Research On The Molecular Mechanism Of Polyamines Metaboolism Regulating HBV Replication

Objective: Hepatitis B virus(HBV)is a DNA virus with its genome size of 3.2 kb that specifically infects human liver cells and replicates in a reverse transcription manner.Polyamines are small positively charged molecules commonly found in mammalian cells,which mainly regulate nucleic acid structure,cell proliferation and signal transduction.Recent studies have found that polyamines are involved in regulating the life cycle of several viruses,including virus genome replication,nucleic acid packaging and protein synthesis.Meanwhile,ornithine decarboxylase(ODC1)is a rate-limiting enzyme in polyamine biosynthetic pathway,and DFMO,an irreversibly inhibitor of ODC1,was reported to inhibit the replication of DNA viruses such as cytomegalovirus(CMV),and RNA viruses such as Zika virus(ZIKV)and Dengue virus(DENV).However,it is unclear whether polyamine metabolism is involved in the regulation of HBV replication and whether DFMO can be used as a new drug against HBV replication.Therefore,in this research,we aimed to investigate the correlation between polyamine metabolism and HBV replication.Firstly,we explored the expression of ODC1 and spermidine synthase(SRM)in the presence of HBV,and further identified ODC1,a key polyamine metabolism enzyme,regulating virus replication through gene function experiments.Secondly,we found that DFMO,an ODC1 inhibitor,targeting the ubiquitination of HBc protein to reduce the expression of HBc and the level of viral particles,thereby limiting the replication of viral DNA.These results not only help to clarify the correlation between polyamine metabolism and HBV,the molecular mechanism of key polyamine metabolism enzymes ODC1 and metabolic substrates regulating HBV replication,but also highlighted that the importance of the polyamine biosynthetic pathway toward HBV replication,as well as its potential as a target in the development of further antivirals or currently available drugs,such as DFMO.Methods:Part 1: the correlation between polyamine metabolism and HBV replication(1)The relationship between HBV replication and expression of polyamine metabolism-related genes was first investigated in HBV stable expressing cells Hep AD38 and HBV infected cells Hep G2-NTCP.Real-time PCR and Western blot were used to detect the effects of virus replication on the expression of polyamine metabolism-related genes ODC1,SRM and SMS,which explored the correlation between polyamine metabolism genes and virus replication.(2)Hep AD38 cells were transfected with si RNA targeting ODC1 and SRM,then total RNA,total protein and virus core particle DNA were extracted.ELISA,real-time PCR,Western blot and Capsids were used to detect changes of HBV markers to explore whether and how ODC1 and SRM regulate viral replication.(3)We employed DFMO,a specific inhibitor of ODC1,to test its anti-HBV effect.MTS assay was used to determine toxicity of DFMO on Hep AD38 and Hep G2.2.15 cells,then Hep AD38 and Hep G2.2.15 cells were treated with DFMO,HBV markers were measured by ELISA,real-time PCR,Western blot and Capsids methods.(4)We explored the effect of polyamine metabolites on virus replication.Hep AD38 were pretreated with DFMO,followed with adding exogenous polyamine mixtures or spermine and spermidine,then HBV markers detected by ELISA,real-time PCR,Western blot,and Capsids assays.Part 2: the molecular mechanism of DFMO inhibiting HBV replication(1)Hep G2 cells tranfected plasmids 3x Flag-HBc,3x Flag-HBs and3 x Flag-HBx,respectively,and then treated with DFMO.Real-time PCR and Western blot were used to detect the m RNA and protein levels of HBc,HBs and HBx.To investigate whether DFMO affect the translation of viral proteins,el F5A1 and el F5A2 were knockdowned by si RNA in Hep AD38 cells,then viral DNA was detected by real-time PCR and Western blot which used to analyze the level of viral proteins.(2)To investigate whether DFMO regulates ubiquitination of HBc protein to inhibit HBV replication.Hep AD38 cells and Hep G2 cells were treated with DFMO,and then added with CHX and MG132,respectively.Western blot was used to determine the levels of HBc protein.Results:Part 1: the correlation between polyamine metabolism and HBV replication(1)In HBV stable expressing cells Hep AD38,HBV replication was inducted by removing the tetracycline.The expression of ODC1 and SRM gradually increased both at m RNA and protein levels with HBV core protein(HBc)expression.Furthermore,it was also found that the expression of ODC1 and SRM increased both at m RNA and protein levels by HBV infection in Hep G2-NTCP cells,indicating that rate-limiting enzymes in intracellular polyamine biosynthetic pathway were related to HBV replication.(2)Silencing of ODC1 and SRM with si RNA in Hep AD38 cells,HBV DNA replication levels decreased by about 40% and 33%,respectively.The levels of protein and virus particles were also significantly reduced.However,it had no obvious effect on HBs Ag levels in the supernatant,indicating that silencing of ODC1 and SRM led to inhibit HBV replication.(3)DFMO treatment had no impact on cells viability even in high concentration of 200?M.ELISA and real-time RT-PCR revealed that DFMO did not affect the secretion of HBs Ag in the supernatant and the level of HBV 3.5 kb m RNA.However,HBV DNA,HBV protein and virus particle levels were significantly reduced,indicating that DFMO inhibits HBV DNA synthesis mostly by reducing HBc protein levels.(4)We treated Hep AD38 cells with exogenous polyamine mixture and single polyamines(spermine and spermidine),the effects of polyamines on HBV replication were detected using real-time PCR,Western blot and Capsids.Our results indicated that polyamines promote HBV replication.Part 2: the molecular mechanism of DFMO inhibiting HBV replication(1)Hep G2 cells were transfected with HBx,HBc and HBs plasmids and then treated with DFMO.Real-time PCR and Western blot methods revealed that DFMO significantly reduced HBc protein levels,but had no significant effect on HBc RNA levels.However,there was no significant effect in levels of HBx and HBs,suggesting that DFMO inhibited HBc expression at protein level.Further silencing of el F5A1 and el F5A2 by si RNA in Hep AD38 cells,there were not significant changes in HBV replication,indicating that DFMO did not affect the translation of HBc protein.(2)CHX and MG132 were added to DFMO treated Hep G2 and Hep AD38 cells,respectively.The result of Western blot showed that DFMO reduced the level of HBc protein by affecting on the ubiquitination of HBc.Conclusion: The expression of polyamine metabolism-related genes ODC1 are related to HBV replication,ODC1 and polyamines significantly regulates the HBc protein levels and DNA replication.The ODC1 inhibitor DFMO has obvious antiviral activity that inhibits the replication of HBV DNA and HBc protein.DFMO inhibits HBc protein levels via promoting its ubiquitination.