Construction Of Replication-competent HBV Viral Vectors With Tracers And Characterization Of Their Biological Features

Hepatitis B is a global health problem affecting more than 350 million people worldwide.So far,there are many important scientific issues unresolved,e.g.understanding of the life cycle of HBV replication process is still limited;the molecular mechanisms of HBV invasion into hepatocytes;the pathogenesis of chronic hepatitis B,cirrhosis and liver cancer has not yet been fully elucidated.HBV infection and replication model are the indispensable tools to explore a new target for anti-HBV drugs,lessening anti-HBV drug resistance,screening new treatment of anti-viral drugs and evaluation of new immunotherapy and vaccines.By inserting the exogenous gene,many replication-competent viral vectors were constructed playing an important role in the molecular biology research on HBV.If the HBV is transformed into a replication-competent viral vector carrying tracer,that will be able to greatly improve the efficiency of development of new anti-HBV drugs,and contribute to clarify many unresolved scientific issues in HBV research.Concerning the challenges above,we used the replication-competent HBV viral vectors constructed by our laboratory,combined with the latest progress of the fluorescent molecules and luciferase reporter gene,to construct replication-competent HBV viral vectors expressing reporter gene miniSOG,standard Nanoluc^TM luciferase（Nluc）and Secretory Nanoluc^TM Luciferase（SecNluc）separately.The biological characteristics of recombinant HBV and its ability of infection was detected.Objective: To utilize tracing features of miniSOG,Nluc and secNluc,formed replication-competent recombinant HBV viral vectors which can be traced,and detected the biological characteristics of the recombinant HBV vectors and its ability of infection.Methods: 1 The full-length sequence of miniSOG,Nluc and secNluc were obtained by PCR.2 Then the sequences of miniSOG,Nluc and secNluc were inserted into the replication-competent HBV vector pCH-repwhich was previously built in our laboratory,constructing the following three plasmids: pCH-miniSOG,pCH-Nluc,and pCH-secNluc.The wild-type HBV vector pTRE-HBV-C7-5 carrying tetracycline regulatory elements and the hygromycin resistance gene which was built previously in our laboratory as the parent plasmid,the fragment of HBV was replaced by HBV-miniSOG,HBV-secNluc which was from pCH-miniSOG and pCH-secNluc,formed two vetors: pTRE-HBV-miniSOG and pTRE-HBV-secNluc were obtained.3 HepG2,293 and Huh7 cell lines were transfected with pCH-miniSOG respectively.The intracellular fluorescence intensity was observed 72h post-transfection,and compared with pCH-hrGFP vector.4 HepG2 cells were transfected with six plasmids（pCH-3093;pCH-BsdR;pCH-Nluc;pCH-secNluc;pCH-miniSOG;pCH-hrGFP）,respectively.The total RNA was extracted 72 h post-transfection.The HBV RNA of replication-competent HBV vectors were detected by Northern blot.5 HepG2 cells were transfected with six plamids mentioned above,Western blot for envelopproteins was performed using a mixture of 9H9 and 4/7B antibody against HBsAg.Native western blot for HBV core capsids was performed using mc-312 antibody.6 HepG2 cells were transfected with six plamids mentioned above,the levels of HBsAg and HBeAg in the supernatant were determined by immuno-chemi-luminometric assay.7 HepG2 cells were transfected with six plasmids above-mentioned and pTRE-3093,pTRE-HBV-miniSOG,pTRE-HBV-secNluc,pTRE-HBV-hrGFP,respectively.The DNA of extracellular and cytoplasm was extracted,and the HBV replication intermediates were detected by Southern blotting.8 HepG2 cells were transfected with the plasmids which were mentioned above.Samples were taken at 72 h post-transfection.Aliquots of prepurified extracellular virals were treated by Dpn I,DNase1 and Nuclease,viral titers were measured using a commercial HBV DNA kit on a SLAN^TM Real-Time qulity PCR system.9 HepG2 cells were transfected with pCH-Nluc,pCH-secNluc.The luciferase activity in HepG2 cells was detected by Nano-Glo ? luciferase assay reagent at indicate times（24h,48h,72h,96h,120h）.10 HepG2 cells were transfected with pCH-Nluc and the viral particles were harvested,which were used to infect HepaRG cells.The luciferase activity in HepaRG cells at indicated time points（the0 2nd,4th,6th,8th and 10 th day post infection）was monitored to evaluate the ability of its infection.Results:1 The full-length sequences of miniSOG,Nluc and secNluc were amplified 2 the replication-competent HBV plasmids with tracer genes were successfully constructed,namely pCH-miniSOG,pCH-Nluc,pCH-secNluc,pTRE-HBV-miniSOG,pTRE-HBV-secNluc.The construction was confirmed by agarose gel eletrophoresis,restriction endonuclease digestion and sequencing.2 The fluorescence of replication-competent HBV vectors expressing green fluorescent protein.Expression of miniSOG and hrGFP could be observed in the three cell lines 72 post-transfaction,but the fluorescence intensity of miniSOG was weaker than hrGFP.3 The RNA deriving from replication-competent HBV vectors.The RNA from replication-competent HBV vectors mentioned above（method-4）,indicating the transcription of HBV pgRNA and sgRNA.The length of pg RNA from HBV vectors carrying transgene were larger than that of wildtype HBV.4 The expression of envelopproteins from replication-competent HBV vectors.It showed that all of the replication-competent HBV vectors mentioned above（method-4）produced similar amount of large,middle and small protein.5 The expression of core protein from replication-competent HBV vectors.It showed that all of the replication-competent HBV vectors mentioned above（method-4）produced similar amount of core capsids.6 The levels of HBsAg and HBeAg in the cell culture supernatants.Both the HBsAg and HBeAg levels in the cell culture supernatants after transfection of replication-competent HBV vectors mentioned above（method-4）were similar with that of supernatants after wildtype HBV vector transfection.No statistically significant difference between levels of every two groups could be found by LSD t-test following one-way ANOVA with SPSS 17.0（P>0.05）.7 Detecting the cytoplasmic and extracellular HBV DNA levels by southern blot.The replication levels of the BsdR（399bp）and miniSOG（312bp）replication-competent HBV vectors were similar with that of wildtype HBV vector.But replication of the Nluc（513bp）,secNluc（597bp）,hrGFP（720bp）vector were less than that of wildtype HBV vector.8 The level of the secreted HBV DNA.Compared with wild-type HBV,the secretion levels in culture medium of cells transfected with the replication-competent HBV vectors mentioned above were decreased.However,HBV DNA of upto 10⁶ copies/mL could be found in medium of cells transfected with the recombinant vectors.9 Luciferase assays.HepG2 cells were transfected with the plasmids of pCH-Nluc,pCH-secNluc.There are different amount of the expression of luciferase at different times.The luciferase activity gradually increased at the time points of 24 h,48h,72 h after infection,the Luminescence of Nluc could be upto 4.5ⅹ10⁹ and the Luminescence of secNluc could be upto 2ⅹ10⁹ at the time of 72h;the luciferase activity had a downward trend at the time point of 96 h,but still at a high level.10 Infection of HepaRG cells by recombinant HBV.The luciferase activity peaked at the 6th day post-infection,with relative luciferase unit of upto 10⁶.The luciferase activity was remained at the 8th day,and it began to decline at 10 th day.Conclusion: Recombinant HBV vectors with tracer genes were successfully constructed,namely pCH-miniSOG,pCH-Nluc,pCH-secNluc,pTRE-HBV-miniSOG and pTRE-HBV-secNLuc.By characterization of recombinant HBV vectors,we proved that the recombinant HBV vectors could produce replication-competent recombinant HBV with sufficient packaging,secretion and infection capability after transfection into HepG2 cells.Expression of exogenous gene was also confirmed.Owing to the unique feature of tracing conveniently,quickly and quantificationally,the recombinant HBV vectors with tracer genes could be used to accelerate screening of anti-HBV agents and underlying of molecular essence of HBV.