The Role Of AKT Signaling In HBV Infection, Viral Replication And Antiviral Therapy

ObjectiveMore than 20 billion people worldwide have been infected with hepatitis B virus (HBV).Most of these individuals experience short, transient infections, yet more than 350 million adults with weakened immune systems develop chronic hepatitis, and thus chronic HBV infection is still a major global health burden. Although HBV replication is only mildly cytopathic, cellular immune responses directed against the virus can produce substantial liver damage and result in chronic hepatitis, cirrhosis, and hepatocellular carcinoma, which together account for 1 million death worldwide annually. The virions contain a 3.2 kb relaxed circular (RC) DNA with four overlapping ORFs, namely P, C, S and X. Following interaction with (an) unknown receptor(s), the entry of relaxed circular DNA (rcDNA) in the nucleus, the completion of plus strand DNA synthesis [HBV DNA(+)], the removal of the polymerase primer for minus strand DNA synthesis [HBV DNA(-)] and of the RNA primer for HBV DNA(+) synthesis, the ligation of both DNA strand extremities, and its incorporation in the nucleosome to form a non-integrated mini-chromosome. Sub-genomic and pre-genomic RNA (pgRNA) molecules are transcribed from this cccDNA template, serving as the template for reverse transcription and the all four mRNAs for the viral proteins (core, polymerase, surface, X). Now, seven drugs have been licensed by the United States FDA (Food and Drug Administration) for the treatment of CHB:interferon-alpha and pegylated interferon-alpha, three nucleoside analogs (lamivudine, entecavir and telbivudine) and two nucleotide analog prodrugs (adefovir dipivoxil and tenofovir disoproxil fumarate). However, Interferon-alpha and pegylated interferon-alpha are easy to cause side effects, and nucleoside analogues are used mostly for long term treatment and easily lead to resistance development.Revealing the relationship between AKT signaling pathway and replication of HBV is incentive to further testify the antiviral effect of inhibitors of AKT on Hepatitis B Virus (HBV).MethodsThe protein expression of AKT-s473 was tested by western blot (WB) in the LO2, HepG2, HepG2.215, Bel and SMMC-7721 cell lines. HepG2 cell lines, Huh7 cell lines and athymic nude mice were transfected with HBV plasmid, the stably expressing cell line were respectively selected. The protein expression of AKT-S473 in normal cell, stably expressing cell line and the liver tissue of chronic hepatitis B patients were tested by WB. The protein expression of AKT-S473 in liver tissue of the infected the nude mice was tested by immunohistochemistry (IHC).The HepG2.215 cell lines were respectively transfected with pEGFP-CA-AKT and pEGFP-NA-AKT plasmid, the stably expressing cell line were carefully selected. The protein expression of AKT-S473 in the stably expressing cell line were tested WB, the amount of HBV DNA were tested by droplet digital polymerase chain reaction (ddPCR).The HBV DNA and covalently closed circle DNA (cccDNA) in the HepG2.215 cell lines and the liver tissue of the infected nude mice, which were previously treated with the inhibitors of AKT and/or Tenofovir for days, were tested by ddPCR. At the same time, the protein expression of AKT-S473 in the cell lines and the liver tissue of infected the nude mice was tested by WB and IHC.ResultsThe protein expression of AKT-S473 in HepG2.215 cell lines was higher than other cells lines as well as the HepG2 cell lines, Huh7 cell lines and athymic nude mice, which were transfected with HBV plasmid, were higher expressed of AKT-S473.The amount of HBV DNA in the stably overexpression cell lines of AKT was lower than the controlled HepG2.215 cell lines. On the contrary, the amount of HBV DNA in the stably overexpression cell lines of AKT was higher than the controlled HepG2.215 cell lines.The amount of HBV DNA in HepG2.215 cell lines which were treated with ADZ5363 (1 μM), AKTi1/2 (0.5 μM) and Rapamycin (1 μM) for 3 days was respectively reduced by 52%,44% and 39%. What is more, the former inhibitors which combined with Tenofovir (0.15 μM) could respectively reduce the amount of HBV DNA in HepG2.215 cell lines by 88%,89% and 62% in 3 days. In addition, the inhibitors of AKT more efficiently reduced the HBV pregenomic RNA (pgRNA) and cccDNA than Tenofovir alone. The amount of HBV DNA in the infected nude mice which were treated with ADZ5363/Tenofovir, AKTil/2/Tenofovir and Rapamycin/Tenofovir for 3 weeks was respectively reduced by 74%,72% and 61%. The amount of HBV s antigens in serum was fallen after 1 month.ConclusionAKT was activated by the infection of HBV further to accelerate the replication of HBV. The antiviral effect of inhibitors of AKT is enhanced by combination with nucleoside analogues. The inhibitors of AKT with uniquely effect on reducing the HBV cccDNA suppose a novel radical therapy to chronic hepatitis B patients.