The Preliminary Study Of Production And Molecular Mechanisms Of Type â…  Interferon In HaCaT Cells Infected With Hantaan Virus

Hantavirus are members of the genus Hantavirues, family Bunyaviridae. The virusgenome consists of three segments of negative-stranded RNA.The large (L) segmentencodes a viral RNA-dependent RNA polymerase, the medium (M) segment encodes aglycoprotein precursor, and the small (S) segment encodes a nucleocapsid protein.Hemorrhagic fever with renal syndrome (HFRS) caused by Hantaan virus (HTNV), Seoulvirus (SEOV) and Puumala virus (PUUV) is prevalent in Asia and Europe. Hantaviruspulmonary syndrome (HPS) caused by Andes virus (ANDV), Sin Nombre virus (SNV)and New York-1virus (NY-1V) is prevalent in the Americas. Both HFRS and HPS arecharacterized with acute infectious diseases and higher mortality. About90%cases ofHFRS are reported in China with Clinical symptoms of fever, bleeding, hypotension andacute renal injury.The pathogenesis and pathological process of HFRS are complicated. Previous reports suggest that, on the one hand, the virus can directly infect organs and tissuesresulting in structural and functional abnormalities, on the other hand, viral infections canactivate the body’s innate immune response and the adaptive immune response resulting inchanges in the internal environment, so that inflammatory mediators, vasoactive factorsand release of various cytokines lead to internal environment and organ function disorderseventually.Rodents are thought to be the major reservoirs of hantaviruses. The spread of thevirus is diverse, and were generally considered to be related with rodent droppings andsecretions through wounds, aerosols and food. Slightly bitten by poisonous rodents alsocan cause infection, but the cytological basis of bites virus infection and the detailedmolecular events is fully unclear. Keratinocytes (KC) is a major component of cell in theepidermis, and it is also considered as a very important non-professional antigenpresenting cells (APC). It can secrete many cytokines, such as interferon, interleukin(IL-1α，IL-1β，IL-3，IL-7，IL-8，IL-10，IL-l8, etc.), clone stimulating factor (G-CSF andGM-CSF), tumor necrosis factor, growth factors and chemokines to curb foreign pathogeninfection, KC also play important role in immune defense. We speculate that, after bittenby poisonous rodents, infected KC might induce immune response and inflammatoryresponse, which may provide a possibility to control the spread of virus or virus infection.Therefore, we selected immortalized human keratinocytes cell line HaCaT as the researchmodel, and observed whether the HaCaT cells could be infected with HTNV and theinterferon response in this cells could be activated. Further, the possible mechanisms oftypeⅠinterferon response were also explored. The above effort will provide experimentalbasis about KC acting as a potential target cells and its molecular events.Main methods and experimental results are as follows.1. HTNV could infect HaCaT cells and proliferate in the cellsHaCaT cells were infected with HTNV at MOI=1. After6h and24h respectively, thecells were collected and lysed, then HTNV NP was detected by Western blot. The resultsshowed that HTNV NP specific bands observed at6h and24h postinfection were consistent with the expected size of the NP, and the band at24h postinfection was morestrong. While the specific bands did not appear in the control group. Theimmunofluorescence test showed that specific green fluorescent in the cytoplasm could beclearly observed, which means the specific expression of NP, and no specific greenfluorescent was observed in the cytoplasm of the control group. It is suggested thatHTNV could infect HaCaT cells and proliferate in the cells.2. IFNβ could be induced in HTNV infected HaCaT cellsHaCaT cells were infected with HTNV at MOI=1, the cells were collected and lysedrespectively at1d and3d postinfection, and IFNα and IFNβ mRNA were detected byqRT-PCR. The results showed that the levels of IFNβ mRNA in HaCaT cells at1d and3dpostinfection were significantly higher than uninfected control group, and the level of3dwas significantly higher than that at1d. While the levels of IFNα mRNA in HaCaT cells at1d and3d postinfection were very low, and there was not statistically significant incomparison with control group. The results indicated that IFNβ-based typeⅠinterferoncould be activated in HaCaT cells infected with HTNV.3. Activation of IFNβ promoter in HTNV infected HaCaT cellspRL-TK and IF△116recombinant plasmids containing IFNβ promoter mediated byLipofectamine2000were co-transfected of HaCaT cells which were infected by HTNVafter6h, and luciferase was detected after24h. The results showed that luciferase activitywas significantly enhanced in HaCaT cells infected with HTNV. The results suggested thatIFNβ promoter could be significantly activate when the cells infected with HTNV.4. Activation of effector molecules of type Ⅰ interferon response pathway inHTNV-infected HaCaT cellsTo clarify the typeⅠinterferon molecular mechanism in HTNV-infected HaCaT cells,PRR, IRF, STAT and ISGs were detected in HTNV-infected HaCaT cells at different timeby qRT-PCR.The results of PRRs showed that the level of RIG-I was significantly higher than thecontrol group at1d and3d, the level of MDA5was significantly higher than RIG-I and TLR3at3d. TLR3was not statistically significant compared to control group. The resultsindicated that RIG-I may be involved in the process of HTNV recognition and mayactivate antiviral signal transduction. Then RIG-I and MDA5may play important role inintracellular signaling pathway. TLR3may not participate in HTNV recognition inHTNV-infected HaCaT cells.The results of IRF3and IRF7showed that the levels of IRF3and IRF7weresignificantly higher than the control group at3d. And the level of IRF7was higher thanIRF3at3d. IRF3, IRF7and nuclear factor κB (NF-κB)/p65were detected inHTNV-infected HaCaT cells at4h,6h and24h by immunofluorescence. The resultsshowed that IRF3specific green fluorescent in the nucleus could be clearly observed inHaCaT cells at6h postinfection and could not be observed at4h and24h; IRF7andNF-κB/p65were not observed in HTNV infected HaCaT cells at three time points; Anddual-luciferase assay also suggesting that the NF-κB-luc changed little in HTNV infectedHaCaT cells; And dual-luciferase assay also suggested that the NF-κB-luc changed little inHTNV infected HaCaT cells. The results suggested that IRF3other than NF-κB/p65translocated to the nucleus and regulated IFNβ gene.The results of STAT1and IRF9showed that the levels of STAT1and IRF9weresignificantly higher than uninfected control group in the HaCaT cells at3d postinfection.Meanwhile, the level of IRF9was higher than STAT1at3d; Moreover, STAT1wasdetected in HTNV infected HaCaT cells at4h,6h and24h by immunofluorescence. Theresults showed that the green fluorescent in the nucleus could be clearly observed inHaCaT at24h postinfection and could not be observed at4h and6h; And dual-luciferaseassay also suggested that STAT1was activated in HaCaT cells followed by HTNVinfection. The results were further validated that STAT1was translocated into the nucleuswhere it activated transcription of ISG.The results of ISG showed that the levels of MxA, OAS1and IFIT1weresignificantly higher than uninfected control group at1d,3d and5d postinfection. IFIT3was also significantly increased than uninfected control group at3d and5d. The resultsindicated that MxA and OAS1might play a major role in host defense in HTNV infected HaCaT cells in early infection. And IFIT1and IFIT3might play an important role in lateinfection.5. CCL5and CXCL10could be induced in HTNV infected HaCaT cellsHaCaT cells were infected with HTNV at MOI=1and CCL5and CXCL10weredetected by qRT-PCR. The results showed that CCL5and CXCL10were induced inHaCaT cells at1d,3d and5d postinfection, and the level of CXCL10at1d weresignificantly higher than other groups. The results suggested that after binding to itsreceptor, CCL5might be mainly involved in the late course of the viral clearance andinflammatory responses, while CXCL10might participate in antiviral response in theinitial stages of infection.In summary, HTNV could successfully infect human keratinocyte cell line HaCaTcells, and could induce high level of IFNβ and ISG. At the same time, HaCaT cellsinfected with HTNV could produce chemokines, and these chemokines might be involvedin viral clearance and inflammation. This study provides an experimental basis for furtherresearching on the mechanisms of innate immune response of HTNV-infected KC.