The Function Of SIRT7 In Hepatitis B Virus Transcription And Replication

Chronic hepatitis B virus(HBV)infection remains a major public health problem with more than 240 million people chronically infected worldwide,and about 6.5 billion people die from liver diseases caused by chronic HBV infection each year.The epidemic situation of HBV in China is also very serious,at present there are more than 20 million patients with chronic hepatitis B in China,and the number of virus carriers as high as 86million.Despite the availability of effective vaccines make the new infection obviously controlled,there is a potential risk of carriers turning into chronic HBV patients,and the number of hepatitis B patients will still be very large in the coming period.Therefore,the prevention and treatment of hepatitis B virus is still very difficult.At present,interferon and nucleoside analogues are the main drugs used in the treatment of hepatitis B virus.However,interferon is effective in less than 30%of the patients,and the side effects are large,the application is limited;nucleoside analogues have a long course of treatment,prone to viral resistance and virological rebound after withdrawal.Therefore,in order to develop new treatment strategies and further improve the therapeutic effect of CHB patients,it is necessary to fully understand the replication and regulation mechanism of the virus.Upon enter into hepatocytes,The HBV genomic relaxed circular DNA(rcDNA)enter into hepatocyte nucleus and convert into a covalently closed circular(ccc)DNA molecule,the cccDNA organized into minichromosome by histones and non-histone proteins,and serves as the template for transcription of HBV RNAs.During this complex process,many host factors are involved in.sirtuins is a highly conservative nicotinamide adenine dinucleotide(NAD+)dependent enzyme family,which is widely involved in a variety of physiological processes,such as genomic stability,stress resistance,energy metabolism,tumorigenesis and so on.Our previous study found that overexpression of SIRT3 and SIRT7 in HBV infected cell models significantly decreased the level of HBV RNAs,suggesting that SIRT3 and SIRT7 may be involved in HBV replication and transcription.In this study,we focused on the specific mechanism of SIRT7 regulating HBV replication and transcription.To study the effect of SIRT7 on HBV replication and transcription,including the following aspects:(1)Overexpressed of SIRT1-7 in HBV infected cell model HepG2-NTCP.Real-time PCR was used to detect the effect of sirtuins on the expression of HBV RNAs.(2)Transducted of SIRT7 shRNA interference lentivirus in HBV infected HepG2-NTCP and PHH cells.Western blot was used to detect SIRT7 interference efficiency;Real-time PCR,Northern blot,Southern blot,Taq-man probe PCR and ELISA were used to detect the effects of interfering with endogenous SIRT7 on HBV RNAs transcription,HBV core DNA expression,HBV cccDNA expression and HBV antigens secretion,respectively.(3)Transducted of SIRT7 and its enzyme activity mutant SIRT7~H187Y/S111A187Y/S111A overexpression lentivirus in HBV infected HepG2-NTCP and PHH cells,respectively.And detected a series of HBV transcription and replication related virological indicators.In this part of the study,we found that ectopic overexpressed SIRT3and SIRT7 in HBV infected cells could significantly inhibit the expression of HBV RNAs.Interference with SIRT7 resulted in markedly increase of the transcription level of HBV RNAs,the expression level of HBV core DNA and the secretion of HBV antigens in HBV infected cells.although it did not affect the expression level of HBV cccDNA,it significantly promoted the transcriptional activity of cccDNA.Overexpression of SIRT7significantly inhibited HBV RNAs transcription,HBV core DNA expression,HBV antigens secretion and HBV cccDNA transcription activity,but its enzyme activity mutant SIRT7~H187Y/S111A187Y/S111A had no significant effect on the above virological indicators.These results suggested that SIRT7 may depend on its enzyme activity to inhibit the transcriptional activity of cccDNA,thus inhibiting HBV replication and transcription.It has been reported that SIRT7 contains multiple enzymatic activities,includingcatalyzeH3K18deacetylationandcatalyzeH3K122desuccinylation.We asked whether SIRT7 could deacetylation or desuccinylation of histones on HBV cccDNA microchromosomes,thereby inhibiting the transcriptional activity of cccDNA?Based on the above conjecture and previous results,we elaborated the molecular mechanism of SIRT7 regulating HBV replicationand transcription:(1)Cell immunofluorescence assay was used to analyze the localization of SIRT7.(2)CHIP assay was used to analyze whether SIRT7 regulated the acetylation level of H3/H4 bound to cccDNA.(3)CHIP-seq assay was used to analyze whether there was succinylation modification on HBV cccDNA,and CHIP assay was used to analyze whether SIRT7 regulated the succinylation level of histone on cccDNA.(4)CHIP assay was used to analyze whether SIRT7 affected the binding of methylase and RNA polymerase II to cccDNA.(5)IP,CHIP and immunofluorescence assay were used to analyze whether SIRT7 binding to cccDNA depended on viral proteins(HBc,HBx).Our results showed that SIRT7 mainly located in the nucleolus,but there was also a large number of distributions in the nucleoplasm.CHIP assay showed that SIRT7 could deacetylate of H3 on cccDNA,but could not deacetylate of H3K18.CHIP-seq assay showed that H3K122succinylationmodificationexistedonHBVcccDNA microchromosome,and CHIP assay showed that SIRT7 could obviously desuccinate H3K122 on cccDNA.After overexpressing of SIRT7,methylase SUV39H1 bound to cccDNA increased and SETD2 bound to cccDNA decreased,which led to the increase of inhibitory marker H3K9me3 and the decrease of activation marker H3K36me3 on cccDNA.The epigenetic changes caused the decrease of RNA polymerase II bind to cccDNA,and the cccDNA transcriptional activity was suppressed.At the same time,we found that SIRT7 could interact with HBc,and SIRT7might binding to cccDNA under the HBc guidance.In this study,we found a new histone modification on the cccDNA microchromosome:histone succinylation.SIRT7,as a histone desuccinylase,could epigenetic restrict HBV cccDNA transcription activity through cooperatively with histone methyltransferase.Our study increased the understanding of histone modifications on cccDNA minichromosome and might contribute to the development of new strategy in the treatment of HBV infection.