The Mechanism Of Human APOBEC3G Inhibits Hepatitis B Virus Replication

Hepatitis B virus (HBV) is a DNA virus that can cause acute and chronic liver disease. The outcome of HBV infection and the degree of liver disease is the result of complicated interactions between the virus and the immune response of the host, the innate immune system is the first defense against virus by a host.Apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 protein G (APOBEC3G)is a novel component of the innate immune system, which is a potent inhibitor of infection by a wide range of retroviruses. Early reports have shown that APOBEC3G is packaged into human immunodeficiency virus (HIV-1) virions through binding Gag, which deaminate cytidine bases in single-stranded DNA formed during reverse transcription of the RNA template. Recently, it was reported that APOBEC3G can restrict the replication of HBV and reduce the expression of HBs Ag and HBe Ag, but the exact mechanism of APOBEC3G anti-HBV is unknown.In this study, we constructed stable expressing-APOBEC3G cell line, and detected whether NF-κB involved in the process of APOBEC3G-mediated antiviral activity. We used the fluorescence resonance energy transfer (FRET) approach in living cells and surface plasmon resonance (SPR) technology to explore the relationship between APOBEC3G and HBV core protein (HBc). We constructed different mutants of APOBEC3G and investigated the effect of they mediated antiviral activity against HBV in cell cultures. The interaction between the mutants and HBc was also detected.This study includes three parts. PartⅠHuman APOBEC3G inhibits hepatitis B virus replication and NF-κB involves in the process of antiviral activityObjectivesTo clarify that APOBEC3G can suppress the replication of HBV DNA and the expression of HBs Ag and HBe Ag. To construct stable expressing-APOBEC3G cell line, and to detect whether NF-κB involves in the process of APOBEC3G-mediated antiviral activity, to study the mechanism of APOBEC3G anti-HBV.MethodsWe transfected the HepG2 cells with APOBEC3G plasmid (pcDNA-APO3G) and pcDNA3.1(-)-myc-his was transfected as control, named HepG2A and HepG2-3.1 cells. To confirm the expression of APOBEC3G in transfected HepG2 cells, RT-PCR and western blotting were carried out. RT-CES system was used to detect the status of the HepG2A and HepG2-3.1 cells. HepG2A and HepG2-3.1 cells were then transfected with GFP-expressing plasmid (pEGFPN-1) respectively. The transfected efficienfy was analyzed by fluorescent microscope and FACS. The HepG2A and HepG2-3.1 cells were transfected with 1.3 copy wild-type HBV genome vector (p1.37). Viral replicative DNA intermediates were analyzed by real-time and the expression of HBsAg and HBeAg was detected via radioimmunoassay. The HepG2A and HepG2-3.1 cells were transfected with pNF-KB-Luc alone, or together with p1.37, luciferase activities were measured. The HepG2A and HepG2-3.1 cells were transfected with p1.37, cells were then incubated with IMD-0354 ((1μM) or without, and HBV DNA level was detected by real time PCR. The expression of IL-6, IL-8, IL-1βand IL-1αwas quantified by real-time RT-PCR in HepG2A and HepG2-3.1 cells.Results1. A stable APOBEC3G-expressing cell line was successfully established. The 298bp band was brighter in HepG2A cells than that in HepG2 cells. The molecular weight of APOBEC3G was detected about 46KD in HepG2A cells.2. The level of HBV DNA and the expression of HBs Ag and HBe Ag were dramatically reduced in HepG2 cells cotransfected with pcDNA-APO3G and p1.37, but the inhibition efficiency was less than that of stable overexpressing APOBEC3G.3. The status of the HepG2 cells was not affected by overexpressing APOBEC3G which was monitored by real-time cell electronic sensing system (RT-CES system).4. There was no difference in average GFP-positive cell ratio between HepG2-3.1 and HepG2A cells by fluorescent microscope and FACS.5. NF-κB was activated by APOBEC3G and this activation was strongly enhanced by co-expression of HBV.6. The antiviral effect of APOBEC3G was strongly suppressed after treatment with IMD-0354. A reduction of 90% with HBV DNA was observed in HepG2A, while cells cultured with IMD-0354, a reduction of 42% with HBV DNA was observed.7. The levels of IL-6, IL-8, IL-1βwere upregluated by APOBEC3G, but the expression of IL-1αwas not affected.Conclusions1. APOBEC3G can inhibit the replication of HBV DNA and the expression of HBs Ag and HBe Ag.2. NF-κB involves in the process of antiviral activity of APOBEC3G.3. APOBEC3G may be through activating NF-κB to induce inflammatory mediators to inhibit the replication of HBV.Part II APOBEC3G directly binds hepatitis B virus core protein in cell and cell free systemsObjectivesTo explore the relationship between APOBEC3G and the HBV core protein (HBc), and examine whether RNA or other cellular protein affects the APOBEC3G-HBc interaction.MethodsAPOBEC3G and HBc coding sequence were amplified by PCR, the PCR products were inserted into HindⅢ/Kpnl restriction sites of pECFP-N1 or pEYFP-N1, named pA3G-CFP and pHBc-YFP. Plasmids pA3G-CFP and pHBc-YFP were prepared and transiently cotransfected into HepG2 cells. The localization of the APOBEC3G-CFP and HBc-YFP fusion proteins were detected in cotransfected HepG2 cells by confocal fluorescence microscopy. To determine whether APOBEC3G interacted with HBc in living cells, the FRET approach was used. APOBEC3G with a haemagglutinin (HA) tag at the C-terminus (pA3G-HA) was constructed by cloning APOBEC3G into the HindⅢand EcoRI restriction sites of pcDNA3.1 (+). To construct an assembly defective mutant of HBc, Y132A mutation was completed by using overlap extension PCR. The final PCR products of HBc-Y132A-HA was digested with HindⅢ/EcoRI and inserted into pcDNA3.1 (+), yield pHBc-Y132A-HA. pA3G-HA or pHBc-Y132A-HA was transfected into 293T cells. After 48 hours, cells were harvested and HA-fusion proteins were purified by EZviewa Red Anti-HA Affinity Gel. The purified HBc-Y132A-HA protein was immbolized on CM5 sensor chips, surface plasmon resonance (SPR) measurement was carried out in cell-free condition. To investigate whether RNA affects the A3G-HBc interaction, cellular RNA or virus RNA was added to the APOBEC3G protein solution before flow through the SPR chip.Results1. Plasmids pA3G-CFP, pHBc-YFP, pA3G-HA and pHBc-Y132A-HA were successfully construced.2. Localization of the APOBEC3G-CFP and HBc-YFP fusion proteins were detected in cotransfected HepG2 cells by confocal fluorescence microscopy. Very bright and punctuated APOBEC3G-CFP was observed in the cytoplasm while HBc-YFP was not only distributed evenly in the cytoplasm, but also in the nucleus. In a number of APOBEC3G-CFP and HBc-YFP co-expressing cells, extensive co-localization was observed.3. We detected FRET signal between APOBEC3G-CFP and HBc-YFP in FRET tube. We also measured a FRET signal in cytoplasm when APOBEC3G-CFP and HBc-YFP were co-expressed in HepG2 cells.4. APOBEC3G-HA and HBc-Y132A-HA proteins were obtained. And there were significant SPR signals generated between the APOBEC3G-HA and HBc-Y132A-HA proteins.5. The signal obtained from APOBEC3G-HA and HBc-Y132A-HA was not affected when total RNA of HepG2 or HepG2.2.15 cells was added.Conclusions1. APOBEC3G can bind to HBc in the cytoplasm of HepG2 cells.2. APOBEC3G can bind to HBc in cell free systems, this binding was neither enhanced by the HepG2 cell RNA nor the virus RNA.Part III Different domains of APOBEC3G inhibits hepatitis B virus replicationObjectivesTo investigate the effect of APOBEC3G different domains mediated antiviral activity against HBV in cell cultures and find the binding domain of APOBEC3G to HBc.MethodsN-terminal and C-terminal cytosine deaminase domain expression plasmids and both active sites deficient plasmids were constructed, named as pCD1, pCD2 and pDE12. HepG2 cells were cotransfected with pCD1, pCD2, pDE12 and 1.37. Viral replicative DNA intermediates were analyzed by real-time PCR. Plasmids pCD1-CFP, pCD2-CFP, pDE12-CFP was constructed and transiently transfected into HepG2 cells, respectively. The localization of the fluorescent fusion proteins were detected in transfected HepG2 cells by confocal fluorescence microscopy. pCD1-CFP, pCD2-CFP, pDE12-CFP and pHBc-YFP were cotransfected into HepG2 cells, acceptor photobleaching method was used to detect the relationship between them.Results1.Plasmids pCD1, pCD2, pDE 12 and pCD 1-CFP, pCD2-CFP and pDE 12-CFP were successfully construced.2. Localization of CD1-CFP, CD2-CFP and DE12-CFP fusion proteins were detected in transfected HepG2 cells by confocal fluorescence microscopy. Very bright and punctuated CD1-CFP was observed in the cytoplasm while CD2-CFP was not only distributed evenly in the cytoplasm, but also in the nucleus. DE12-CFP was detected in perinuclear.3. The N-terminal or C-terminal cytosine deaminase domain alone could inhibit HBV replication in HepG2 cells. The inhibition efficiency of CD2 was even more than that of APOBEC3G. DE12 has little inhibitory effect to HBV.4. Fret signal was obtained between CD1-CFP, CD2-CFP and HBc-YFP respectively, but the signal between CD1-CFP and HBc-YFP was stronger. There was no FRET signal between DE12-CFP and HBc-YFP.Conclusions1. The N-terminal or C-terminal cytosine deaminase domain alone can inhibit HBV DNA replication.2. The N-terminal or C-terminal cytosine deaminase domain can bind to HBc, but the N-terminal domain is the main structure.Conclusion of full text1.APOBEC3G can inhibit the replication of HBV DNA and the expression of HBs Ag and HBe Ag, and NF-κB may involve in the process of antiviral activity of APOBEC3G.2. APOBEC3G can bind to HBc in cell and cell free systems, and APOBEC3G imay be incorporated into HBV viral particles via direct binding with HBc protein.3. The N-terminal or C-terminal cytosine deaminase domain alone can inhibit HBV DNA replication, the mechanism of APOBEC3G antiretroviral action is not single.