| Background and objective:Hepatitis B virus(HBV)is an unclosed circular double-stranded DNA virus.When hepatitis B virus particles invade host cells,their nucleic acids are transported into the nucleus to complete the gap to form a template covalently closed circular DNA(cccDNA),which is also the earliest replication intermediate.Under the action of antiviral drugs or host autoimmunity,HBV replication is inhibited,but HBV cccDNA is difficult to clear.Therefore,when external biological or chemical factors act on host cells,HBV replication may be enhanced by affecting host transcription activating factor of cccDNA,resulting in HBV reactivation,even serious liver damage.The process of HBV replication,packaging and secretion is complex and tightly regulated.cccDNA as a replication template,transcription produces different lengths of mRNA nucleation,reverse transcription produces offspring virus nucleic acid or encodes structural and functional proteins of the virus,and assembles offspring virions.The infectious intact hepatitis B virus particles(Dane particles)are composed of two parts:envelope(containing HBsAg,glycoprotein and cell lipid)and core particles(containing nucleic acid and core protein).During secretion,the virus particles move from the endoplasmic reticulum to the cell surface,and modify the sugar chain of HBsAg in the process of transport to increase the compatibility between the virus membrane and the cell membrane,so that the progeny virus particles can be successfully secreted outside the cell.Due to the diversity of assembly,there are a variety of progeny viral particles secreted in cells,including spherical virions containing HBsAg(including complete Dane particles and spherical particles packaged with other viral components),naked core particles,tubular particles,small particles and so on.Cisplatin is one of the commonly used chemotherapeutic drugs in anti-tumor therapy.At present,it is widely used in the treatment of a variety of solid tumors.In the process of clinical application,it is found that a large number of cases have liver damage caused by active hepatitis B virus,so cisplatin is also one of the chemotherapeutic drugs warned by FDA to have the risk of HBV reactivation.Some studies have suggested that the mechanism of HBV reactivation induced by cisplatin is related to host immunity.Cisplatin suppresses the cellular immune signal pathway and makes virus replication lack of immune surveillance,which indirectly leads to the reactivation of HBV.However,in recent years,some studies have begun to pay attention to the direct regulatory effect of drugs on virus replication by affecting host factors.For example,cisplatin can induce autophagy hollow cell formation by activating ROS/JNK,which provides a tertiary structure for HBsAg assembly to promote viral HBV assembly;cisplatin can also enhance HBV mRNA transcription and replication by up-regulating the expression of host transcription factor HNF4 αunder endoplasmic stress.These studies suggest that cisplatin plays an important role in the regulation of HBV life cycle which contributs to cisplatin-induced HBV reactivation.Although these studies suggest that cisplatin regulates the replication,assembly and secretion of HBV,the specific link it works on is still worth further study with both cell and animal models.On one hand,the mechanism of the effect of cisplatin on the assembly and secretion of HBV is not completely clear.As an important structural protein of the virus,the N-glycosylation modification of HBsAg is very important to maintain its normal secretory function.Whether cisplatin can affect the glycosylation modification of HBsAg and then affect its secretion remains unclarified.On the other hand,how cisplatin promotes virus replication remains unelucidated.As an important place for viral protein synthesis,modification and progeny virus assembly,endoplasmic reticulum(ER)would be influced by accumulated viral proteins when the secretion of HBsAg is impaired.Afterwards,it would induce endoplasmic reticulum stress,up-regulate endoplasmic reticulum stress marker protein GRp78,and activate downstream transcription factors.Therefore,whether cisplatin promotes HBV replication through affecting the secretion of HBsAg and leads to endoplasmic reticulum stress also needs further study.To sum up,this study is to be carried out through three parts.First of all,we intend to use hydrodynamic HBV replicating mouse(Hydrodynamic mouse)model,HBV transgenic mouse model and HBV replicating hepatoma cell model to study the effects of cisplatin on HBV replication and HBsAg assembly and secretion in vitro and in vivo.Secondly,we will deeply study the mechanism of cisplatin affecting HBsAg secretion.By screening the key factors affecting HBsAg glycosylation modification,we will further clarify whether cisplatin affects HBsAg glycosylation modification through this key factor,thus regulating its packaging and secretion.Finally,we will use the above models to further study the changes of endoplasmic reticulum stress in host cells and the host transcription factors that enhance HBV transcription in stress-related pathways,and to explore the mechanism of cisplatin promoting HBV replication.The purpose of this study is to clarify the role and possible mechanism of cisplatin in HBV reactivation,so as to identify the key host factors that cisplatin may depend on in HBV reactivation,so as to facilitate the development of targeted interventions and provide a new management strategy for the safe application of cisplatin-containing chemotherapy in patients with chronic HBV infection.Materials and Methods:1.To study the effects of cisplatin on HBV replication and packaging and secretion of progeny virus particles.The hydrodynamic HBV replication mouse model was treated with cisplatin.The indexes of HBcAg and HBsAg in liver tissue of mice were detected by immunohistochemical(IHC)to observe the effect of cisplatin on the levels of HBcAg and HBsAg in liver,and the effect of cisplatin on the level of HBsAg in serum was detected by ELISA,so as to understand the effect of cisplatin on virus replication and secretion in vivo model.HBV transgenic mice were treated with cisplatin.The levels of HBcAg and HBsAg in liver tissue of mice were detected by IHC,and the level of HBsAg in peripheral blood of mice was detected by ELISA to further clarify the effect of cisplatin on HBV replication and secretion.The level of HBeAg in peripheral blood of mice was further detected by ELISA to verify whether the effect of cisplatin on virus secretion was mainly related to HBsAg.On the basis of preorder study,HBV replication hepatoma cell model was treated with cisplatin.The levels of extracellular HBV virus particles containing HBsAg and HBV replication intermediates were detected by antibody enzyme-linked immunosorbent assay((EIA))and DNA hybridization(Southern blot)to further clarify whether cisplatin increased the level of intracellular replication intermediates and mainly affected the assembly and secretion of HBsAg spherical particles.2.To study the mechanism of cisplatin affecting the secretion of progeny viral particles by affecting HBsAg glycosylation.As an important structural protein of the virus,HBsAg has glycosylation modification sites.The N-glycosylation level of HBsAg is very important to maintain the normal biological function of HBsAg.We speculate that cisplatin may affect the glycosylation of HBsAg,thus affecting its packaging secretion.First of all,using HBV replicative hepatoma cells,after cisplatin treatment,the changes of 10 glycosyltransferases were detected by PCR,and the key glycosylation enzymes that may be affected by cisplatin were screened and studied.Then,using HBV replicating hepatoma cell model and hydrodynamic HBV replicating mouse model,after cisplatin treatment,the effect of cisplatin on glycosylase level was detected by Western Blot and IHC respectively to study the regulation of cisplatin on glycosylase.Then,the sublocalization of the selected glycosylase was studied by immunofluorescence confocal study,and its correlation with the key organelles modified by viral protein synthesis was analyzed.The interaction between HBsAg and the selected glycosylase was studied by co-immunoprecipitation to further clarify whether the screened glycosylase was related to the modification of HBsAg.Finally,the over expression plasmid was used to up-regulate the level of selected glycosylases in HBV replicating hepatoma cells,and chemical inhibitors were used to inhibit the glycosylation of HBsAg.Western blotting was used to detect the glycosylation level of HBsAg,EI A was used to detect the level of extracellular HBsAg spherical particles,and DNA blotting was used to detect the level of replication intermediates in cells.To further confirm the effect of selected glycosylase on the glycosylation of HBsAg and the packaging secretion of HBsAg spherical particles,and to explore the effect of packaging secretion of HBsAg spherical particles on the level of HBV replication.3.To study the mechanism of cisplatin affecting HBV replication.As an important place for virus protein synthesis and modification,virus assembly and secretion,endoplasmic reticulum is easily affected by abnormal virus secretion.Therefore,abnormal secretion of HBsAg may regulate host factors through endoplasmic reticulum stress,thus affecting HBV replication.First of all,the expression of endoplasmic reticulum stress marker protein GRp78 in HBV replicating hepatoma cells was detected by Western blotting to observe the effect of cisplatin treatment on endoplasmic reticulum stress.Then,in the cells without HBV replication,we observed the effect of cisplatin treatment on the level of GRp78 in cells,and studied whether the effect of cisplatin treatment on endoplasmic reticulum stress was caused by direct action or by affecting HBsAg.Secondly,using HBV replicative hepatoma cells,Western blotting was used to detect the expression of host transcription factors PGC-1 α and HNF4 α downstream of GRp78,which can enhance HBV transcription,to understand whether endoplasmic reticulum stress can up-regulate the level of host transcription factors that enhance HBV transcription.Finally,using HBV transgenic mouse model,after cisplatin treatment,the levels of glycosylase,GRp78,PGC-1 α and HNF4 α in liver tissue were detected by Western blotting,and the levels of glycosylase,HBsAg,GRp78,PGC-1 α,HNF4 α and HBcAg in liver tissue were detected by IHC.To verify whether cisplatin affects HBsAg glycosylation and packaging secretion by inhibiting selected glycosylases,resulting in endoplasmic reticulum stress caused by intracellular accumulation of HBsAg,upregulation of GRp78,and further up-regulation of PGC-1α and HNF4α levels,and finally enhanced HBV replication.Result:1.Cisplatin promotes HBV replication,but may affect the packaging and secretion of progeny viral particles.Firstly,we used hydrodynamic HBV replication mouse model to verify the"reactivation effect of cisplatin on HBV" found in clinical diagnosis and treatment.The results of IHC showed that the expression levels of HBcAg and HBsAg in liver tissues of mice treated with cisplatin were up-regulated compared with those of mice without cisplatin treatment,suggesting that cisplatin enhanced the replication of HBV in the liver.Although IHC results showed that the level of HBsAg in liver tissue of mice treated with cisplatin also increased,because the level of HBsAg in tissue was greatly affected by virus secretion,we detected the level of HBsAg in peripheral blood of mice by ELISA.The results showed that there was no significant change in peripheral blood HBsAg.It is suggested that although cisplatin promotes virus replication,it may affect the secretion of HBsAg.In order to further confirm that the effect of cisplatin on the secretion of HBsAg comes from the changes of HBsAg itself,rather than the secretory dysfunction of cells,and to eliminate the effect of the model,we used cisplatin to treat HBV transgenic mice and detected two secretory proteins:HBeAg and HBsAg in serum of HBcAg,in liver tissue of mice.The results showed that after cisplatin treatment,with the activation of HBV replication and the increase of HBcAg,the level of serum HBeAg increased significantly,but there was no significant change in HBsAg.It is suggested that cisplatin may affect the secretion of HBsAg,but not HBeAg,and cisplatin does not affect the secretory function of host cells.Then,we verify it in the cell model.Virus particles containing HBsAg in peripheral blood include spherical particles(including intact offspring virus particles),tubular particles and small particles.ELISA can detect all three kinds of particles.Only spherical particles were detected by EIA method.In order to further clarify whether cisplatin affects viral replication and what kind of HBsAg particles may affect the secretion of HBV particles,we used a HBV replicating hepatoma cell model to study the effects of cisplatin on HBV replication and viral particle secretion.The results showed that after cisplatin treatment,the level of replication intermediates in cells increased,while the secretion of spherical HBsAg particles in the supernatant decreased.It is suggested that cisplatin promotes HBV replication,but affects the packaging and secretion of spherical viral particles containing HBsAg.2.Cisplatin affected the N-glycosylation level of HBsAg by inhibiting RPN2 and affected the packaging and secretion of HBsAg spherical particles.Our structural prediction shows that there are glycosylation modification sites in HBsAg.Previous studies have also proved that the level of N-glycosylation is very important to maintain the normal biological function of HBsAg,promote its packaging offspring virus,form spherical particles and secrete cells.We speculate that cisplatin may affect the glycosylation of HBsAg,thus affecting its packaging secretion.We first detected the changes of 10 glycosyltransferases in HBV replicative hepatoma cells treated with cisplatin by PCR.The results showed that the mRNA of RPN2 was related to the concentration of cisplatin.The mRNA expression level of RPN2 decreased with the increase of cisplatin concentration.It is suggested that cisplatin can inhibit the mRNA expression of RPN2 and affect the transcription of RPN2.Then,we used HBV replicative hepatoma cell model and hydrodynamic HBV replicative mouse model to detect the effect of cisplatin on the level of RPN2 in HBV replicative hepatoma cells and mouse hepatocytes.The results showed that the level of RPN2 in HBV replicative hepatoma cells and mouse hepatocytes decreased significantly after cisplatin treatment,suggesting that cisplatin could inhibit the expression of RPN2.In order to further study whether RPN2 is the key enzyme affecting HBsAg,we observed the cellular localization of RPN2 by immunofluorescence confocal microscopy and found that RPN2 was clustered in endoplasmic reticulum(ER),which is the key site of viral protein synthesis.Further co-immunoprecipitation results also showed that there was interaction between HBsAg and RPN2.It is suggested that RPN2 may be the key enzyme that regulates the glycosylation of HBsAg and affects its packaging and secretion.In order to confirm the role of RPN2,we used overexpression plasmids to upregulate the level of RPN2 in HBV replicative hepatoma cells,and found that after overexpression of RPN2,the glycosylation level of HBsAg increased and extracellular HBsAg spherical particles increased,while when chemical inhibitors were used to inhibit the glycosylation of HBsAg,the secretion of HBsAg spherical particles decreased even though RPN2 was still in a state of high expression.It is suggested that RPN2 promotes the packaging and secretion of HBsAg spherical particles by promoting the glycosylation of HBsAg.Interestingly,we found that the level of replication intermediates in cells did not change significantly after RPN2 overexpression,if it did not affect the glycosylation of HBsAg,but increased when chemical inhibitors were used to inhibit HBsAg glycosylation.It is suggested that the enhancement of intracellular HBV replication in cisplatin-treated cells and mice may be related to the regulation of HBsAg glycosylation,which is worthy of further exploration.3.Cisplatin impact HBsAg secretion and activates virus replication through endoplasmic reticulum stress response.In the previous study,we found that the levels of HBV replication intermediates and core protein expression were significantly increased after cisplatin treatment.The mechanism may be related to the secretion disturbance caused by the regulation of HBsAg glycosylation.The deficiency of HBsAg secretion leads to a large number of aggregation in the endoplasmic reticulum.Therefore,we first detected the expression level of endoplasmic reticulum stress marker protein and GRp78 in HBV replicating hepatoma cells to observe the effect of cisplatin treatment on endoplasmic reticulum stress.The results showed that cisplatin treatment could increase the expression level of GRp78 and cause endoplasmic reticulum stress.However,in cells without HBV replication,cisplatin treatment did not affect intracellular GRp78 levels,suggesting that the effect of cisplatin treatment on endoplasmic reticulum stress is achieved by affecting HBsAg.Next,we detected the expression of PGC-1α and HNF4α,which are the core factors related to HBV transcription and replication,downstream of GRp78.We found that when cisplatin induced the increase of GRp78 expression,the expression of PGC1α and HNF4a also increased accordingly.Conclusion:1.Cisplatin can enhance HBV replication in cells and reduce the secretion of extracellular HBsAg spherical particles.2.N-Glycosylation is very important for HBsAg to maintain its normal biological function.RPN2 is an important protein catalyzing the glycosylation of HBsAg.Cisplatin affects the glycosylation of HBsAg by inhibiting the level of RPN2.3.Cisplatin induces endoplasmic reticulum stress and induces endoplasmic reticulum stress response by inhibiting HBsAg glycosylation leading to endoplasmic reticulum stress.GRp78,a marker of endoplasmic reticulum stress response,is upregulated andHBV transcription and replication is activated by increasing the levels of PGC-1α and HNF4α. |
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