Construction And Application Of Novel Split Protein Reporting System And Hepatitis B Virus Infection And CccDNA Drug Screening Model

Reporting systems represented by fluorescent proteins and luciferase are widely used in cell biology research and functional drug screening,providing powerful tools for the visualization and multi-dimensional quantitative monitoring of research objects.Splitreporter protein system is a new technology developed based on functional protein fragmentation and complementary reconstruction.Compared with intact reporter protein,the split reporter proteins have the advantages of shorter coding sequence,easy fusion with the target proteins,and higher application flexibility.It is particularly suitable for insertion into pathogens with small genome,coding gene overlap and limited foreign gene tolerance.However,there are some shortcomings such as low efficiency of protein function recovery,slow response time,and low sensitivity,which need to be further optimized.Hepatitis B virus(HBV)is a pathogen that could cause severe liver diseases.The lack of an efficient virus reporting system suitable for high-throughput screening leads to slow progress in the research and development of novel drugs.The HBV genome is about 3.2 kb in length,and the open reading frames of coding genes are highly overlapping.Inserting coding sequence of an intact reporter protein into HBV genome often leads to functional defects in infection and replication,which poses great challenges to the construction of HBV infection reporting system.In addition,covalently closed circular DNA(cccDNA)produced by HBV infection and replication is the root cause of the virus’s long-term carrying and difficulty of clearance by existing drugs.How to easily indicate the level of cccDNA in cells is also a difficult problem in the field of HBV model research.The main purpose of this study is to develop new cell model that can efficiently indicate HBV infection or intracellular cccDNA levels and is suitable for high-throughput drug screening based on an optimized split protein reporting system,providing new tools for the development of new drugs for hepatitis B treatment.We thought that the split reporter protein fragment coding gene is small enough and had the potential to be applied to HBV reporting system.In the early exploration,we tried to apply split-GFP:GFP1-10/GFP11 reporting system to indicate the expression of HBV.However,it was not successful due to the low sensitivity and signal-to-noise ratio.In order to improve the performance and applicability of split-GFP reporting system,the sequence and components of the system were systematically optimized in this study.After evaluating the function of 30 nanobodies against GFP,it was found that 9 of them can replace GFP11 and restore the fluorescence of GFP1-10 fragment.Among them,GBP1 has the best effect.The fusion of GBP1 to GFP11 can significantly enhance the fluorescence intensity and speed of fluorescence recovery.Optimizing the GFP1-10 sequence by point mutation to obtain variant Mdd26 can further improve the detection sensitivity and signal-to-noise ratio of split GFP reporting system.We have confirmed the good performance of the nanobody enhanced split-GFP reporter system through a series of experiments in this study,including localization of specific proteins in cells,indicating Herpes Simplex Virus Type 1(HSV-1)infection,characterization of Respiratory Syncytial Virus(RSV)infection-mediated cell fusion,screening of small molecule drugs that promote myoblast fusion,indicating cleavage of site-specific protease,etc.Nevertheless,there is still a problem of great interference to HBV virus when this system is applied to the construction of HBV reporting system.To solve this problem,we further explored the potential of applying split luciferase LgBiT/HiBiT system in HBV reporting model.It was showed that split luciferase LgBiT/HiBiT had higher signal-to-noise ratio when compared to split-GFP.By inserting the short fragment of luciferase HiBiT(11 aa)or its optimized fragments into different positions of HBV and tried different HBV modification methods,we finally obtained a replicative HBV mutant that can secrete HiBiT(C132Hibit).The mutant supports HBV replication regulated by Doxycycline(Dox).The mutant was stably transfected into HepG2 cell and obtained infectious recombinant virus(HBVC132Hibit)in supernatant.Infecting HepG2 cells stably expressing NTCP(HepG2hNTCP-2B1)with HBV-C132Hibit can detect an increase in HiBiT bioluminescence signal in the supernatant of infected cells and the infection can be blocked by neutralizing antibody against HBV.In terms of the cccDNA report model,by integrating HiBiT sequences into different positions of the HBV genome,we successfully constructed a Dox-regulated HepaRG-Hibit16 stable integrated cell model for de novo synthesis of cccDNA and a HepG2-Rcccl stable integrated cell controlled by Cre recombinase model.The cccDNA level in cells was significantly positive correlated with HiBiT level in cell culture supernantant,indicating that the HiBiT signal of HepaRG-Hibit16 and HepG2-Rcccl can be detected to indicate changes in cccDNA levels in cells after drug treatment.The detection of split luciferase HiBiT requires only one step of operation and the reaction takes 5 minutes,which greatly improves the feasibility of the infection system and cccDNA reporting system for large-scale automated drug screening.Using the cccDNA reporting models,we screened 189 drugs and selected 11 compounds that had up-regulation or inhibitory effects on HiBiT signal for verification.In the HepG2-hNTCP-2B 1 infection model,corresponding upregulation or suppress effect to cccDNA levels was observed.Among them,Palovarotene(a clinical trial drug for the treatment of fibrodysplasia ossificans progressiva)can simultaneously reduced the HiBiT level in both models.Further verification found that Palovarotene can significantly decreased antigen level and cccDNA level of HBV in replication model HepAD38 or infection model HepG2hNTCP-2B1,HepaRG-M14A and human primary hepatocytes,and no obvious cytotoxicity was observed.It means that Palovarotene has the potential to be developed as a new anti-HBV drug precursor.In summary,this study constructed cccDNA report models and a recombinant virus infection model suitable for high-throughput screening of HBV inhibitors.The practicability and screening efficiency are superior to other existing detection methods.It was found for the first time that the drug Palovarotene has new function to inhibit HBV infection and reduce cccDNA level.In addition,various nanobody enhanced split fluorescent protein reporting systems were developed in this study,which had significantly improved detection sensitivity and signal-to-noise ratio,and based on this,a variety of original reporting models indicating different cell physiological phenomena or virus infection were constructed,which can promote the development of new drugs in related fields,and also provide optional toolboxes for other application scenarios.