells.8.Real-time quantitative PCR method was used to detect the virus adsorption and entry after ZIKV infection of 4.1R^+/+MEF and 4.1R^-/-MEF cells.The results show that the deletion of protein 4.1R does not affect the adsorption and binding of ZIKV to MEF cells,while can affect the entry of ZIKV into MEF cells.9.Electron microscopy detected the virus entry and replication after ZIKV infection of 4.1R^+/+MEF cells and 4.1R^-/-MEF cells.The results showed that at 4 h,24 h and 48 h after ZIKV infection,the positive control Vero and 4.1R^+/+MEF cells could see complete virus particles in the cells.Virus particles can be seen in Vero and4.1R^+/+MEF cells 4 hours after virus infection,indicating that the virus can enter Vero and 4.1R^+/+MEF cells.At 24 h and 48 h of virus infection,the amount of virus in the cells increased,indicating that the virus can replicate and proliferate in Vero and 4.1R^+/+MEF cells.However,after 4.1R^-/-MEF cells were infected with ZIKV,no virus particles were seen in the cells.10.Using anti-4.1R antibody to co-precipitate 4.1R and ZIKV E protein,it can be seen that protein 4.1R and ZIKV E protein are co-immunoprecipitated in the cell.The above results indicate that protein 4.1R may interact with ZIKV E protein to affect ZIKV entry into MEF cells.ConclusionFirst,the expression of protein 4.1R in 4.1R^+/+MEF and 4.1R^-/-MEF cells was identified at the gene level and protein level,and the p EGFP-4.1R vector and p CMV3-E-HA vector were successfully constructed.Three different methods proved that the deletion of protein 4.1R did not affect the dengue virus infection of MEF cells.Next,it was proved at the RNA level and protein level that ZIKV could infect 4.1R^+/+MEF and4.1R^-/-MEF cells,and the absence of protein 4.1R could affect ZIKV-infected MEF cells.A preliminary study of its mechanism of action found that the absence of protein4.1R does not affect the adsorption and binding of ZIKV and MEF cells,but affects ZIKV entry into MEF cells;protein 4.1R may affect ZIKV entry into MEF cells by interacting with ZIKV E protein.