Preliminary Study On The Role And Mechanism Of MAPK Inhibitors In Enhancing HBV Infection In Vitro

Chronic infection of hepatitis B virus(HBV)is still one of the public health concerns affecting human health.Although the popularity of preventive vaccines has significantly reduced the new infection of hepatitis B virus,there are still more than 240 million chronic hepatitis B patients worldwide,Since existing therapeutic drugs can only inhibit the replication of viruses but fail to cure chronic hepatitis B,it is urgent and necessary to develop more effective new drugs.New drug development may rely on a deep understanding of HBV biology and the interaction between HBV and host.All of these concerns depend on the establishment and development of the in vitro cell models and in vivo animal models that can mimic the whole life cycle of HBV.Cell culture model is the most commonly used and convenient platform for the study of HBV infection and replication,interaction between HBV and host,screening and evaluation of anti-HBV drugs in vitro.Our group previously established two cell models,HepG2-N10 and HepG2-hNTCP(2B1),respectly.One of them,HepG2-N10 is a stable replication cell line that stably integrates the 1.1 length of HBV genome in HepG2,which can support HBV replication and used for drug screening and evaluation.On the other,HepG2-hNTCP(2B1)can support HBV infection and cccDNA formation,but high virus dose is required for infection and the low infection positive rate,only 20%?30%.In addition,PEG is involved in infection.In this study,we screened the MAPK inhibitor library to optimize the HepG2-hNTCP cell infection model,improve its infection efficiency and get rid of PEG constraints.At the same time,we searched for factors that restricted the rat liver cells from supporting HBV infection,and then constructed a mouse model with a healthy immune system that supports HBV infection.In this study,140 MAPK inhibitors were screened on the replication model HepG2-N10 and the infection model HepG2-hNTCP.The results obtained by the two models were significantly different.Moreover,antigen-level correlation analysis did not find a significant correlation between the results obtained by the two models.Since the infection model can support HBV infection and its virus replicates in a state are similar to natural infection,which is more in line with the physiology of HBV.Therefore,it can be explained that the results of the replication model for drug screening and evaluation require further verification.In this study,140 MAPK inhibitors were screened in the 2B1 cell model,and found that 30 MAPK inhibitors could enhance HBV replication by added on the sixth day after infection.Further analysis of the effects of these 30 MAPK inhibitors on the HBV infection by added before infection and sustained,showed that 12 MAPK inhibitors significantly promote HBV infection.In addition,SCH772984,Trametinib,PD184352 and Cobimetinib,could significantly increase the level of HBV infection by treated only 24 hours before infection,including HBeAg increased by more than 5 times and HBsAg increased by more than 10 times.In addition,this study investigated the mechanism for promoting HBV infection.According to whether NTCP expression is up-regulated,these MAPK inhibitors were classified into NTCP pathway-dependent and non-NTCP-dependent.B-Raf IN1 is a NTCP pathway-dependent MAPK inhibitor,which can enhance the infection of HBV,especially for cccDNA.In order to investigate the mechanism of B-Raf IN1 for promoting HBV infection and the host genes involved in HBV infection,the transcriptome of 2B1 cells treated with B-Raf IN1 were sequenced.The results showed that the expression of NTCP was up-regulated with B-Raf IN1 addition,and the transcriptional level of the host genes related to HBV infection and replication were also increased in varying degrees.We sletcted 1 up-regulated gene to test its effect on HBV infection of 2B1,and it has been found that overexpression of JunB enhances HBV infection in 2B1 cells and inhibits HBV infection after knockdown.Therefore,it was hypothesized that the promotion of HBV infection with B-Raf IN1 addtion was associated with enhanced expression of JunB.However,JunB itself is a transcription factor and does not directly act on HBV.Therefore,it is still necessary to find factors that are more critical to HBV infection.Other host genes associated with HBV infection are still being validated.Furthermore,for non-NTCP-dependent MAPK inhibitors,we hypothesized that there are other receptors on the surface of 2B1 cells that bind to HBV Therefore,we performed HBsAg binding assay and found that B-Raf IN-1 d,Pimasertib,Selumetinib,PD0325901,SCH772984,Trametinib,TAK632 and Cobimetinib can significantly enhance HBsAg binding,indicating that these MAPK inhibitors induce a certain protein on the cell surface that could binds to HBsAg,thus promoting HBV infection.Furthermore,we screened several of them for transcriptional sequencing,and the host genes associated with HBV infection are being verified.In summary,several MAPK inhibitors that can significantly promote HBV infection were screened out in HepG2-hNTCP cell model and their mechanisms were preliminarily explored.It provides a possibility for the construction of a better in vitro infection cell model.