The Effects Of Nuclear Receptor Interaction Protein (NRIP) On Hepatitis B X Protein (HBX)through Proteinprotein Interaction

Hepatitis B virus(HBV)is a hepatotropic DNA virus.HBV infection is closely related to the occurrence and development of liver diseases such as hepatitis and cirrhosis,and may eventually lead to hepatocellular carcinoma(HCC).At present,the pathogenesis of HBV has not been fully elucidated,so further study of the pathogenesis of various diseases caused by HBV infection is profound and significant for the prevention and treatment of HBV infection-related diseases.The HBV genome is 3.2kb,partially double-stranded,circular DNA,including four open reading frames of P,C,S and X,encoding the polymerase,the core antigen(HBcAg),the surface antigen(HBsAg)and X protein(HBx).HBx gene encode a154-amino acid regulatory protein with a molecular mass of 16.5 kDa,which is one of the important factors of HBV disease,and the protein is unstable with a half-life of30-40 min.Nuclear receptor interacting protein(NRIP),which is able to interact with members of nuclear receptor families such as androgen receptors,estrogen receptors,progesterone receptors and glucocorticoid receptors,can enhance a series of transcription activities mediated by nuclear receptor via the ligand-dependent pathway.NRIP it is able to combine with DDB1 of the DDB1-CUL4-ROC1 E3 ligase thro?gh a WD40 repeating sequence and participates in the activities of recruiting the substrate for ubiquitin-proteasome system.A series of proteins capable of interacting with HBx protein,including NRIP,had been screened by yeast two-hybrid system previously.However,the effect and mechanism of NRIP on HBx is still unknown.In this study,we verified that NRIP could interact with HBx in eukaryotic cells and further explored the effect of NRIP on the protein stability and function of HBx.The first part of this paper was designed to validate the interaction between HBx and NRIP.Based on the results of the preliminary screening of yeast two-hybrid,weconstructed HBx and NRIP expression vectors pHBx-Flag and pNRIP-Myc respectively,and both were transfected into hepatoma cells Huh7 cells and we confirmed the interaction between HBx and NRIP in eukaryotic cells by co-immunoprecipitation(Co-IP).The second part of this paper aimed to study the effect of NRIP on the stability of HBx.Firstly,the effect of NRIP on HBx protein expression was examined.HBx and NRIP were co-transfected into Huh7 cells,and the protein level of HBx increased along with the increase of intracellular NRIP transfection.Whereas overexpression of HBx protein in Huh7 cells did not affect the protein level of NRIP.And then we explored the mechanism of NRIP affecting HBx protein levels.RT-PCR result showed that overexpression of NRIP had no significant effect on the mRNA level of HBx.We speculated that the regulation of HBx protein by HBIP was achieved by interfering with the degradation pathway of HBx.After treatment with MG132,NRIP further enhanced the level of HBx protein,implying that the effect of the NRIP protein on HBx protein is mediated by inhibition of its ubiquitin-proteasome pathway degradation.