Effect Of A Novel Nucleoside Analogue β-L-D4A On Hepatitis B Virus

Objective Traditionally approved therapies against HBV include treatment with alpha interferon and lamivudine. However, the efficacy of alpha interferon is limited by undesirable side effects, low sustained response rate and high cost. While lamivudine is well-tolerated and less costly, resistance emerges in approximately 20% of patients receiving monotherapy per year. Adefovir-dipivoxil prodrug and entecavir are recently approved anti-HBV therapeutics. While these two agents produce multilog suppression of serum HBV DNA, they cause only modest rates of HBV surface antigen seroconversion and, thus, require longterm administration to control disease in most patients. ADV retains clinical effi cacy against la mivudine-resistant mutants, but selects the mutations rtN236T and rtA181V In addition, treatment with ADV is reported to be associated with doselimitingnephrotoxicity. Entecavir shows reduced susceptibility to lamivudineresistant mutants in vitro and clinically, and an accelerated development of entecavir resistance mutations (rtI169T, rtM250V, rtS202I and rtT184S/G) in lamivudine-resistant patients. Therefore, more effective and safer therapies are needed for the treatment of chronic HBV infections.β-L-D4A is an L-enantiomer of the natural nucleoside deoxyadenosine. In this report, we detected the effi cacy ofβ-LD4A against HBV replication in vitro.Methods 2.2.15 cells were maintained in DMEM (supplemented with 10% fetal bovine serum, 200 g/mL G418, 2 mmol/L glutamine, 50 U of penicillin per milliliter and 50μg of streptomycin per milliliter) at 37℃and 5% CO₂. The medium was changed every three days. 2.2.15 cells were digested by parenzyme, seeded at a density of 1×10⁵ cells per well on 24 well plates and maintained in 1.5 mL DMEM at 37℃and 5% CO₂ for 48 h. Then, 2.2.15 cells were treated with freshly prepared medium containingβ-L-D4A or 3TC. The following concentrations were used: 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 umol/L. Negative control cells were treated with DMEM only. Media were changed every three days; on day 8, both the supernatants and cells were collected and stored at -20℃. Then, the cells were lysed at room temperature with lysis buffer (50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L MgCl₂, 0.2% Nonidet P-40). Removal of cellular debris was accomplished by a 5-min microcentrifuge spin at 16 000 r/min. Lysates were then incubated with 20% PEG 8000 and 1 mol/L NaCl at 4℃for 2 h, followed by a 30 min microcentrifuge spin at 12000g. The resulting pellets containing HBV core particles were resuspended in solution added with proteinase K ( 10 mM Tris [PH7.6], 10 mM EDTA, 0.25% SDS, 1mg/ml proteinase K) at 56℃for 2h, followed by extraction with phenol chloroform and ethanol precipitation. Nucleic acids were dissolved in TE, electrophoresed through 1% agarose, transferred to a positively charged nylon membrane and hybridized to a probe, which was prepared from a full length HBV DNA genome template excised from plasmid P3.6â…¡and DIG labeled using a random primer; Then, the membrane was incubated with anti-DIG-ap-conjugateand developed with NBP/BCIP. The concentration of extracted HBV DNA was also determined by real time fluorescent quantitative Polymerase Chain Reaction. The inhibition ratio was calculated according to the following formula: inhibition ratio = (HBV DNA concentration of negative control HBV DNA concentration of experimental group)/HBV DNA concentration of negative control×100%. Then, the 50% inhibition concentration was calculated according to the Reed Muench method. 2.2.15 cells were treated with antiviral compounds and the collected supernatants were detected using an ELISA Kit for the detection of HBsAg according to the manufacturer's instructions, and A450 was observed. MTT assays were used to detect the survival rates of 2.2.15 cells. Briefly, HepG2 cells were seeded at a density of 1×10⁵ cells per well on 96 well plates and treated with media containingβ-L-D4A or 3TC of different concentrations, the following concentrations were used: 31.25, 62.5, 125, 250, 500, 1000umol/L. Three days later, the supernatants were discarded and 2ul of MTT (5mg/ml) was added per well. After incubation at 37℃and 5% CO₂ for 4h, the supernatants were discarded and 150μl DMSO was added per well. The survival ratio of HepG2 cells (%) = (A490 of experimental group-A490of negative control)/ A490 of negative group^* 100%. Then, the 50% toxicity dose (TD50) was calculated according to the Reed Muench method. All statistics were analyzed using SPSS10.0, and differences were considered signifi cant when the P value was < 0.05.Results To investigate the potential ofβ-L-D4A to inhibit HBV viral replication, HBV DNA was isolated from treated 2.2.15 cells and detected by Southern hybridization. The results showed that the production of HBV DNA lessened following treatment of cells withβ-L-D4A; this effect became more notable as the dose ofβ-L-D4A increased. A similar phenomenon was observed in cells treated with 3TC. The concentration of HBV DNA in 2.2.15 cells was determined by real-time fluorescent quantitative PCR.β-L-D4A had an apparent inhibitory effect on the concentration of HBV DNA (P > 0.05 when compared to that of 3TC). Calculated according to the Reed Muench method, the IC50 values ofβ-L-D4Aand 3TC were 0.61 umol/L and 0.30 umol/L, respectively.β-L-D4A inhibits the secretion of HBsAg following treatment of 2.2.15 cells with antiviral compounds for eight days, HBsAg was detected in the medium. When the concentration ofβ-L-D4A was 100 umol/L, the HBsAg inhibition ratio was above 50%. However, the HBsAg inhibition ratio with 3TC was less than 40% even at the highest concentration of 100 umol/L. Cellular toxicity ofβ-L-D4A The cellular toxicity of both compounds was not evident at drug concentrations below 125 umol/L. The survival ratios were less than 50% when the drug concentrations were as high as 1000 umol/L. Calculated according to the Reed-Muench method, the TD50 values ofβ-L-D4A and 3TC were 417 umol/L and 243 umol/L, respectively. This means that these two compounds are high in efficacy and low in cellular toxicity.Conclusions HBV infection is one of the most prevalent viral diseases in the world and more than 350 million people are chronically infected, with about 1 million deaths annually. In order to screen anti-HBV compounds, researchers often purify HBV DNA polymerase. However, the purification procedure is very complicated and the quantity of polymerase is not satisfying. 2.2.15 cells can stably replicate HBV DNA and express HBsAg thus, they can be used as an ideal cell model to screen the in vitro antiviral effi cacy of medicines. In this study, we found thatβ-L-D4A has a dose dependent inhibitory effect on HBV DNA replication that is apparent when the concentration is above 1 umol/L. However, the inhibition of the secretion of HBsAg was not as pronounced as that of HBV DNA replication. Whenβ-L-D4A was at the highest concentration of 100 umol/L, the HBsAg inhibition ratio was slightly above 50%.β-L-D4A is a new type of L-nucleoside for which the natural counterpart is deoxyadenosine. Although there have been reports about L-nucleosidessince 1960s, it was only after about 30 years that the merits of L-nucleoside became widely recognized. Previous studies revealed the bioactivities and securities of L-nucleosideanalogues were superior to their counterpart enantiomers.β-L3/(2R, 5S)-1,3-oxathiolane-cytidine (lamivudine, 3TC), a typical L-nucleoside, was reported to be about 50 times more effective than its D-enantiomer. The mechanism of action ofβ-L-D4A is speculated to be inhibition of HBV DNA polymerase and termination of DNA replication. In order to confirm this speculation, further study is necessary. It also remains to be seen whetherβ-L-D4A is effective against lamivudine resistant HBV mutants and whether it will lead to HBV mutants resistant toβ-L-D4A. Besides, much work remains to be done to gain a better understanding of the metabolism, toxicology and pharmacokinetic parameters of this compound. Our studies to date suggestβ-L-D4A deserves further development as a potential treatment for chronic HBV infection. Objective: Infection with HBV affects an estimated 2 billion people worldwide. Primary HBV infection can become persistent and continues for many years. The infection progresses into chronic hepatitis with high risk of developing liver cirrhosis and/or hepatocellular carcinoma. HBV is a hepatotropic virus with a 3.2 kb partial doublestranded circular genome. Hepadnaviruses have a complex replication cycle and HBV polymerase is involved in all phases of the replication process. After infection, a partially double-stranded DNA genome is converted into covalently closed circular DNA from which a 3.5 kb, greater-thangenome-length, (+) strand pregenomic mRNA (pgRNA) is transcribed. HBV polymerase exerts its RNA-dependent DNA polymerase activity (reverse transcriptase) to create a full-length (-) strand DNA by first reverse transcribing the pgRNA inside the newly synthesized nucleocapsid. Next, the HBV polymerase synthesizes incomplete (+) strand DNA from the (-) strand DNA template. The initiation mechanism of RT is unique. While HBV polymerase uses a specific cellular tRNA as a primer, HBV polymerase utilizes a tyrosine residue located within its own N-terminus as the acceptor for the initiating deoxynucleotide residue. Priming is templated by a bulge sequence within a stemloop structure called s located near the 5'-end of the pgRNA. This priming step yields a discrete 3- to 4-deoxyribonucleotide oligomer that is covalently linked to the polymerase. The polymeraseprimer adduct subsequently translocates to the 3'-end of the pgRNA and binds to a complementary sequence in an element called direct repeat 1 (DR1), where the elongation of (-) strand DNA is initiated. Finally, HBV polymerase also mediates DNA-dependent (+) strand DNA synthesis, which is primed by an RNA primer resulting from incomplete degradation of pgRNA by an RNaseH activity at the C-terminus of polymerase.HBV polymerase is non-covalently linked to the HBV core protein that harbors RNA and DNA binding activities and plays an essential role in hepadnavirus replication and propagation. DR1 and the s stem-loop structure prove important for the function of HBV polymerase. HBV polymerase has intrinsic RNAdependent RT, DNA-dependent DNA polymerase as well as RNaseH activity. The multifunctional properties of the HBV polymerase make this enzyme an attractive target for nucleoside antiviral therapy. A large number of antiviral agents have been evaluated as potential therapeutics for the chronic hepatitis. Current efforts have mostly focused on nucleoside analogs as inhibitors of the multifunctional viral polymerase. The nucleoside analogβ-L-D4A-TP is structurally similar to the natural D-enantiomer of 2'-deoxyadenosine-5'-triphosphate with the exception of its configuration and inner carbon-carbon double bond content. Previous studies revealed thatβ-L-D4A-TP is also a very effective and selective agent against HBV in Hep G2 2.2.15 cells. The compound rapidly reduced the amount of viral DNA and hepatitis B surface antigen (HBsAg) in serum of transgenic mouse without significant drug-induced liver and kidney toxicity. The aim of the present study was to study the inhibition mechanism of the nucleoside analogβ-L-D4A-TP. A polymerase reaction in vitro with the recombinant HBV nucleocapsids was conducted and then the viral DNA and viral DNA-polymerase complex formed in the polymerase reaction were assayed to determine whether reverse transcriptase activity of HBV polymerase was inhibited byβ-L-D4A-TP.Methods All HBV sequences were cloned from the plasmid p3.6â…¡containing complete genome of HBV strain adr, and introduced into the pFastBac Dual donor plasmid (Invitrogen) by the standard molecular cloning techniques. The desired fragments HBV polymerase gene followed by 3' DR1 and 3'εstem-loop structure (PE) (nt 2279 to 3215 and 1 to 1918) and 5'εstem-loop structure plus HBV core protein gene (EC) (nt 1809 to 2483) were amplified by PCR. Both constructs were sequentially subcloned into pFastBac Dual. DH10Bac competent cells were transformed with the recombinant plasmid pFastbac Dual-polymerase-core. Recombinant bacmid was isolated, purified and then Sf9 cells were transfected with recombinant bacmid using Lipofectin reagent (Gibco-BRL) according to the manufacturer's protocol. Sf9 cells were grown at the density of 2×10⁶ cells/well in 6 well tissue culture plates and infected with the recombinant baculovirus at a MOI= 10. The cells were incubated for 72 hrs until the signs of viral infection appeared. Then the cells were harvested by centrifugation, washed 3 times with PBS, and the cell pellet was stored at -80℃. Thawed cell pellet was disrupted on ice for 30 mins in 1/10 of original volume of NP-40 lysis buffer. After low-speed centrifugation, aliquots of 1ml lysate were 华中科技大学博-2学位论文immunoprecipitated with 40å±±of mouse monocbnal antibody anti—HBV core proteincoupled to CNBr actiVated Sepharose CL一4B at 4。C for 12 hrs.Folbwing low speedcentrif.ugation,the Sepharose with immunocomplexed nucleocapsids,i.e.immunobeadswere washe d three times with PBS and prepared to polymerase reaction in Vitro.Forpolymerase reaction,40 Ul of immunobeads were resuspended in 1 00 Ul reaction mixturecontaining 50 mmol/l 1'ris reaction buffer,pH 7.5,supplemented with 75 mmol/l NH4Cl,20mmol/l MgCl 2,0.5%Tween-20,0.1 mmol/l p—mercaptoethanol,50 gmol/l three dNTPs(dATP,dGTP,and dCTP),15 gmol/l dTTP,35 umol/l digoxiglenin(DIG)一1 1一dUTP andvarious concentrations of 6一L—D4A—TP.The sample with 3TC—TP was used as a positiVecontroL while the sample without the dmg was used as a nega tiVe contr01.The reactionmixture was incubated at 37。C for 12 hrs.Assay of Viral DNA formed in the polymerasereaction.The produc馏oft}1e p01ymerase reaction were analy^ed by agarose gel electrophoresis.The immunobeads undergoing polymerase reaction were rinsed 3 times with 50 mmol/l1.ris washing buffer to remove the unincorporated dNTPs.The immunobeads wereresuspended in fiVe VOlumes 0.1 mol/l glycine elution buffer for 1 5 mins on ice,and thencentrifuged at 3,000 11)m for 5 mins at 4。C.200å±±of supernatant was neutraliZed with 13Ul of 0.8 mol/l 1.ris neutralization buffer.The nucleocapsids bound to immunobeads werereleased t0 the solution and collected. This procedure was repeated twice. Purm ednucleocapsids were digested with proteinase K folbwed by several extractions with phenol—chbroform and precipitation with ethan01.DNA partially labeled in polymerase reactionwas separated by agarose gel electrophoresis followed by transfer to positiVely chargedHybond—N membrane by capillary transfer.The membranes were incubated with 1 50mU/ml anti—DIG—ap—conjugate at 37。C for 2 hrs,detected by alkaline—phosphatase—catalyå²”d cobr reaction with NBT/BClP.For analysis of polymerase—linked RT productsby SDS—PAGE,the immunobeads undergoing polymerase reaction were nnsed as describedabove,disrupted by being boiled for 7 min in protein sample buffer.The polymerase—linked RT products were bbtted t0 positiVely charged membrane.The membrane wasstained with NBT/BClP as described above.The real—time fluorescent quanti切tiVe PCR forquanti切tiVe determination of HBV DNA was performed for monitoring of antiViral effectof reagents bbcking HBV replication. Amount of extracted DNA in nucleocapsids38 subjected to polymerase reaction was determined by this assay. Inhibition rate was calculated according formula. Fifty percent inhibitory concentration (EC50) was calculated by Reed-Muench formula.Results We expressed in vitro replication-competent HBV nucleocapsids in Sf9 cells using the Bac-to-Bac baculovirus expression system, which is an efficient site-specific transposition system to generate baculovirus. The nucleocapsids were purified with anti-HBVcore protein antibody coupled to Sepharose from the cleared lysate of Sf9 cells infected with recombinant baculoviruses. Purified polymerase and core proteins were verified by Western blot analysis. To investigate the potential ofβ-L-D4A-TP to inhibit replication of viral DNA in nucleocapsids, recombinant HBV nucleocapsids were subjected to the polymerase reaction that yielded abundant DIG-labeled HBV DNA. DIG-11-dUTP was used as the label and the drugs were titrated against the unlabeled dATP substrate for the A analog,β-L-D4A-TP or against dCTP for 3TC-TP. In comparison with untreated control we observed that the amount of DNA from nucleocapsids treated with the drugs was decreasing in a dose-dependent manner. These results suggested that 3TC-TP as well asβ-L-D4A-TP inhibited HBV replication and reduced the amount of DNA in nucleocapsids. We confirmed that both drugs inhibited the RT activity of the polymerase in the following study. HBV polymerase is different from the other RTs because it can use its tyrosine residue as the primer to initiate deoxynucleotide residue and synthesize (-) strand DNA covalently linked to polymerase. After polymerase reaction in vitro, HBV nucleocapsids were disrupted by boiling in sample buffer and subjected to SDS-PAGE. DNA polymerase adducts moved together and a heavily labeled smear appeared at the beginning of the stained polymerase band. Presumably, the labeling at the positionof the polymerase band represented covalent linkage of a single nucleotide to polymerase in the nucleotide priming reaction, and the labeling of the higher-molecular-weight material represented the extension of this product by RT. DNA synthesized in polymerase reaction gradually diminished as the concentrations ofβ-L-D4A-TP and 3TC-TP increased. This result indicated that the drugs indeed inhibited the RT activity of polymerase and caused the termination of the nascent DNA chains. For quantitative analysi s of the formation HBV DNA, we used a real-time fluorescent PCR The curves of inhibition rate in the presence of tested drugs in different concentrations showed that inhibition of the HBV DNA synthesis in the presence ofβ-L-D4A-TP was more intensive when compared with the inhibition of HBV DNA synthesis in the presence of 3TC-TP at the same concentration. The EC50 forβ-L-D4A-TP and 3TC-TP was 35.30 and 44.57μmol/l, respectively.Conclusions In an attempt to explain the mechanism of the inhibition of HBV replication by the nucleoside analogβ-L-D4A-TP we conducted the polymerase reaction with recombinant HBV nucleocapsids. The presented data suggested thatβ-L-D4A-TP inhibited the replication of HBV DNA by inactivation of RT activity of HBV polymerase in a concentration-dependent manner, although other modes of inhibition were not excluded. It was presumed thatβ-L-D4A-TP acted at least in part as a chain terminator that was irreversibly incorporated into the nascent DNA chain. This mechanism was similar to that previously demonstrated for other nucleotide analog RT inhibitors that lack a 3'-hydroxyl group required for nucleotide addition and were DNA chain terminators such as 3TC-TP. The kinetics ofβ-L-D4A-TP inhibition of the RT activity was the result of an apparent competitive inhibition with respect to dATP.β-L-D4A-TP was more effective than 3TC-TP, reducing synthesis of viral DNA by 50% at an inhibitor concentration of 35.30 umol/l, followed by 3TC-TP, which inhibited synthesis by 50% when present at a concentration of 44.57μmol/l. Due to the serious resistance of 3TC, it is urgent to look for new anti-HBV drugs. Our studies provide an experimental basis for the clinical application ofβ-L-D4A-TPin the future.