Construction And Characterization Of A Full-length Human Bocavirus 1 (HBo V1)clone And The Regulation Of NF-κB Signal Pathway By Its NS1 And NS1-70 Proteins

This thesis contains two research projects during my master-doctor study.The first one focus on the construction of full length clone of HBo V1wh(p IHBo V1wh).And the second part focus on the mechanism of HBo V1 NS1 and NS1-70 proteins inhibit NF-κB signal pathway.In the first research project,we construted a full-length HBo V1 clone(p IHBo V1wh-GFP)inserted with a heterogenous EGFP gene successfully.In this study,we have constructed the infectious clone of HBo V1wh(p IHBo V1wh)using the method described previously.And demonstrated its ability to transcription,replication,encapsidation of the ss DNA genome and generate the HBo V1 virions from transfected HEK293 T cells.We also developed a novel HBo V1 clone(p IHBo V1wh-GFP)inserted with a heterogenous EGFP gene after the ATG of the genome and connected with NS1 by a linker.The p IHBo Vwh-GFP clone is capable of transcription,translation,replication and producing progeny virions(HBo V1wh-GFP)from transfected HEK293 T cells.What is more important,the packaged ss DNA genome of HBo V1wh-GFP is intact and not truncated.Since EGFP protein is high sensitive and convenience to detect,HBo V1wh-GFP is a useful tool to seek new cells that can be infected by HBo V1.Considering that HBo V1 can be used as a viral vector for lung gene therapy,the ability to encapsidate lager genome will further augment its usefulness.In summary,we have established two useful clones,p IHBo V1 wh and p IHBo Vwh-GFP.And they will facilitate the basic virology,pathogenesis,immune response and gene therapy research of HBo V1.In the second project,we investigated that HBo V1 NS1 and NS1-70 proteins could inhibit the activation of NF-κB signal pathway.Human bocavirus 1(HBo V1),a parvovirus,is a single-stranded DNA etiologic agent causing acute respiratory tract infections in young children worldwide.Nuclear factor kappa B(NF-κB)transcription factors play crucial roles in clearance of invading viruses through activation of many physiological processes.Previous investigation showed that HBo V1 infection could significantly upregulate the level of TNF-α which is a strong NF-κB stimulator.Here we investigated whether HBo V1 proteins modulate TNF-α–mediated activation of the NF-κB signaling pathway.We showed that HBo V NS1 and NS1-70 proteins blocked NF-κB activation in response to TNF-α.Overexpression of TNF receptor-associated factor 2(TRAF2)-,IκB kinase alpha(IKKα)-,IκB kinase beta(IKKβ)-,constitutively active mutant of IKKβ(IKKβ SS/EE)-,or p65-induced NF-κB activation was inhibited by NS1 and NS1-70.Furthermore,NS1 and NS1-70 didn’t interfere with TNF-α-mediated IκBα phosphorylation and degradation,nor p65 nuclear translocation.Coimmunoprecipitation assays confirmed the interaction of both NS1 and NS1-70 with p65.Of note,NS1 but not NS1-70 inhibited TNF-α-mediated p65 phosphorylation at ser536.Our findings together indicate that HBo V1 NS1 and NS1-70 inhibit NF-κB activation.This is the first time that HBo V1 has been shown to inhibit NF-κB activation,revealing a potential immune-evasion mechanism that is likely important for HBo V1 pathogenesis.