The Role And Mechanism Of Fludarabine In Natural Antiviral Immune Response

Background and objective:Fludarabine(FDL),a fluorinated vidarabine,is an adenine analogue.It is mainly used to treat low-indolence lymphoma and lymphoproliferative disorders and is a standard first-line treatment drug for chronic lymphocytic leukemia.At present,the anti-tumor mechanism of FDL has been extensively studied.FDL mainly suppresses the proliferation of tumor cells by inhibiting either the synthesis of DNA and RNA,or the translation of proteins,or the activity of related enzymes,thus promoting apoptosis of cells.It is reported that the precursor of FDL,vidarabine,resists DNA viruses.The derivatives of FDL also has a strong inhibitory activity against dengue viruses.However,it has not been reported whether FDL itself participates in the regulation of broad-spectrum natural antiviral immunity.Therefore,this study mainly focuses on exploring the role of FDL in natural antiviral immunity and its potential molecular mechanism.It aims to further understand the mechanism of FDL in broad-spectrum antiviral immunity and provide new experimental basis for the development of small molecular compounds to resist viral infection in clinical practice.Methods:(1)The effects of FDL on the viruses:different cells treated with FDL for 12 h were infected with different viruses.Relative levels of viral RNA or proteins were evaluated by Realtime PCR or Western Blot.(2)The influence of FDL on interferons(IFNs):the production of IFNs and the activity of secreted IFNs was detected by Realtime PCR or the Dual luciferase reporter assay or ELISA.(3)The FDL-mediated regulation of IFNs production:cells treated with FDL for 12 h were analyzed by Western Blot or Immunoprecipitation(IP).(4)The regulation of STAT1 by FDL:the specific regulation of FDL on STAT1 was explored by Western Blot,Realtime PCR and Immunoprecipitation(IP).(5)The interaction between FDL and STAT1:cells transfected with different STAT1 deletion mutants were treated with FDL for 12 h and then anaylzed by Western Blot.Results:(1)FDL obviously inhibits the replication and invasion of various viruses.(2)FDL promotes the production of IFNs and the secreted IFNs induced by FDL have antiviral activity.(3)FDL promotes the activation of RIG-I by increasing K63-linked ubiquitination of RIG-I;FDL does not noticeably affect the levels of several key signaling proteins in the RIG-I-MAVS signaling pathway.(4)FDL increases the ubiquitination of STAT1 and promotes the degradation of STAT1 by inhibiting the interaction between USP13 and STAT1,which may partially limit the antiviral activity of FDL.(5)The amino acid 704-750 domain of STAT1 is required for FDL-mediated downregulation of STAT1 proteins.Conclusion:FDL enhances K63-linked ubiquitination of RIG-I and increases the production of IFNs to promote natural antiviral immunity.However,FDL increases the ubiquitination of STAT1 and promotes the degradation of STAT1 by inhibiting the interaction between USP13 and STAT1,which may partially limit the antiviral activity of FDL.How to inhibit FDL-mediated downregulation of STAT1 proteins is worth further exploration in the future,which will provide scientific evidence for enhancing the antiviral activity of FDL.