The Effect And Mechanism Study Of Na~+/K~+ ATPase?3 Subunit Inhibiting Hepatitis B Virus Replication

Hepatitis B virus(HBV)infection causes more than 250 million people worldwide to suffer from multiple liver diseases.1.4 million people die every year from HBV-related diseases,including cirrhosis,liver failure and hepatocellular carcinoma.Currently,only nucleotide analogs and interferon(IFN)are approved for the treatment of patients with HBV infection.The HBV genome is a 3.2 kb partially double-stranded relaxed circular DNA genome(rc DNA).After infection,the HBV genome is transmitted to the nucleus and converted into covalently closed circular DNA(ccc DNA).ccc DNA in turn forms a minichromosome and is used as a template for transcription of different viral transcripts,including 3.5 kb pre C mRNA and pregenomic RNA(pg RNA),two envelope mRNAs(2.4kb and 2.1kb)and X mRNA(0.7kb).In viral transcripts,pre C mRNA encodes the pre-core protein.pg RNA can be translated into viral HBc and Pol proteins,and can also be used as a template for HBV genome replication.The 2.4kb and 2.1kb envelope mRNAs encode LHBs,MHBs and SHBs proteins.In addition,0.7kb X mRNA can be translated to produce HBx protein.The four promoters S1P,S2P,XP and CP regulate and control the transcription of viral pre-genomic RNA and proteins.After the viral RNA is converted into HBV protein in the host cytoplasm,the viral pg RNA is encapsulated into the core particle.Inside the core particle,pg RNA is further reverse transcribed into viral DNA(ss DNA and ds DNA).Then,the mature virus particles containing HBV DNA are wrapped and released from the host cell.Hepatitis B virus particles and subviral particles are the main virus particles produced during the replication of hepatitis B virus.Hepatitis B e antigen(HBe Ag)is another form of HBc Ag that translocates into the cavity of the endoplasmic reticulum for proteolytic processing,and is secreted as a soluble protein.However,the function of HBe Ag is still unclear,but HBs Ag and HBe Ag are considered to be very important for HBV transmission and clinical diagnosis.ATP1B3(also called CD298),one of the three regulatory?subunits of Na~+/K~+ATPase,was first identified and identified in 1998.Previous studies have shown that ATP1B3 not only participates in the activity of the Na~+/K~+pump,but also regulates the independent Na~+/K~+ATPase activity of T cell activation.Recent studies have shown that the?subunit of Na~+/K~+ATPase is related to certain viral infections.For example,ATP1B1 has been identified as a chaperone of the UL136 protein of human cytomegalovirus(HCMV)and the M2 protein of influenza A and B viruses.In addition,ATP1B3 can also reduce the restriction of BST-2 mediated human immunodeficiency virus 1 production in Hela cells.And recent studies have shown that ATP1B3 interacts with the 3A protein of Enterovirus 71(EV71)and inhibits EV71 replication by enhancing the production of type I interferon.At the same time,BST-2 was identified as an antiviral protein that can induce IFN,which prevents the release of various enveloped viruses,such as HIV,Lassa,Marburg and Ebola.Existing research proves that BST-2 binds newly born HBV virus particles to the plasma membrane.Meantime,yeast two-hybrid screening identified the interaction between ATP1B3 and BST-2.So is ATP1B3 involved in the spread of HBV?And is the relationship between ATP1B3and BST-2 related to the spread of HBV?This study explores the effect of ATP1B3 on HBV replication,and draws the following experimental results and conclusions:1.ATP1B3 can inhibit the secretion of HBV.In this study,HBV infectious cloning plasmid was transfected into Hep G2 cells,and ATP1B3 expression plasmid was transfected at the same time,and the HBs Ag content in the supernatant was detected by ELISA.Compared with the control group,the HBs Ag content of the experimental group was significantly reduced.Overexpression of ATP1B3 in Hep G2.2.15 and Hep AD38 cells can also reduce the production of HBs Ag in the cell culture supernatant.In contrast,the use of small interfering RNA and short hairpin RNA in cells to mediate the silencing of ATP1B3 can increase the content of HBs Ag in the supernatant.At the same time,ATP1B3 did not down-regulate the content of HBV replication intermediates(HBV DNA)and transcripts(HBV RNA)in cells.It shows that ATP1B3 can impair the expression of viral proteins,but not the replication and transcription of the virus.The above results indicate that ATP1B3 can inhibit the secretion of HBV.2.ATP1B3 activates the NF-?B signaling pathway in Hep G2 cells.The effect of ATP1B3 on NF-?B activation in Hep G cells was detected by dual luciferase reporter gene.It was observed that ATP1B3 activated NF-?B in a dose-dependent manner.Nuclear and cytoplasmic separation experiments confirmed that ATP1B3 induces the transport of NF-?B subunit P65 into the nucleus.TAK1 protein can activate NF-?B.When TAK1 was transfected into Hep G2 with sh RNA-mediated ATP1B3 silence,the activation of NF-?B was strongly inhibited.The use of NF-?B inhibitor Bay11 proved that inhibiting NF-?B activity can significantly reduce the expression of HBs Ag.It is speculated that ATP1B3 can inhibit HBV by activating NF-?B.3.ATP1B3 induces IFN-?/ISG and IL-6 to inhibit HBV.ATP1B3 up-regulates the expression of IFN-?and IL-6 in Hep G2 cells.The results of real-time quantitative PCR showed that ATP1B3 can obviously induce OAS2 and BST-2,but not the expression of ISG-15 and ISG-56.These findings indicate that the expression of BST-2 induced by ATP1B3 can contribute to the inhibitory function of HBV antigen.Further research found that compared with HEK293T cells,the expression of BST-2 in Hep G2 cells was significantly up-regulated by IFN-?.Therefore,the above studies show that ATP1B3 can induce BST-2 through NF-?B/IFNs,thereby promoting the inhibition of HBV by hepatocytes.4.ATP1B3 cooperates with BST-2 to promote HBs Ag to restrict HBV inhibition.In order to prove the synergistic effect of ATP1B3 and BST-2 in limiting HBV,we studied the inhibitory effect of ATP1B3 on HBV in BST-2 negative cells HEK293T.The results showed that the inhibitory effect of ATP1B3 on the HBs Ag production of BST-2 positive cells Hep G2 was stronger than that of BST-2 negative HEK293T cells,indicating that BST-2 may be a partner of ATP1B3 restricting HBs Ag in hepatocytes.5.ATP1B3 promotes proteasome-dependent intracellular HBs Ag degradation.Western blotting showed that overexpression of ATP1B3 significantly reduced the intracellular protein levels of LHBs and MHBs,SHBs slightly decreased,and Core protein did not show any decrease.CHX tracking analysis confirmed that the expression of ATP1B3 accelerated the degradation of HBs in cells.The proteasome inhibitor MG132 can significantly inhibit the degradation induced by ATP1B3 in a dose-dependent manner.6.ATP1B3 interacts and co-localizes with LHBs and MHBs in cells.The co-localization of ATP1B3 and HBV protein was studied by immunofluorescence analysis.As a transmembrane protein,ATP1B3 is located on the cell membrane alone.When co-expressed with LHBs and MHBs,ATP1B3 was spotted around the perinuclear region and diffused in the cytoplasm together with LHBs and MHBs,while HBc showed an aggregated cytoplasmic distribution and almost co-localized with ATP1B3.We speculate that ATP1B3 is strongly degraded due to its interaction with HBs to diffuse it from the membrane to the cytoplasm.The deletion of the transmembrane region of ATP1B3 almost causes ATP1B3 to no longer co-localize with LHBs,MHBs and SHBs.We speculate that the transmembrane region may be the functional domain responsible for HBs binding and degradation.Co-immunoprecipitation confirmed the interaction between ATP1B3 and HBs.7.ATP1B3 induces polyubiquitination of LHBs and MHBs.When staining with ubiquitin or targeting protein antibody,when ATP1B3 is present,the degree of polyubiquitination of LHBs and MHBs is stronger than that in the absence of ATP1B3.We also found that a ubiquitin mutant containing only one lysine at position 48(K48)is sufficient for ATP1B3-mediated ubiquitination of HBs.After using the negative ubiquitin mutant,the degradation of LHBs and MHBs mediated by ATP1B3 was completely blocked,which further confirmed that the degradation of HBV envelope protein mediated by ATP1B3 was a proteasome-dependent manner.In summary,this study used molecular biology,virology and other research methods to prove the inhibitory effect of ATP1B3 on HBV,and revealed that ATP1B3passes nuclear factor?B(NF-?B)/IFN-?and NF-?B/interleukin The-6(IL-6)pathway becomes a new host restrictor for HBV replication.In addition,this study proved that ATP1B3 induces its binding protein BST-2 to antagonize HBs Ag in Hep G2 cells.At the same time,ATP1B3 promotes the proteasome-dependent degradation of HBV envelope protein,and promotes accelerated degradation of HBs through ubiquitination of lysine at position 48.We concluded that ATP1B3,as a new type of antiviral factor,can use multiple mechanisms to combat HBV.Our work highlights ATP1B3 as a potential therapeutic target for HBV infection.