Study On The Up-regulation Expression Of The Receptor SCARB2 Caused By Infection Of Enterovirus 71

Hand,foot and mouth disease(HFMD)has been widespread in the world in the past 20 years,especially in the Asia-Pacific region.In recent years,there are approximately 2 million reported HFMD cases in China each year,ranking first among all infectious diseases and showing a yearly growth trend.Although there are many kinds of pathogens of HMFD,most severe and death cases are still caused by enterovirus 71(EV71)infection.Scavenger receptor class B,member 2(SCARB2),a receptor that mediates high-density lipoprotein endocytosis,is the main receptor for EV71 infection.Due to the similarity between EV71 capsid structure and SCARB2's ligand,SCARB2 can combine with EV71 capsid surface and mediates the endocytosis of virus,thereby enabling the infection of EV71 in host cells.However,this is not the only interaction between EV71 and SCARB2.Our group has found that the infection of CVA16 virus leads to the up-regulation of SCARB2 expression in host cells.The transcriptome(mRNA and miRNA)of CVA16-infected 293 T cells was studied by second-generation sequencing.The most significant change in the transcriptome was SCARB2,with around 10 folds up-regulation.This is an interesting and significant phenomenon.Does EV71 infection also induce the increase of SCARB2 expression? What are the underlying mechanisms and significance? To answer these scientific questions,we start this study.This thesis is consist of four parts.The first part verifies the importance of SCARB2 for EV71.Down-regulation of SCARB2 expression by siRNA interference caused decreased EV71 replication after infection;while,up-regulation of SCARB2 by over-expression caused increased EV71 replication after infection.These results indicated that SCARB2 was crucial to EV71 infection.The second part was to identify whether EV71 infection caused up-regulation of SCARB2 expression.The experiment was divided into two parts,in vitro cellexperiments and in vivo animal experiments.In cell experiments,RD,HT29 and 293 cells were infected with EV71 then detected EV71 and SCARB2 expression.In animal experiments,1 day old newborn mice were intracranially inoculated with EV71,then brain tissues were collected to detect EV71 and SCARB2 expression.The results all showed that EV71 infection caused up-regulation of SCARB2 expression,which peaked at 24 h after infection,and the expression level was about 15 fold higher than that of before infection.The third part is to explore the mechanism of up-regulation of receptor SCARB2.Literature studies have shown that the up-regulation of SCARB2 occurs in some pathological conditions.For example,in the rat model of cerulein-stimulated pancreatitis,the expression of SCARB2 in the pancreas is up-regulated,and may be regulated by ERK1/2 and JNK signaling pathways.In addition,it has been reported that EV71 infection leads to the phosphorylation of ERK1/2 and JNK,and the activation of thee signaling pathways.In this context,we verified the up-regulation mechanism of the receptor SCARB2.Using ERK1/2 and JNK signaling pathway inhibitors,we observed that SCARB2 up-regulation caused by EV71 infection could be inhibited in cell experiments.These results preliminarily proved that the mechanisms of SCARB2 up-regulation caused by EV71 infection were: EV71 infection induced the phosphorylation of ERK1/2 and JNK signaling pathways,followed by the regulation of SCARB2 expression.This study found a phenomenon that the viral infection and its receptor expression are mutually promoting.What are the roles and significance to virus and host cell? Why is such interaction mechanism developed? In the fourth part,the role and significance of receptor SCARB2 upregulation caused by EV71 infection was explored.On the one hand,we detected potential promotion roles of EV71 infection caused the up-regulation of SCARB2 to subsequent and progeny virus infection.The method is to inhibit the up-regulation of SCARB2 after EV71 infection,followed by detection of the replication level of progeny EV71 in second round infection;or followed by detection of the expression level of an EV71-Luciferase reporter pseudovirus which was used to infect cells in second round.These results showed that the level of EV71 replication or luciferase expression in the inhibitiongroup was significantly lower than that in control group.It can be seen that the up-regulation of the receptor SCARB2 caused by EV71 infection promoted the infection of progeny or subsequent viruses in the same cell.On the other hand,it can be seen from the literature that SCARB2 also plays roles in antibacterial and antiviral innate immune responses,thus the up-regulation of SCARB2 may be a cell defense response that is produced to promote anti-EV71 innate immune response.Therefore,we subsequently studied the potential roles of EV71 infection caused up-regulation of SCARB2 to innate immunity.We inhibited the up-regulation of SCARB2 after EV71 infection,then detected the expression levels of interferon-inducible genes(ISG54,ISG56)in cells.The results showed that EV71 infection in the control group induced increased transcription of ISG54 and ISG56,while inhibition of SCARB2 up-regulation induced decreased transcription of ISG54 and ISG56.It indicates that up-regulation of the receptor SCARB2 caused by EV71 infection promotes the antiviral innate immune response of the cells.This study suggests that up-regulation of SCARB2 expression has a dual promotion of both EV71 infection and host innate immunity,which may be a new mechanism of virus-host interaction.However,further studies are needed to explore the relationship among them.Taken together,the present thesis shows that SCARB2 plays an key role in EV71 infection.The expression of SCARB2 is increased after infection of EV71.We suggest that the mechanisms of the up-regulation of SCARB2 are: EV71 infection leads to phosphorylation of ERK1/2 and JNK signaling pathways,which in turn regulate SCARB2 expression.The up-regulation of SCARB2 caused by EV71 infection promotes the infection of subsequent and progeny virus in same cells,as well as the anti-viral innate immune response of host cells.This thesis provides a novel insight into the study of the interaction between virus and host.