| APOBEC3G (apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G) has an activity of cytidine deaminase, and can induce nucleoside mutations from deoxycytidine (C) to deoxyuridine (U) in the viral genome, which results in termination of the entire viral replicative cycle. Therefore, APOBEC3G has a strong anti-retroviral activity. APOBEC3G has been identified able to restrict replication of human immunodeficiency virus type 1 (HIV-1) lacking the viral accessory protein Vif. Recently, it has been revealed that APOBEC3G also can inhibit hepatitis B virus (HBV) replication in cultured human hepatocytes. However, the exact mechanism of APOBEC3G inhibiting HBV replication, and the other biological functions and mechanism of APOBEC3G in human cells are largely unknown. In addition, the expression level of APOBEC3G is normally low in the human liver. Thus, whether there will be differences of the APOBEC3G expression in peripheral blood and liver cells among different types of clinical outcomes in the patients with chronic HBV infection, and whether APOBEC3G played a role in anti-HBV in the bodies of the patients with chronic HBV infection are also unclear. On the basis of above-mentioned research background, this study firstly aims to construct a eukaryotic expression vector containing APOBEC3G, and to study the effects of APOBEC3G on replication and expression of HBV in vitro, and observe the effects of APOBEC3G on biological behaviors of HepG2.2.15 cells after highly-expressed APOBEC3G, including cell growth, migration ability, cell cycle and apoptosis; and then, aims to explore the expression level and intracellular localization of APOBEC3G in peripheral blood mononuclear cell (PBMC) and liver tissue in different chronic HBV infected patients; finally, aims to investigate whether JAK-STAT (Janus kinase-signal transducers and activators of transcription) pathway participating in the regulation of APOBEC3G gene transcription and to explore the molecular mechanisms of interferon resistance in the patients with chronic hepatitis B (CHB).(1) Effects of APOBEC3G gene on biological behaviors of HepG2.2.15 cells: The APOBEC3G gene was extracted from PBMC by RT-PCR. A recombinant Eukaryotic expression vector pcDNA3.1-APOBEC3G was constructed by molecular cloning and transfected into HepG2.2.15 cells by lipofectamine. The levels of HBsAg and HBeAg in the media of transfected HepG2.2.15 cells were detected by ELISA. The levels of HBV DNA and HBV mRNA were determined by real-time PCR. Limiting dilution monoclonal screening was used to establish HepG2.2.15 cells in which APOBEC3G protein was stably expressed. Western-blot was used to identify and screen HepG2.2.15 cells in which APOBEC3G protein was highly expressed. The changes of cell cycle and apoptosis were detected by using flow cytometry (FCM). The proliferation of cells was analyzed by MTT assay. The migration and movement of cells was determined with rowing trace. The results showed that APOBEC3G eukaryotic expression vector was constructed successfully by identification of restrictive endonuclease. The levels of HBsAg, HBeAg, HBV DNA and HBV mRNA in HepG2.2.15 cells transfected APOBEC3G recombinant plasmid were significantly lower than that of HepG2.2.15 cells which were not transfected APOBEC3G. Compared among HepG2.2.15-APOBEC3G cells transfected with pcDNA3.1-APOBEC3G HepG2.2.15-pcDNA3.1 cells transfected with blank vector and HepG2.2.15 cells (control), there were no significant changes in cell cycle (the proportions of each phase of cells were, for G0/G1:(64.27±1.26)% vs (64.18±1.24)% vs (64.09±1.30)%, P>0.05; for S:(23.16±1.38)% vs (22.67±1.41)% vs (23.27±1.43)%, P>0.05; for G2/M:(11.36±1.26)% vs (11.84±1.31)% vs (11.37±1.22)%, P>0.05, respectively), in apoptosis, in cell growth, and in the ability of cell migration (the inhibition rates of cell migration at 24h and 48h were (7.5±3.7)% vs (10.2±5.5)%, P>0.05; (13.4±6.5)% vs (17.8±9.2)%, P>0.05, respectively). These experimental results concluded that APOBEC3G could obviously inhibit replication and expression of HBV in vitro. Highly-expressed APOBEC3G has no obvious effects on biological behaviors of HepG2.2.15 cells. The successful construction of APOBEC3G eukaryotic expression vector lays the foundation for further researching the mechanism of anti-HBV effects by APOBEC3G.(2) Expression and localization of APOBEC3G in PBMC and liver tissue in different chronic HBV infected patients:By using the western-blot and confocal laser scanning microscope (CLSM), the expression levels and intracellular localization of APOBEC3G in PBMC and liver tissue were detected in different types of patients with chronic HBV infection, including chronic hepatitis B, chronic severe hepatitis, liver cirrhosis, and liver cancer. The results showed that the expression level of APOBEC3G in PBMC of healthy people was very low. The relative expression levels of APOBEC3G in PBMC of patients with chronic hepatitis B, chronic severe hepatitis, liver cirrhosis, or liver cancer were 4.12±0.21,4.07±0.28,4.16±0.36 or 4.21±0.39 respectively, all of which had stronger APOBEC3G expression levels compared with healthy people. However, there was no statistical difference on APOBEC3G expression in PBMC among these different chronic HBV infected patients (Q=0.931, 0.744,1.675,1.675,2.606 or 0.931, respectively, all of P values>0.05). In addition, there was also no statistical difference on APOBEC3G in liver tissue between chronic hepatitis B patients and hepatocellular carcinoma patients (4.40±0.34 vs 4.34±0.43, Q=0.588, P>0.05). CLSM observations indicated that the localization of APOBEC3G protein was in cytoplasms of PBMC and liver cells and was not in the nuclei. These experimental results suggested that chronic HBV infection could increase the expression of APOBEC3G, but high expression of APOBEC3G may be only the satellite phenomenon of chronic HBV infection. Whether APOBEC3G is involved in the innative immune response to chronic HBV infection is still uncertain.(3) Interferon-alpha induces high expressions of APOBEC3G and STAT-1 in vitro and in vivo:Using real-time RT-PCR and Western-blot, the changes of APOBEC3G and STAT-1 expression levels in HepG2.2.15 cells, which were treated with IFN-αin different concentration or at various time periods, were detected. In addition, the differences of STAT-1 and APOBEC3G expression in the liver tissues were also observed in the patients with different anti-viral responses to IFN-a. The results showed that IFN-a suppressed HBV replication and expression markedly in HepG2.2.15 cells, and simultaneously enhanced APOBEC3G expression as a dose- or time- dependent manner within a certain range. Moreover, a corresponding gradual increase of STAT-1 expression levels was also observed. The expression levels of STAT-1 and APOBEC3G in the liver of the CHB patients with complete response to IFN-a are significantly higher than that of the patients with non-response to IFN-a treatment. These experimental results suggested that inducing intracellular APOBEC3G expression may be one of anti-HBV mechanisms of IFN-a, and IFN-α-induced APOBEC3G expression may be via JAK-STAT signaling pathway. Moreover, interferon resistance may be related with the down-regulation of STAT-1 expression in the patients who had non-response to IFN-a treatment. |
PDF Full Text Request