NIRF Can Significantly Inhibit the Expression of Hepatitis B Virus Antigens in vitro and in vivo

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 1032 NIRF * NIRF Np95 / ICBP-90 like RING finger protein NIRF HBc hepatitis B virus core protein HBV paav-hbv1 3 BALB / C NIRF ELISA HBsAg HBeAg Western HBcAg NIRF HBV NIRF R329 NIRF Can Significantly Inhibit the Expression of Hepatitis B Virus Antigens in vitro and in vivo HU Bin 1 ZHOU Dan-Lin 1 XUAN Yan-Yan 1 1 DUAN Chang-Zhu 2 * 1 Department of Cell Biology and Medical Genetics 2 Key Laboratory of Clinical Laboratory Diagnostics of Ministry of Education Chongqing Medical University Chongqing China Abstract NIRF Np95 / ICBP-90 like RING finger protein is a protein that has been suggested to function in carcinogenesis and epigenetic modifications from recent research Reports have shown that NIRF interacted with the hepatitis B virus core protein HBc however the effects of NIRF on the secretion of hepatitis B virus HBV antigens remained unclear Here we established both the cell culture and animal models of HBV infection by the transfection of paav-hbv1 3 plasmid into HepG2 cells or venous hydrodynamical injection of the plasmid in BALB / C mice to explore the connection between NIRF and HBV antigen expression in vitro and in vivo The secretion of HBsAg and HBeAg in the cultural supernatants of the transfected cells or the sera of the injected mice were analyzed by ELISA The HBcAg secretion was assayed by Western blot or immunohistochemical analysis The results demonstrated that the expression and secretion of HBV antigens were decreased following NIRF overexpression both in vitro and in vivo Our findings suggested that the inhibition of NIRF on HBV antigens might provide a better understanding of HBV infection for developing new therapeutic strategies against hepatitis B virus Key words hepatitis B virus NIRF Np95 / ICBP-90 like RING finger protein antigens No CSCT No 2008BB5400 No KJ * Tel duanchzhu@ cqmu edu cn Received August Accepted September Supported by National Natural Science Foundation of China No Chongqing Science and Technology Commission Foundation CSTC No 2008BB5400 Chongqing Education Commission Foundation No KJ * Corresponding author Tel duanchzhu@ cqmu edu cn

2 11 NIRF min hepatitis B virus HBV 2 μg 250 μl DMEM 8 μl 1 2 Lipofectamine 2000 Invitrogen min h 24 h 45% 4 5 HBV 4 PBS 3 Np95 / ICBP-90 like RING finger protein NIRF ICBP90 Np p min 15 min NIRF _ 1 3 Western N Tudor PHD SRA / YDG RING-finger 5 1 Rb 7-9 NIRF min cyclin D1 E1 8 % 12 % 10 Cdk2-CyclinE2 PCNP PEST-containing nuclear protein 2 h FLAG 11 p53 HBcAg Millipore β-actin p NIRF TBST 8 min 3 IgG 1 h TBST 8 min 3 HBc A B 1 1 NIRF PVDF Quantity One NIRF HBc HBc HBV 6 ~ 8 BALB / C 18 g ± 2 g μg 0 2 ml PBS 8 s 24 h HBV d 2 4 % ELISA HBsAg HBeAg 1 1 paav-hbv1 3 HBsAg HBeAg BamH Ⅰ EcoRⅠ Promega 50 μl FLAG NIRF 50 μl min % 80 μl 30 min Ep r / SDS-PAGE 5 % ELISA A B 50 μl min 50 μl 450 nm HepG2 10 % A DMEM HyClone h 2 3 % H 2 O 2 10 min ml 250 μl DMEM 4 μg PBS 5 min 3

3 min 24 h ELISA HBsAg HBsAg HBcAg HBeAg 3 d 4 PBS A pflag-nirf paav- HBV1 3 A Fig min PBS 5 min 3 DAB 70 % 80 % 95 % 100 % 3 min 3 min 1 7 x 珋 ± SD SPSS12 0 t P < 0 05 P < paav-hbv1 3 Fig 1 BamH Ⅰ kb 4 EcoR Ⅰ kb 2A HepG2 NIRF Fig 1 Identify of recombinant plasmid paav- HBV1 3 M Marker DL10000 TaKaRa Japan 1 paav-hbv1 3 digested by BamH Ⅰ 2 paav-hbv1 3 digested by EcoR Ⅰ Fig 2 Inhibitory effect of NIRF on HBV antigens in vitro A HepG2 cells were co-transfected with paav- HBV1 3 pflag-nirf or pflag and the pflag expression plasmid was used as a control The cultural supernatants were collected every day Levels of HBsAg and HBeAg of the cells were determined by ELISA All data represent x 珋 ± SD n = 3 The statistical analysis was performed by t-test * P < 0 05 B HepG2 cells were 2 2 NIRF HBV transfected or co-transfected with the specific plasmid as the NIRF HBV graph showed The cells were harvested at day 3 Western paav-hbv1 3 pflag-nirf pflag blotting was performed with FLAG or HBcAg antibodies HepG2 while β-actin was used as the loading control

4 11 NIRF 1029 HBsAg HBeAg 3 d HBV 6 ~ 8 Western HBcAg BALB / C paav- HBcAg HBV1 3 4 d ELISA Fig 2B NIRF HBsAg HBeAg Fig 3A HBsAg HBeAg HBcAg d HBsAg HBeAg ELISA 2 3 HBV 30 d NIRF HBV Fig 3B Fig 3 Establishment of HBV transfected immunosuppressed mouse A The plasmid paav-hbv1 3 was injected hydrodynamically into the tails vein of BALB / C mouse in 8 s The mouse sera was collected everyday The level of HBsAg and HBeAg were measured using ELISA and were positive at the 4 th day after hydrodynamically injection All data represent mean ± SD n = 3 The statistical analysis was performed by t-test ** P < 0 01 B Pathology detection for mouse livers tissue days of control group 2 30 days after transfected with HBV plasmid 2 4 NIRF HBV Fig 4B HBcAg HBV Fig 4C pflag-nirf NIRF HBV HBsAg HBcAg 7 d 4 d ELISA HBsAg HBcAg HBsAg HBeAg pflag-nirf NIRF HBcAg HBsAg HBeAg Fig 4A HBsAg NIRF 5 d HBsAg HBeAg ELISA HBV NIRF 3 HBsAg HBeAg HBV NIRF 4 d paav-hbv1 3 NIRF HBc HBsAg HBcAg HBsAg 15 NIRF

5 Fig 4 Inhibitory effect of NIRF on HBV antigens in vivo A The plasmid paav-hbv1 3 and pflag-nirf were co-injected hydrodynamically into the tails vein of BALB / C mouse in 8 s The sera were collected at indicated time and levels of HBsAg and HBeAg were measured using ELISA The pflag expressing plasmid was used as a control All data represent 珋 x ± SD n = 3 The statistical analysis was performed by t-test * P < 0 05 B Immunohistochemistry for HBsAg of mouse livers after HBV and NIRF plasmid injected DAB staining days of control group 2 HBsAg expression on 4 days after plasmid paav-hbv1 3 injection 3 7 days of control group 4 Significantly reduce of HBsAg on 7 days after NIRF had co-injected with paav-hbv1 3 plasmid C Immunohistochemistry for HBcAg of mouse livers after HBV and NIRF plasmid injected DAB staining days of control group 2 HBcAg expression on 4 days after plasmid paav-hbv1 3 injection 3 7 days of control group 4 Significantly reduce of HBcAg on 7 days after NIRF had co-injected with paav-hbv1 3 plasmid

6 11 NIRF 1031 HBV HBV NIRF HBV NIRF HBV HBV NIRF HBV NIRF References HBV HBsAg HBeAg HBcAg NIRF 1 Ganem D Prince A M HBV HBV Huang C57BL /6 HBV HBV paav-hbv1 3 HBV 3 Dandri M 1 3 HBV ayw HBV d HBsAg HBeAg HBsAg HBeAg CD4 + T CD8 + T 22 NIRF HBV 4 d HBsAg HBeAg HBcAg HBV CORF p21 HBc HBcAg INSIGHT Ⅱ HBc HBc HBc NIRF E Mori T HBc HBc 1 E3 ligase NIRF HBc 24 HBc 25 HETC Nedd4 γ2-adaptin HBc 96 Nedd4 γ2-adaptin J Shu-Ping Mori T and p53 J Prog Biochem Biophys 2006 HBsAg HBeAg HBcAg 13 NIRF HBV NIRF Yang Y et al Hepatitis B virus infection natural history and clinical consequences J N Engl J Med Lee B O Tucker A Frelin L et al Interaction of the hepatitis B core antigen and the innate immune system J J Immunol Burda M R T r k E et al Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus J Hepatology NIRF J Chang L Qian G H Duan C Z Construction of eukaryotic expression vector for human NIRF gene and bioinformatics of deduced expressed protein J Chin J Biol Tan T L Chen W N A proteomics analysis of cellular proteins associated with HBV genotype-specific HBX potential in identification of early diagnositic markers for HCC J J Clin Virol Mori T Li Y Hata H et al NIRF a novel RING finger protein is involved in cell-cycle regulation J Biochem Biophys Res Commun Li Y Mori T Hata H et al NIRF induces G1 arrest and associates with Cdk2 J Biochem Biophys Res Commun Zhang J Gao Q Li P et al S phase-dependent interaction with DNMT1 dictates the role of UHRF1 but not UHRF2 in DNA methylation maintenance J Cell Res Pichler G Wolf P Schmidt C S et al Cooperative DNA and histone binding UHRF2 links the two major repressive epigenetic pathways J J Cell Biochem Ikeda D D Fukushima T et al NIRF constitutes a nodal point in cell cycle network and is a candidate tumor suppressor J Cell Cycle Mori T Li Y Hata H et al NIRF is a ubiquitin ligase that is capable of ubiquitinating PCNP a PEST-containing nuclear protein J FEBS Lett Mori T NIRF p53 Duan Chang-Zhu Pu et al Studies on the interactions between NIRF HBV J Jin F M Qian G H Preliminary study on the interaction between human NIRF protein and HBV core protein J Life Sci Res

7 Qian G Jin F Chang L et al NIRF a novel ubiquitin ligase interacts with hepatitis B virus core protein and promotes its degradation J Biotechnol Lett NIRF p53 NIRF 22 Fabrizi F Dixit V Martin P et al p53 J immunological response to hepatitis B virus vaccine in dialysis Wang X H Bai L Duan C Z Identification of the NIRF- patients meta-analysis of clinical studies J J Viral Hepat interacting domain with the p53 in NIRF-mediated p53 ubiquitination J Chin J Biochem Mol Biol Dandri M Volz T K Lügehetamann M et al Animal models for the study of HBV replication and its variants J J Clin Virol Suppl 1 S Huang L R Wu H L Chen P J et al An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection J Proc Natl Acad Sci U S A core protein J J Virol HBV 26 Chen C Matesic L E The Nedd4-like family of E3 ubiquitin J Guo Y J Wang W Sun S H et al Immunosuppressant dexamethasone can significantly extend the expression of hepatitis B virus antigens in the HBV mouse model by hydrodynamic transfection method J Chin J Virol Kabra R Knight K K Zhou R et al Nedd4-2 induces J Ying R S Fan X endocytosis and degradation of proteolytically cleaved epithelial G Li N et al Development of hepatitis B virus infection animal model by hydrodynamics J World Chin J Digestol Liu F Song Y Liu D Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA J Gene Ther Yang P L Althage A Chung J et al Hydrodynamic injection of viral DNA A mouse model of acute hepatitis B virus infection J Proc Natl Acad Sci U S A Hepatitis C virus and the 23 Bai L Wang X Jin F et al UHRF2 another E3 ubiquitin ligase for p53 J Biochem Biophys Res Commun HBc NIRF J Yang Y Jin F M Bai L et al Identification of protein-protein interaction between HBc and NIRF in vitro J J Biol Shirakura M Murakami K Ichimura T et al E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus ligases and cancer J Cancer Metsstasis Rev Lambert C D ring T Prange R Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III Vps4 and γ2- adaptin J J Virol Na + channels J J Biol Chem Rost M Mann S Lambert C et al γ2-adaptin a novel ubiquitininteracting adaptor and Nedd4 ubiquitin ligase control hepatitis B virus maturation J J Biol Chem