| Objective:Hepatitis B Virus(HBV)is the leading cause of liver disease worldwide and is a high risk factor for cirrhosis and hepatocellular carcinoma(HCC),which has become a social and public health problem that seriously harms hundreds of millions of people worldwide.The causes of chronic HBV infection are complex and not yet fully known,with some patients suffering from long-term HBV infection or even progression to cirrhosis or HCC,and occasionally even acute deterioration.Although studies have shown that the widely used HBsAg vaccine is effective in protecting the body from HBV infection,tens of millions of people worldwide are still infected with HBV every year.After HBV infects the host,firstly it forms a covalent,closed,ring DNA(covalently closed circular DNA,cccDNA)in the nucleus of the liver cell,and then continuously transcripts and produces viral RNA,which includes a replicant RNA intermediate,pre-genome RNA(pgRNA),which encodes two viral proteins,HBV core protein(HBc)and HBV polymerase(Pol).At present,it is recognized that effective anti-HBV drugs can be divided into two categories,including:the first category is interferon,this kind of drug can achieve serological removal of the virus,but the main defect is only part of HBV has antiviral effect,the overall response rate is less than 40%.Long-term use is prone to side effects and drug resistance.The second category is nucleoside analogobiates,such as Ramifedin(LAM)and Entekavir,they can quickly and efficiently inhibit HBV DNA replication,and anti-infection effect is better than interferon,which have become the current first choice to treat hepatitis B.The disadvantage is that once stopped taking drugs,the virus is easy to rebound,but also prone to drug resistance,genotype mutation and susceptible to loss.Studies have shown that resistance to nucleotide analogs may be associated with mutations in the HBV P gene.And cccDNA can remain resting in liver cells and is difficult to remove,which is the most important obstacle to chronic hepatitis B that is still difficult to cure.Finding effective strategies for the complete removal of cccDNA has long been a focus of researchers around the world.At the same time,finding a new strategy to solve the problem of resistance to nucleotide analogs is also the focus and difficulty in the field of research in Hepatitis B.Studies have found that when the host infects HBV,it would lead to chronic active inflammation,which causes inflammatory factors to activate lipoprotein B mRNA-editing enzyme catalytic polypeptide 3(APOBEC3s)cytosine desinease,a class of enzymes that are expressed only in small amounts in normal liver tissue but significantly increased after HBV infection.It has been found that its editing function can inhibit the replication of HBV.Other studies have found that inflammatory tissues can promote the mutation of the genomes of HBV and host cells by inducing the expression of APOBEC3s cytosine deaminase,which in turn leads to an increase in the frequency of HBV gene mutation or loss,especially the X gene(HBx)mutation of HBV genotype C.This is a landmark molecular event for the occurrence of hepatitis B resistance and disease progression.In our previous studies,we also found that abnormal expression of APOBEC3s cytosine deaminase was associated with poor prognosis of HCC.Interferon as a commonly used hepatitis B treatment drug,it is known that it can be widely cited as the mutation of various genes into the virus,and nucleoside drugs also have the risk of causing genetic mutation resistance,so the development of non-nucleoside drugs need to be actively included in the agenda,which is also a necessary solution to fight chronic hepatitis B in the future.This study sheds light on the effects of IFN-? and cytokine TGF-?1 on APOBEC3s cytosine deaminase causing HBV genomic variation,and explains the possible causes of drug resistance caused by interferon-nucleotide analogues.And for the first time,it was found that,as a new anti-HBV replication compound,it has a strong inhibitory effect on the replication of wild HBV strains and LAM resistant HBV mutant strains.Also ATA does not affect the transcription of HBV or the formation of RNA-DNA hybrid molecules in the viral enclosure.The results of this study provide a new strategy and theoretical basis for the exploration and development of new anti-HBV drugs,as well as the possibility of curing Hepatitis B.Methods:1.Research ObjectIn this study,a variety of liver cells and liver cancer cells are cultured and transfected,includes:human liver cancer cells(HepG2),human liver ancestral cells(HepaRG),human liver cancer cell strain HepG2 derivative cells(HepG2.2.15).2.Cell CultureHepaRG And HepG2 cells were cultured by William's E media containing 10%inactivation FBS,2mML-glutamine,200U/ml penicillin,200 mg/ml streptomycin,and supplemented by 5?g/ml insulin,20 ng/ml skin growth factor,50?m hydrogenated pine and 2%DMSO to induce differentiation of cells.Place culture in a cell incubation at 37?,5%CO2.Passed down from generation to generation every 2 days.HepG2.2.15 cells were cultured using DMEM media containing 10%inactivation FBS,10mM HEPES,200U/ml penicillin,and 200 mg/ml streptomycin.Place culture in a cell incubation at 37?,5%CO2.Passed down from generation to generation every 2 days.3.Cell transfectionBefore transfection,the cells were inoculated in a 6-well plate at a density of 0.8×106/mL(cell fusion is about 60%-80%),and the HBV virus plasmids were transfectioned with prime transfection reagents(Polyplus-transfection),with 200?L jetPRIME buffer and 4?L jetPRIME,respectively,vortex mixing,short-term centrifugation,room temperature static incubation 10min,200?L of configured transfective reagents slowly drip into the cell culture,gently shake the plate so that the reagents are evenly distributed,continue to culture in the incubator,4 to 6 hours after 50%fresh cell culture,24 or 48 hours after,test the transfective effect and collect culture for use.4.Virus purification and infectionFirstly,according to the above transfection method to prepare virus particles,with 0.45?m filter to obtain culture.Add 6%polyglycolic 8000(PEG8k)precipitated virus particles and re-suspend with PBS containing 25%FBS.The cells were infected according to MOI=1.0,1200×g centrifugal force centrifugation for 2 hours to obtain purified viral precipitation.1×107 HBV genome equivalent copies corresponding to 1 X 105 liver cells were vaccinated proportion of the cell infection,3 hours later,screening with penicillin,adjust the final concentration to 1?g/mL.5.Genome DNA(gDNA)extraction and differential DNA denaturation PCR(3D-PCR)According to the QIAamp mini kit instruction manual,complete gDNA extraction,get gDNA samples,and then follow the 3D-PCR amplification kit instructions,set amplification conditions,complete two rounds of PCR amplification.6.Natural agarose gel electrophoresis(NAGE)The resulting viral particles will be purified by polyglycolic precipitation method and analyzed directly with agarose gel electrophoresis.Firstly,the buffer is made and then glued,the appropriate amount of sample is mixed with the 6x sample buffer,the control voltage is maintained at 110V,the current is more than 40mA electrophoresis about 40min;and the ultraviolet is photographed and observed.The gel in turn with the following reagent treatment for alkali degeneration,gently shake at room temperature to ensure that the solution covers the gel:0.25M NaOH 15min;Pour 20x SSC solution into the transfer groove,a solid phase support in the slot,in the solid phase support from the bottom up in turn:filter paper,gel,membrane,filter paper,absorbent paper,400-800g heavy.Transfer for 4-18 hours.(The membrane can be pre-hybridized and interbreeded immediately,or stored at 4? for use.)Subsequent expressions of viral RC-DNA and HBc can be detected with HBV DNA probes and anti-HBc Antibody,respectively.7.Enzyme-linked immunosorbent test(ELISA)Firstly,prepare 25mL lx washbuffer,balance the 96-well plate to room temperature,then dilute the cell culture sample with 5000x dilution of the culture fluid,and multiply the standard product by the kit instruction manual(the concentration of the standards are 100pg/mL,50pg/mL in turn),25pg/mL,12.5pg/mL,6.3pg/mL,3.1pg/mL),sample,incubate,then add 100?L Stop Solution to each hole,read the detection values and standard curves with an enzyme marker within 20 minutes,and calculate the sample concentration based on dilution multiply.8.Real-time fluorescence quantitative reverse transcription PCR(RT-qPCR)The PCR reaction fluid is first preparation,and then the two-step PCR amplification procedure is completed in accordance with the kit.Next,use PrimeScriptTM RT Reagent Kit with gDNA Eraser kit to remove the DNA contained in the RNA and reverse the RNA to cDNA,and then use the TB Green Premix Ex Taq kit for real-time fluorescence quantification of PCR.With GAPDH as the internal ginseng,the expression of the gene to be tested relative to the GAPDH gene was calculated as the result for subsequent analysis.9.Protein extraction and Western Blot analysisUltrasound shatters with RIPA Lysis and Extraction Buffer re-suspended cells.4?,12000rpm/min centrifugal 15min,transfer to the fresh tube,add 4xSDS PAGE loading buffer and mixed.Bring the sample to a boil for 5min at 99? to denature the protein,then place it in a refrigerator at-20?.According to the SDS-PAGE gel preparation kit instructions for the preparation of gels,constant electrophoresis 1 hour;After one wash,5%BSA room temperature was closed for 1 hour,PBST was washed after 1 hour of room temperature incubation,PBST was washed after 30 minutes of second anti-room temperature incubation,and ECL luminescence imaging was analyzed with GAPDH protein as internal ginseng.10.Statistical analysisThe data were statistically analyzed and graphed using SPSS 19.0 and GraphPad 8.0.The measurement data was analyzed using the Students' T test,and it is statistically different by P<0.05.Results:1.Study on HBV gene mutation induced by APOBECsFirstly,HepG2 cells were transfected with protons expressing pHBV1.5 and cultured them for 3 days,treating cells with IFN-? or TGF-?1.The total RNA and virus particles of the viral genome were then recovered to detect the expression of viral genomic DNA and HBV capsid protein,and it was found that both were significantly inhibited,indicating that IFN-? and TGF-?1 were both specifically inhibiting HBV replication.And the virus genome DNA amplification and sequencing,it was found that IFN-? and TGF-?1 can induce a large number of G-to-A and C-to-T mutations,the type of mutation conforms to the physiological characteristics of the APOBECs family.Therefore,we further expressed the APOBECs family to detect the replication and mutation of HBV.By recovering quantitative viral transcripts of total cell RNA,no APOBECs were found to affect virus transcription.Then by collecting viral particles and extracting viral genome DNA,the mutation levels were detected in 3D-PCR,and it was found that all but A3B in APOBECs were able to trigger hyper-mutations in the HBV genome,while at similar protein expression levels,A3G and A3F triggered more mutations.In addition,the main function of uracil-N-glycosylase(UNG)is to remove dUs from single-stranded DNA molecules.Since IFN-? and TGF-?1 can induce liver cells to upwardly express APOBECs,we speculate that IFN-? and TGF-?1 induce hyper-mutation of HBV to be partially repaired by UNG,it may be that the virus is using the host to repair some of its own mutations to maintain life,thus avoiding death due to too many mutations.After sequencing viral DNA,it was found that the number of G-to-A and C-to-T gene mutations increased significantly after the addition of UFI that specifically inhibit the expression of UNG in the liver compared to IFN-? and TGF-?1,which revealed that the virus gene super-mutations caused by IFN-? and TGF-?1 could be partially repaired by UNG.2.Study on the mechanism of combined using IFN and LAM to induce HBV drug resistanceFirstly,cells were induced to differentiate,then cleavaged.And recover the total RNA and HBV virus particles.The results showed that all RNA fragments were significantly lowered by IFN-?,and the expression of HBV's DNA and viral caspid were significantly reduced,resulting in IFN-? inhibiting the transcription and replication of HBV.Next,we further detect mutations in viral genomic DNA,which can be seen that IFN-? significantly induces HBV genome DNA super-mutation,the type is also G-to-A and C-to-C,and sequencing results can be seen in the mutation of HBV's X gene and P gene.Studies have suggested that mutations of P gene may be the main cause of HBV resistance.In the same way,we also studied the effects of LAM on HBV replication and found that LAM significantly inhibited the synthesis of HBV DNA without causing any mutations in viral DNA.Because LAM drug-resistance mutation strain(YIDD)can be introduced through the G-to-A point mutation at P gene.Previous studies have also shown that A3G can induce super-mutation in P gene.Next,we used A3G over-expression protons and pHBV1.5 to transfer HepG2 cells,treating different groups of cells with or without LAM(30?M)and isolating the recovered viral particles.The study found that both A3G and LAM inhibited the synthesis of HBV DNA,and the inhibition of both was more obvious than that of single use.Through further HBV DNA amplification sequencing,it can be seen that LAM itself cannot promote the virus super-mutations.Only A3G can promote the virus super-mutation,while LAM and A3G at the same time,LAM promotes more YIDD mutations.Thus,the deaminase activity of A3G can induce YIDD mutation of wild virus gene by producing G-to-A gene mutations,and by the selection of LAM,the deaminase activity directly induced the continuous accumulation of YIDD mutations,forming a directional mutation of LAM resistance.3.Study on the mechanism of ATA to inhibit HBV replicationIn this section,we have first discovered a compound with extensive antiviral activity that can fight many RNA or DNA viruses,which is called ATA.And this study confirmed that ATA has a more obvious inhibitory effect on HBV replication.Firstly,by treating HBV-infected HepaRG cells with different concentrations of ATA and determining the release of HBeAg,it's seen that ATA can continuously inhibit HBV's infection and replication,but also continuously inhibit the expression of HBeAg.As the concentration of ATA drugs increases,the inhibition is enhanced,indicating that ATA's inhibiting activity is dependent on its drug concentration.Next,we further verified the molecular mechanism of ATA by using HepG2.2.15.Treated HepG2.2.15 with AT A,and determined NC-DNA and HBc by NAGE,respectively.Results showed that ATA has its drug dose-dependent inhibition effect on NC-DNA synthesis,but there is no obvious inhibition effect on HBcAg expression.HBV transcription is no difference,suggesting that ATA can inhibit HBV replication without affecting the transcription of the virus.Based on previous studies,ATA is considered as a broad-spectrum nuclease inhibitor,so we assume that ATA may inhibit the replication of HBV by blocking its Pol RNase H activity.Based on the above hypothesis,we constructed a mutant HBV particle containing D737V point mutation to detect the expression level of DNA of HBV virus particles,and the results show that the DNA expression of mutant HBV virus particles is not inhibited by ATA,HBcAg expression is not significantly changed,and the DNA expression level of wild HBV virus particles is significantly reduced.It' s explained that ATA inhibits HBV by inhibiting the RNase H activity of DNA polymerase.Extracting HBV total RNA to detect its transcription level is known,ATA has no effect on the expression of mutant virus transcripts.It is explained that ATA inhibits HBV replication by interfering with the activity of the virus Pol RNase H and suspending the HBV RNA/DNA hybridization process,without inhibiting virus transcription.Finally,in order to clarify the effect of ATA on YIDD,YVDD mutation drug-resistant HBV replication,YIDD,YVDD and YMDD 3 different genotype HBV expression protons were transfered to cells.After treated by LAM or ATA,the expression of intrinsic viral genomic DNA and virus HBcAg were detected.It was found that both ATA and LAM significantly inhibited the expression of wild HBV DNA,while ATA also significantly decreased the expression of viral DNA with IIDD and YVDD mutations.There was no difference between the groups in the expression of HBcAg.It can be seen that ATA also has a certain inhibitory effect on the replication of LAM drug-resistant HBV.ATA is expected to be a new compound against HBV' s drug resistance.Conclusions:1.IFN-? and cytokine TGF-?1 can inhibit HBV replication,induce HBV genome hyper-mutation,and increase the expression of APOBECs in liver cells,thereby inducing hyper-mutation of HBV genome.This gene hyper-mutation induced by IFN-? and TGF-?1,can be partially repaired by UNG.2.APOBEC3G can trigger wild HBV to produce YIDD gene mutations,while combined using of IFN-? and LAM could induce the accumulation of the LAM-resistant mutation.3.ATA can inhibit HBV replication by inhibiting HBV Pol RNase H activity without affecting HBV transcription.4.ATA also inhibits the replication of LAM-resistant HBV. |
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