Suppression Of Hepatitis B Virus By Cytidine Deaminase APOBEC3DE And APOBEC3B

The chronic hepatitis B virus infection is a serious health problem worldwide. According to WHO statistics that about 400 million people are chronically infected with HBV and are at high risk of developing liver cirrhosis and hepatocellular carcinoma(HCC). An estimated 1000 000 people die of HBV-related cirrhosis and HCC each year. APOBEC3s have been identified as potent innate cellular defenses against both endogenous retroelements and diverse retroviruses. It has been reported that several APOBEC3s proteins can inhibit HBV replication, and can be induced by IFN, IL2 and TNF in recent years. We have been reported that A3B can suppress HBV replication by sequestering heterogenous nuclear ribonucleoprotein K(hnRNP K) and as a result inhibit HBsAg and HBeAg expression as well as HBV core DNA synthesis. Here, we address the inhibition profiles of expression of HBsAg and HBeAg by APOBEC3s proteins, one of which, A3DE shows the potent inhibitory effects on HBsAg and HBeAg expression, and does not depend on the terminal amino-or carboxyl-deamination domains of the enzyme activity. In contrast, both deamination domains of A3DE are required for HIV-1 inhibition. A3DE may also induce HBV DNA deamination without affecting HBV core-associated DNA. A3DE expression is observed in normal liver tissues and may be up-regulated by IFN-?. IFN-?and TNF-a in liver cell line cells. And the inhibitory effects on HBsAg and HBeAg by A3B also do not depend on the enzyme activity.Part I Inhibition of HBV replication by APOBEC3DE in cell lines in vitroWe have previously reported that APOBEC3B has a potent inhibitory effect against the expression of HBsAg and HBeAg. It has not been fully characterized whether other human APOBEC3s family proteins have a similar effect. To examine whether other human APOBEC3s proteins have similar activity, HepG2 cells were co-transfected with an HBV-expressing plasmid plus an expression vector for various human APOBEC3s proteins or a control plasmid pcDNA3.1(1:1). HBsAg and HBeAg were measured by ECLIA in the culture supernatants 48 h after transfection. The levels of HBsAg and HBeAg from the cells transfected with HBV plus A3B or A3DE were markedly lower than those from cells transfected with HBV plus the control vector. On the other hand. A3A and A3C had little effect on the expression of HBsAg or HBeAg. HBsAg levels in A3B-and A3DE-co-transfected samples were 18.48±1.88%and 11.06±0.44%of the control respectively. HBeAg levels in A3B-and A3DE-co-transfected samples were 28.37±1.18%and 22.87±0.9%of the control, respectively. A3A had little inhibitory effect on HBsAg and HBeAg expression (86.38±7.38%.88.03±2.31%. respectively), while A3C had no inhibitory effect on HBsAg and HBeAg expression (110.4±5.3%,91.82±4.94%. respectively).A3F, A3G and A3H had only moderate effects on the expression of HBsAg and HbeAg which were 39.53±5.97%.44.34±3.23%.65.52±11.96%and 40.44±1.0552.74±0.81.57.1±5.72%. respectively.To determine if the two cytidine deaminase domains of A3DE were required for inhibiting the expression of either HBsAg and/or HBeAg. A3DE mutant constructs were developed mutating critical glutamic acids (within the cytidine deaminase domain) at positions 80 and 264. Compared to empty vector control, expression levels of both HBV proteins were significantly reduced in the presence of wildtype A3DE. Expression levels for both proteins were also substantially reduced when co-expressed with mutant A3DE E80K. E264K, and E80/264K. In fact mutations at each glutamic acid yielded results similar to co-expression with wildtype A3DE. To determine the impact of both N-terminal and C-terminal cytidine deamination domains in A3DE during HIV-1 infection, A3DE mutants were transfected into cells infected with HIV-1 clone NL4-3. While wildtype A3DE reduced HIV-1 infectivity levels to below 5%, A3DE E80K and E80/264K only reduced infectivity levels by approximately 50%. Interestingly, A3DE E264K reduced infectivity approximately 80%compared to infectivity in the presence of empty vector control. In addition, levels of A3DE and viral proteins were detected in cell and viral lysates by western blot. Wildtype A3DE, but none of the A3DE cytidine deaminase mutants, is incorporated into the virion. Levels of budding virus and total viral Gagp55 are reduced in the presence of A3DE cytidine deaminase mutants, but not wildtype A3DE.Using a 3D-PCR and co-transfected approach, we wanted to determine if A3DE could induce cytidine deamination within the HBV genome. We first confirmed that core-associate HBV DNA levels are similar when co-expressed with A3DE compared to control vector. Next, sequencing revealed A3DE is capable of inducing G-A hypermutations in HBV X gene DNA.Part II Expression profiles of A3DE and its regulationWe use Kodak 1D image analysis software to analysis the expression of A3DE in PBMNCs and live cell lines cells. A3DE is expressed in all PBMNCs samples and weaker in liver cell lines, and We find that A3DE expression in HepG2.2.15 is about 1.5 fold higher than that in HepG2. which may suggest that A3DE expression could be induced by HBV infection. A3DE expression in human leukemia cell lines is not less than that in PBMNCs.Then, to determine whether A3DE is also expressed in hepatocytes, we analyzed normal liver tissue from 16 donors by reverse transcription of A3DE mRNA and subsequent cDNA analysis by gel electrophoresis. In all samples, expression of A3DE was detected. However, some individual variation in A3DE expression was observed. It has been reported that there may be a possible association between A3B expression levels and hepatocellular carcinoma (HCC). We were curious whether there may be a link between A3DE and HCC. Unlike A3B, fewer than half of the sample pairs comparing healthy to HCC cells showed elevated expression of A3DE. Therefore, A3DE does not appear to be elevated in HCC cells(p=0.1219).Recent studies reported that A3B and A3G are inducible by IFN-a in liver cells, and A3DE can be induced by IFN in macrophage and monocyte cells, but not in primary CD4?T cells. We tested the ability of IFN-?, IFN-?and TNF-?to up-regulate A3DE expression in various liver cell lines. Using an RT-PCR-based approach, we observed that A3DE was up-regulated by IFN-?, IFN-?and TNF-?in all liver cell lines at multiple time points, and that in QGY7703 was the highest. Variations in IFN-?, IFN-y and TNF-a induced A3DE expression were noted. In HepG2 cells, IFN-a induced A3DE expression was less profound that those in QGY7703.Part III The cytidine deamination domains of A3B are not required for inhibition of HBsAg and HBeAg expressionThe approaches in part III are similar to these in part I. We reported that A3B can suppress HBV replication by sequestering heterogenous nuclear ribonucleoprotein K and as a result inhibit HBsAg and HBeAg expression as well as HBV core DNA synthesis, here, we further investigate the mechanism that A3B suppresses HBV. Firstly. we contruct A3B mutants contructs with point mutating critical cysteine (within the cytidine deaminase domain) at positions 100 and 289. Compared to empty vector control, expression levels of both HBV proteins were significantly reduced in the presence of wildtype A3B and its mutants, which are 18.98±1.81%,17%±0.84%; 20.89%±0.65%and 19.39±1.22%; 30%±2.8%; 27.45±0.58%,33.05%±2.54%and 32.39%±4.24%, as well as the level of HBs mRNA. And using an co-immunoprecipitation appraoch, we make sure there is no interaction difference with hnRNP K between A3B and its mutants.According to part I and II, we give conclusions:A3DE is a potent inhibitor on HBV and regulated A3DE expression may contribute to HBV regulation in vivo; Suppression on HBV by A3DE and A3B does not depend on cytidine deamination.