The Antiviral Activity And Significance Of Wild-type MxA And Some Mutants Against The Replication Of Hepatitis B Virus

Background: The dynamin-like GTPase activities, GTP binding and hydrolysis, are critical for MxA to exert its cellular functions. MxA contains conserved GTP-binding domains in the amino (N)-terminal moiety and the specific effector domain for GTP hydrolysis in the carboxy (C)-terminal portion . Several single amino acid substitutions near the MxA C- or N-terminus were shown to disrupt the GTPase activity. The MxA K83A mutant, which is altered in the GTP-binding motif, had disrupted GTP binding ability but exhibited extrinsic GTPase activity . MxA T103A, which tended to form large homo-oligomers when expressed in transfected cells, differs from wild-type MxA by a threonine in place of an alanine at position 103 within another N-terminal GTP-binding domain. This mutant lacks GTP binding and GTP hydrolysis activity . The C-terminal GTPase effector domain (GED, amino acids 564-662), containing two LZ domains(LZl and LZ2) of MxA, was able to hydrolyze intrinsic or extrinsic GTP. MxA L612K mutant, which is introduced by site-directed mutagenesis of a lysine in place of a leucine at position 612 in the LZ1 domain, abolished the enzymatic activity of the intrinsic or extrinsic GTP hydrolysis but exhibitted GTP binding ability, these particular mutants were utilized in previous studies to assess the importance of GTP binding or GTP hydrolysis for cellular function of MxA.Multiple studies have established that MxA can inhibit a broad spectrum of negative and positive strand RNA viruses. MxA can act at different stages of the virus replication, but GTP-binding ability or GTP hydrolysis seems critical for the antiviral activity. Several in vitro experiments showed that GTPase defective MxA mutants lose the ability to block the RNA viruses. Recently, Kremsdorf et al demonstrated that MxA can inhibit the replication of HBV, a DNA virus, in vitro. In the presence of MxA, HBV viral protein secretion was profoundly reduced, and a nearly complete disappearance of HBV DNA replicative intermediates was observed. Further studies showed that MxA can suppress the HBV production in vivo and may block HBV production by inhibiting the HBV post-transcriptional regulatory element (HPRE)-mediated nucleocytoplasmic export of viral pre-genomic mRNA. However, whether GTP binding or hydrolysis is required for MxA to inhibit the replication of HBV remains elusive. To characterize the role of the GTP binding or GTP hydrolysis in the MxA-mediated inhibition of HBV, we observed the antiviral activity of the wild-type or GTPase defective mutant MxA (K83A, T103A, and L612K) or GTPase-related mutant (E645R),. Because MxA inhibits the transport of viral HBV mRNAs from the nucleus, we constructed the nuclear forms of wild-type and GTPase defective MxA T103A mutant to investigate whether they can robustly block HBV production.Methods:1. The research on the antiviral activity of wild-type MxA protein against HBV: All vectors were identified by double enzyme cutting and sequencing. HepG2 cells were cultured by standard techniques. 5×10⁶cells were seeded into 10 cm dishes. pU19-1.24HBV were cotransfected into HepG2 cells with pcDNA3.1-Flag-MxA at ratioes of 1:1 and 2:1 respectively. The control group was cotransfected wth pU19-1.24HBV and control DNA including pcDNA3.1 and salmon DNA . The transfection efficiency is balanced by the cotransfection of pSEAP. Three days post-transfection, viral replication was studied. The expression of Flag-MxA protein was detected by western blot using the anti-Flag monoclonal antibody. Extracellular HBsAg and HBeAg was detected by Abbott assay. The expression of extra- and intracellular HBV DNA was measured by real-time PCR.2. To investigate the expression and redistribution of MxA during inhibition of HBV: In experimental group, 10μg pcDNA3.1-Flag- MxA were co-transfected into HepG2 cells with 5ug pU19-1.24HBV. In the control group, 10μg pcDNA3.1-Flag-MxA were co-transfected into HepG2 cells with 5μg control DNA (pU19 and salmon DNA). Three days post-transfection, the expression of Flag-MxA protein in the two groups was detected by western blot using the anti-Flag monoclonal antibody. The localization of Flag-MxA protein in the two groups was detected by laser confocal microscope using the anti-Flag monoclonal antibody. The nuclei were counterstained by HO33342.3. The construction of pcDNA3.1-Flag-MxA E645R vector by site- mutagenesis: We constructed the recombinant vector of pcDNA3.1 -Flag-MxA E645R based on the pcDNA3.1-Flag-MxA using site-mutagenesis kit. According to the mannual of the kit, we designed the correspondent primer and the reverse primer to do PCR, the site mutation is further identified by double enzyme cutting and sequencing.4. The inhibitory effect of MxA E645R mutant on HBV replication: pU19-1.24 HBV was cotansfected into HepG2 cells with pcDNA3.1-Flag-MxA, pcDNA3.1-Flag -MxA E645R and control DNA respectively. Three days post- transfection, the expression of MxA protein was detected by western blot using the anti-Flag monoclonal antibody. Extracellular HBsAg and HBeAg was detected by Abbott assay. The expression of extra- and intracellular HBV DNA was measured by real-time PCR.5. The inhibitory effect of GTPase defective mutants on HBV replication: HepG2 cells were cultured through standard way. Subsequently pU19-1.24HBV was cotansfected into HepG2 cells with pcDNA3.1-Flag-MxA, pcDNA3.1-Flag-MxA K83A, pcDNA3.1-Flag-MxA T103A, pcDNA3.1-Flag-MxA L612K and control DNA respectively. Three days post-transfection, the expression of MxA protein was detected by western blot using the anti-Flag monoclonal antibody. Extracellular HBsAg and HBeAg was detected by abbott assay. The expression of extra- and intracellular HBV DNA was measured by real-time PCR.6. pcDNA3.1-HA-TMxA and pcDNA3.1-HA-T103, two nuclear forms of pcDNA3.1-HA-MxA(wild-type) and pcDNA3.1-HA-MxA(T103A), were constructed by standard techniques: pcDNA3.1-HA-MxA(wild-type) and pcDNA3.1-HA-MxA (T103A) were provided by prof. Geog and we sequenced it to ensure it right. According to the reported ways, we sythesized the upstream and downstream 66bp DNA nucleotides of the the simian virus 40 large-T antigen nuclear translocation signal (NLS). These two single sythesized DNA fragments (T fragmant) was turned to double-srands by a simple reaction"55℃, 30sec; 72℃, 30sec". pcDNA3.1-HA-MxA (wild-type) and pcDNA3.1-HA-MxA (T103A) were digested by double enzyme cutting, Hind III and EcoR I and subsequently this product was ligated with NLS fragmant by T4 ligase. The ligated products were transformed into supercells and then identified by sequencing.7. The inhibitory effect of the nuclear forms of MxA protein on HBV replication: We extracted the recombinant vectors of pcDNA3.1-HA-TMxA and pcDNA3.1-HA -T103 using HiSpeed Plasmid Midi Kit; subsequently pU19-1.24HBV were co-transfected into HepG2 cells with control DNA, MxA and T103A (103), TMxA and T103 respectively. Three days post-transfection, the expression of MxA protein was detected by western blot using the anti-HA monoclonal antibody; extracellular HBsAg and HBeAg was detected by abbott assay; the expression of extra- and intracellular HBV DNA was measured by real-time PCR.Results:1. The inhibitory effect of MxA protein on HBV replication: the MxA protein can be detected possitive by western bot when the transfection ratio is 1:1 or 2:1, but negative in the control. When the co-transfection ratio was 1:1, compared with the control group, the supernatant HBeAg in MxA group was decreased by 27%(P < 0.01), while the supernatant HBsAg showed no significant decrease (P > 0.05); the extra- and intracellular HBV DNA was decreased by 1.05 log₁₀ and 0.68 logic respectively. When the co-transfection ratio was 2:1, HBeAg was decreased by 65% in comparison with the control group (P < 0.01), and HBsAg was decreased by 21% (p < 0.05); The extra- and intracellular HBV DNA was decreased by 1.91 log₁₀ and 1.69 log₁₀, respectively(P < 0.01).2. The expression and redistritution of MxA protein during HBV replication: we tested whether the MxA protein was depleted in the presence of wild-type HBV to investigate the potential strategies of HBV to evade the inhibitory effects of MxA. The MxA protein expression showed no marked difference between the experimental and control groups. Immunofluorescent microscopy was used to localize the Flag-tagged MxA proteins in transfected HepG2 cells. In contrast to MxA proteins in the control group which were detected predominantly in the cytoplasm, MxA proteins during HBV replication redistributed partly from the cytoplasm into the nucleus. Approximately 70% of the HepG2 cells co-transfected with pcDNA3.1-Flag-MXA and pU19-1.24HBV contained MxA both in the nucleus and the cytoplasm.3. MxA E645R mutant could downregulate HBV replication: The level of Flag-tagged MxA was analyzed by western blot using anti-Flag antibody. Compared with the control group, HBeAg in the MxA and E645R groups was decreased by 65% and 59% respectively and HBsAg was 21% and 24% respectively; extracellular HBV DNA was decreased by 1.91 log₁₀ and 2.22 log₁₀, respectively; intracellular HBV DNA decreased by 1.69 log₁₀ and 1.70 log₁₀, respectively. Supernatant HBeAg and HBsAg, extra- and intracellular HBV DNA in E645R mutant MxA groups displayed no marked difference in comparison with the wild-type MxA group (P > 0.05).4. Effect of the GTPase defective mutants on HBV DNA and antigens: The level of Flag-tagged MxA was analyzed by western blot using an anti-Flag antibody. We measured the secretion of HBsAg, HBeAg and viral HBV DNA levels three days post-transfection. The mean value for the levels of viral HBeAg and HBsAg in the control was set at 1.0 and used as the reference. Compared with the control, HBeAg in the wild-type MxA, K83A, T103A, and L612K mutant MxA groups was decreased by 73%, 71%, 73% and 70%, respectively (P < 0.01); HBsAg was decreased by 41%, 41%, 44% and 41%, respectively (P < 0.01); the extracellular HBV DNA was decreased by 2.22, 2.11, 2.23 and 2.18 log₁₀, respectively (P < 0.01), and the intracellular HBV DNA was decreased by 1.89, 1.78, 1.72 and 1.93 log₁₀, respectively (P < 0.01). Supernatant HBeAg and HBsAg, extra- and intracellular HBV DNA in the GTPase defective MxA mutants groups (K83A, T103A, and L612K) displayed no marked difference in comparison with the wild-type MxA group(WT)(P>0.05).5. Effect of nuclear MxA on HBV replication: Our data shows, in the two groups cotransfected pU19-1.24HBV with the recombinant vector of pcDNA3.1-TMxA or pcDNA3.1-MxA-T103, the increased nuclear accumulation of MxA protein was observed by confocal microscopy when compared with the another two groups cotransfected pU19-1.24HBV with the cytoplasmic MxA expression vectors. The level of HA-tagged MxA analyzed by western blot using an anti-HA antibody. Compared with the control group, supernatant HBeAg in the HepG2 cells transfected with MxA, TMxA, 103, and T103 was decreased by 28%, 9%, 28% and 10% respectively; the extracellular HBV DNA was decreased by 88%, 44%, 88% and 39%, respectively, and the intracellular HBV DNA was decreased by 81%, 35%, 76% and 42%, respectively. In the two groups transfected with pcDNA-TMxA and pcDNA-T103, the supernatant HBeAg levels, the intra- and extracellular HBV DNA levels were statistically lower in comparison with the control group (P < 0.01) but statistically higher than in the cytoplasmic form of wild-type and T103A mutant groups (P < 0.01). These results show that the nuclear form of MxA proteins do not robustly inhibit the replication of HBV.Conclusion:1. MxA protein can inhibit HBV replication in HepG2 cells.2. MxA was not depleted but partly redistributed from cytoplasm into nucleus during HBV replication.3. GTPase defective mutants(K83A, T103A, and L612K)could block the secretion of the extracellullar HBsAg and HBeAg and reduce the expression of extra- and intracellular HBV DNA in HepG2 cells at levels similar to that by wild-type MxA. GTPase activity, including GTP binding and GTP hydrolysis, is not essential for interferon-inducible MxA to block HBV replication.4. TMxA and T103, two nuclear forms of wild-type MxA and GTPase defective mutant (T103A), just could slightly decrease the expression of extra- and intracellular HBV DNA in HepG2 cells. MxA may just have a minimal action on the replicative cycle of HBV in the nucleus. The inhibition of HBV replication by MxA is possibly only slightly dependent on blocking the HPRE-mediated transport of pregenomic mRNA from the nucleus.