Inhibition Of Hepatitis B Virus By Cytidine Deaminases APOBEC3 And Its' Mechanism

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors. Chronic infection with hepatitis B virus(HBV)is the major high risk factor for HCC in Africa and Asia.People chronically infected with HBV will be at high risk for the development of chronic hepatitis,cirrhosis and HCC.There is great individual difference in spontaneous HBV clearance in vivo among people infected with HBV. While a fraction of those infected with HBV become chronic carders after infection, most are able to induce a strong antiviral response that results in HBV clearance.HBV clearance is mediated by non-cytolytic mechanisms via interferons and other cytokines.Interferon-mediated HBV clearance in vivo is characterized by an initial decline in HBV DNA intermediates,followed by a reduction in HBV gene expression and eventual disappearance of HBV from the liver.The mechanism of interferon-induced clearance of HBV DNA is,however,unclear. The cytidine deaminases apolipoprotein B mRNA editing catalytic polypeptide 3 (APOBEC3),which are unique to mammals,have been identified as potent innate cellular defenses against both endogenous retroelements and diverse retroviruses.But their functions can be suppressed by viral encoded proteins,such as virion-infectivity factor(Vif)for HIV-1.APOBEC3 family proteins consist of at least 7 members (APOBEC3A-3H,A3A-A3H),and most of them can induce C to U mutations in the minus-strand viral DNA during reverse transcription.These mutations could trigger the action of uracil-DNA glycosylases,generating abasic sites.This abasic site-containing DNA may inhibit plus-strand viral DNA synthesis or be degraded by cellular endonucleases.The minus-strand uracil-containing viral DNA that survives the action of uracil-DNA glycosylase can potentially serve as a template for synthesizing the sense-strand DNA,yielding G-to-A hypermutated viral DNA. Antiviral activities of APOBEC3 proteins could also result from G-to-A hypermutations in retroviral genes that generate premature stop codons or heavily mutated nonfunctional viral proteins.It is known that APOBEC3 prefer single-stranded DNA as targets.HBV is an partially double-stranded DNA virus that replicates by reverse transcription of a pregenomic RNA intermediate.Like retroviral DNA,the DNA of HBV exists in single-stranded form during reverse transcription.So whether APOBEC3 could suppress HBV replication just as they do in retrovirus draws researcher's attentions.Recently,exogenous expression of A3G and A3F was reported to reduce HBV core-associated DNA in transfected liver cell lines.Furthermore,a few studies showed that a number of APOBEC3 members had inhibitory effect on HBV replication and could be induced by intefferons,suggesting that they may be involved in the non-cytolytic clearance of HBV.The mechanism of inhibitory effect of APOBEC3 proteins on HBV infection is still unknown.To explore the mechanism of HBV suppression by APOBEC3,we investigated the inhibitory effects of APOBEC3 proteins on HBV by co-transfecting APOBEC3-expressing plasmids plus a HBV-expressing plasmid into cells at first,then identified the proteins binding to APOBEC3 proteins.Further investigation was carried out to study whether the interaction between APOBEC3 and their binding proteins might have impacts on the replication and expression of HBV genes.In addition,the expression profiles of APOBEC3 in a variety of cell lines and tissues and the ability of interferon to induce the expression of APOBEC3 in hepatocytes were examined,and the subcellular localizations of APOBEC3 proteins were observed.Three parts of this paper are as follows:First PartInhibition of HBV replication by APOBEC3 in cell lines in vitroTo profile the antiviral activities of APOBEC3 proteins,HepG2 cells were co-transfected with a HBV-producing plasmid plus A3B or A3G expression vectors or a control plasmid pcDNA3.1.HBsAg and HBeAg were measured by ECLIA in the culture supernatants at 48 h after transfection.The levels of HBsAg and HBeAg from the cells transfected with HBV plus A3B plasmid were markedly lower than those from cells transfected with HBV plus the control vector.HBsAg and HBeAg levels in A3B- co-transfected samples were 32.16±15.43%and 43.07±13.88%of the control, respectively.In contrast,A3G had little effect on their expression.HBsAg and HBeAg levels in A3G-co-transfected samples were 89.54±5.08%and 88.51±5.33%of the control,respectively.To gain insight into the mechanism responsible for A3B-mediated reduction of HBV gene expression,we next analysed HBs RNA expression in transfected HepG2 cells.HBs RNA was evaluated with semi-quantitative reverse transcription polymerase chain reaction(RT-PCR)using total cellular RNA at 48 h after transfection.Consistent with the above results,HBs RNA was reduced in HepG2 cells co-transfected with A3B plasmid when compared with the control pcDNA3.1, indicating that A3B inhibited HBs mRNA expression.To test whether A3B mediated HBs expression at the transcription level,we cloned HBV S gene promoter region into a luciferase reporter vector,pGL2-basic vector,and determined the effect of A3B on HBV S gene promoter activity in 293T cells.We observed that A3B suppressed HBV S gene promoter activity as compared with the pcDNA3.1 vector.Again,A3G had little effect on the promoter activity of HBV S gene.In addition,to address whether A3B could influence other viral promoter activity except for HBV promoter activity,we co-transfected 293T cells with a CMV promoter-driven GFF expression vector and either the A3B expression vector or a control vector.Interestingly,A3B was found to inhibit CMV promoter mediated GFF expression,when compared with the control vector.The inhibitory effect on CMV promoter activity was rather unique for A3B compared with other APOBEC3 proteins. Furthermore,A3B also inhibited SV40 promoter-mediated neomycin phosphotransferaseⅡexpression.In contrast,A3G had little effect on the SV40 promoter activity.A3B expression had no obvious effect on two nonviral promoters including human ribosomal protein L32 promoter(Hu-GFP)and elongation factor 1 promoter(EF-GFP),suggesting that inhibition of A3B on viral promoter is specific and unique among APOBEC3 proteins.A3B could have a broad antiviral activity.To investigate the effect of A3B on core-associated HBV DNA,HBV cores were isolated from cytoplasms of transfected HepG2 cells at 48 h after transfection,and cytoplasmic core associated HBV DNA was examined.As previously reported,four APOBEC3 members(A3B,A3G,A3F and A3C)showed the ability to suppress intracellular core associated HBV DNA synthesis,but their potency varied in hepatocytes.A3B and A3G potently inhibited HBV core DNA synthesis.Compared to the empty vector control,1ug A3B or A3G expression plasmids plus 1ug HBV-producing plasmid reduced the formation of intracellular core-associated HBV DNA to 9.14±4.42%and 21.27±4.46%respectively,and 4ug A3B or A3G expression plasmids plus lug HBV-producing plasmid reduced the formation of intracellular core-associated HBV DNA to 1.50±0.71%and 6.58±3.19%respectively.A3C and A3F showed moderate suppression for HBV core DNA while A3A lacked anti-HBV activity.These results indicated that A3B has potent anti-HBV activity affecting different steps of HBV replication that was different from that of other AFOBEC3 deamineases.A3B has dual inhibitory effects on both the accumulation of core-associated HBV DNA and the expression of HBsAg at protein and RNA levels, whereas A3G has a lesser effect on the expression of HBsAg,although it is a potent inhibitor of the accumulation of core-associated HBV DNA.The mechanism responsible for HBV suppression by A3B may be different from that by A3G. To observe whether APOBEC3 deaminases could be packed into the newly produced HBV particles or not,just as they do in HIV-1 virus particles,HepG2.2.15 cells were transfected with HA-tagged A3B or A3G or A3A expressing plasmids. Virus particles were pelletted from the culture medium and purified using HBsAb-beads.A3B and A3G proteins were detected in the purified virus particles by Western Blot with anti-HA antibody,whereas A3A was not detected.It suggested that A3B and A3G but not A3A could be packed into the newly produced HBV particles.Second PartA3B protein interacts with hnRNP K and inhibits the interaction between hnRNP K and HBV enhancerⅡIn first part,the A3B protein had been identified as a potent innate anti-HBV factor,which might have different antiviral mechanism from A3G.But its binding partners were still unknown.In this part,We therefore made use of coimmunoprecipitation analysis and mass spectrometry(MS)to identify potential binding partners of A3B,and further study was carded out to observe the impacts of these interactions between A3B and its binding partners on the clearance of HBV. HA-tagged A3B was co-immunoprecipitated with anti-HA antibody conjugated to agarose beads from lysates of A3B-HA-transfected 293T cells but not control 293T cells.And this was also confirmed by immunoblotting using the anti-HA antibody. Several protein bands were co-precipitated with A3B-HA,one of which was approximately 60-70 kDa and identified as hnRNP K by MS.Immunoblotting with an hnRNP K-specific antibody confirmed the interaction between A3B and hnRNP K. Furthermore,several other members of the hnRNP family,including hnRNP I,hnRNP C1/C2,hnRNP H/F and hnRNP A/B,were also found to interact with A3B. Interestingly,the interaction between A3B and hnRNP K was effectively unique to A3B among APOBEC3 family proteins.A3A,A3C,A3F and A3G did not interact or interact poorly with hnRNP K compared with A3B.To further confirm the interaction between A3B and hnRNP K,we performed a reciprocal experiment using co-immunoprecipitation analysis with anti-hnRNP K antibody and Western blot analysis with anti-HA antibody for the detection of A3B-HA.The result showed that A3B-HA could also be immunoprecipitated with hnRNP K.hnRNP K is a well known protein,which interacts with a diversity of molecules involved in gene expression and signal transduction,including RNA,DNA,factors involved in chromatin remodeling,transcription,RNA splicing and translation.It is known that the enhancerⅡ(enhⅡ)of HBV plays an important role in regulating the transcription of HBV genes,and hnRNP K can positively modulate the replication efficiency of HBV via binding to the enhⅡof HBV.Therefore,we next investigated the possible cross-talk between A3B and hnRNP K in EnhⅡ-mediated HBV expression.293T cells which express high level of endogenous nuclear hnRNP K protein were transfected with A3B-HA-expressing plasmid or control vector.The total nuclear proteins from these cells were immunoprecipitated with enhⅡdouble-stranded DNA(EnhⅡdsDNA)probes and then detected by Western blot analysis.The hnRNP K protein was detected in total nuclear proteins and EnhⅡdsDNA precipitate from nuclear proteins of cells transfected pcDNA3.1,but not from that of cells transfected with A3B expression vector.These findings suggest that A3B may inhibit the binding of hnRNP K to the erdaⅡof HBV.To confirm this hypothesis, we tested the effect of A3B-HA protein on the binding of recombinant GST-hnRNP K with EnhⅡdsDNA in vitro.Purified GST-hnRNP K protein was first bound to EnhⅡdsDNA probe,and then incubated with 293T cells nuclear protein extract containing A3B-HA protein.The specific EnhⅡdsDNA-binding complex was eluted for immunoblotting analysis.The hnRNP K protein level in EnhⅡdsDNA-hnRNP K complex after treatment of A3B-HA protein obviously decreased.These results strongly suggest that the inhibitory effect of A3B on HBV expression occurs at transcription level through at least in part,reducing the binding of hnRNP K to the enhⅡof HBV.Since hnRNP K is a RNA-binding protein,we next determined whether the interaction between A3B and hnRNP K proteins was mediated by RNA.293 T cell extracts transfected with A3B expression vector were used as a source of RNA and A3B in the binding experiments.Purified GST-hnRNP K proteins were first bound to EnhⅡdsDNA probes,then incubated with RNase A-treated- or untreated- 293 T cell nuclear protein extracts.Thereafter the specific protein complex was eluted for Western blot analysis.The results showed that RNase treatment did not change in the strength of binding hnRNP K to A3B,indicating that the interaction between A3B and hnRNP K is RNA-independent.Third PartExpression profile and subcellular localization of A3B and their regulationTo determine the expression profile and relative expression levels of endogenous A3B,RNA samples from a variety of cell lines and tissues were analysed by semi-quantitative RT-PCR.A3B was detected at low level of expression in the normal liver tissue from all three healthy liver donors,and also in non-tumor liver tissue from two of three patients with metastatic liver tumors,and considerable individual variation in A3B expression was observed.A3B was low expressed in normal human liver cell line QSG-7701. whereas A3B expression were detected in all of the seven investigated hepatoma cell lines,three(HepG2,SMMC7721 and HepG2.2.15)of which expressed high levels of A3B.Furthermore,A3B expression levels were high in various tumor cell lines,such as K562,KU812,U937,AGS,SW480 and SW620.It seemed that the expression levels of A3B in tumor cell lines are higher than in normal one.Individual variation in A3B expression was also observed in periphery blood monocytes(PBMC)from 8 healthy adults.Two subjects had low levels of A3B expression,and three subjects had relatively high levels of A3B expression,whereas two cases had no A3B expression.After PBMC were treated with PHA for 24 hours, A3B expression in PBMC from two subjects originally with low levels of A3B could be upregulated.But one subject still could not be induced to express A3B even after they were treated with PHA.29 pairs of HCC samples and their matched surrounding non-tumor(NT)samples were examined for the expression of APOBEC3.Low levels of A3G or A3F were sporadically detected in some HCC or NT samples.An increased expression of A3G or A3F in HCC samples compared with in NT samples was observed in only a small fraction of the samples.In contrast,A3B transcripts were significantly elevated in 24 of 29 HCC samples(P＜0.01).We furthermore examined HCC samples and matched NT of 10 subjects with active HBV infection(HBsAg and HBcAb positive)among these 29 cases for the expression of A3B and HBV genes.A3B transcripts were significantly elevated in all of these HCC samples compared to their matched NT samples(P＜0.01).In sharply contrast,the expression of HBsAg detected by Western Blot was all reduced in HCC compared to their matched NT samples,and the transcripts of HBV X gene were also the same case.Reduced HBV expression in HCC samples matched well with increased A3B expresssion in these samples,and further confirmed the anti-HBV activity of A3B.Meanwhile,relatively high levels of A3B expression in HCC and some tumor cell lines whereas low levels in normal liver tissue and primary bepatocytes suggested that abnormal activiation and over-expression of A3B could be related to the carcinogenesis of HCC.A recent study has shown that multiple APOBEC proteins,including A3A,A3B, A3F and A3G,can be induced by IFN-αin human macrophages and hepatocytes. Therefore,we examined the ability of interferons to induce the expression of A3B in liver cells.We saw the low levels of A3B expression in primary hepatocytes from two healthy donors,and their expression could be upregulated at different efficiency by interferon-α.Interferon-αincreased significantly the expression of A3B in HepG2, Huh7,QSG-7701,QGY-7703,and BEL-7404 cells at 10-1000 IU/ml for 24 to 48 hours.Interferon-βalso induced A3B expression in HepG2 cells at 1 and 2μg/ml for 12 to 24 hours,albeit at a lower efficiency than did interferon-α.However,induction of A3B expression by interferon-γin our assay system was inefficient.These above results raise the possibility that individual differences in A3B protein expression and IFN inducibility in primary liver cells might contribute to the individual differences in spontaneous HBV clearance in vivo.Immunofluorescence microscopy was used to localize the HA-raged APOBEC3 proteins in transfected hepatoma cells HepG2 and HepG2.2.15.In contrast to A3G and A3F that were detected primarily in the cytoplasm of both cell lines,A3B was found predominantly in the nucleus of HepG2 cells.It is in accordance with the inhibitory effect of A3B on HBV at transcription level.But we found that A3B was localized primarily in the cytoplasm of HepG2.2.15 cells,which continuously replicated the HBV genome.It needs further investigation on the reason of the redistribution of A3B from nucleus to cytoplasm when co-exist with HBV. Conclusions1,A3B is a potent innate anti-HBV factor.The time-or dose-dependent induction by interferon-αand interferon-βcan be observed in the expression of A3B in hepatocytes.The upregulated expression of A3B has dual inhibitory effects on both the accumulation of core-associated HBV DNA and the expression of HBsAg at protein and RNA levels.2,HnRNP K is one of the main proteins interacting with A3B in vivo.The inhibitory effect of A3B on HBV genes expression occurs at transcription level through at least in part,reducing the binding of hnRNP K to the enhⅡdsDNA of HBV.3,The inhibitory effects of A3B on HBV promoter-and CMV promoter-as well as SV40 promoter-mediated gene expressions suggest A3B protein could have a broad antiviral activity when expressed and regulated in appropriate cell types.4,Relatively high levels of A3B expression in HCC and some tumor cell lines whereas low levels in normal liver tissues suggested that abnormal activiation and over expression of A3B could be related to the carcinogenesis of HCC.Reduced HBV expression in HCC samples matched well with increased A3B expresssion in these samples,and further confirmed the anti-HBV activity of A3B.5,Considerable individual variation in A3B expression among primary hepatocytes, normal liver tissues and PBMC,and the different response of primary hepatocytes to interferon treatment could be related to various clinic consequence.