Mechanism Of Infectious Entry Of Enterovirus 71 Into Human Enterocytes

Enterovirus 71(EV71)is a positive-strand RNA virus within the enterovirus genus of the family picornaviridae.It is one of the primary pathogenic agents that cause hand,foot,and mouth disease(HFMD)mainly among young children.EV71 can cause a wide spectrum of typical clinical manifestation,including fever,skin eruptions on hands and feet,and vesicles in the mouth.As the disease progresses,pulmonary edema and neurogenic failure can be observed in some cases.To date,there are no effective treatment strategies to combat EV71 infection.A better understanding of the cellular factors that influence virus infection would pave the way for the development of new drugs against this virus.It is generally assumed that,to infect host cells,viruses need to proceed through a multistep replication cycle including binding,entry,uncoating,synthesis of viral genome and proteins,assemble,and release.In the process of cell entry,receptors binding and virus internalization are considered to be the initial steps.Most viruses usually take advantage of cellular receptors and endocytosis to enter target cells.Previous studies have demonstrated that EV71 uses human scavenger receptor class B member 2(SCARB2)and P-selectin glycoprotein ligand-1(PSGL-1)as its functional receptors.These receptors serve to induce cellular signaling or promote virus endocytosis.Viruses potentially use several different endocytic entry mechanisms in different cells or even a single cell.Of the endocytic pathways taken by viruses,the clathrin-mediated pathway and caveolae-dependent uptake are the most commonly observed endocytosis pathways in the process of virus internalization.In addition,it is reported that several clathrin-and caveolae-independent pathways can be employed to mediate the infectious entry of virus particles.As one of enteroviruses,EV71 is often transmitted via the fecal-oral route.By this route,the intestinal tract is considered as the first barrier against pathogens.Animal experiments have showed that EV71 antigen could be first detected in small intestine of mouse which is orally vaccinated with EV71,indicating that EV71 might initially replicate in the small intestine,followed by viraemia and infections of various organs.Up to date,the mechanism of EV71 penetrating human intestinal cells remains obscure.The objective of this study is to investigate how EV71 exploits cellular factors to enter human enterocytes,and whether viral entry can increase the permeability of gastrointestinal epithelial cells and disrupts the tight junctions that interlock adjacent cells.The human colon carcinoma cell line(Caco-2)was utilized as the target cell for EV71 infection.The receptor of EV71 was identified by performing western blotting,quantitative real time PCR and RNAi analysis.A robust high-throughput si RNA screening assay was performed to identify cellular factors involved in EV71 infection in Caco-2 cells.In combination with the RNAi screen analysis,small interfering RNAs,specific chemical inhibitors,and dominant-negative mutant were used to explore the internalization mechanism and signaling transduction pathways in the process of EV71 entry into Caco-2 cells.Furthermore,to explore the effects of EV71 entry on the permeability of intestinal epithelium and the structure of tight junctions that interlock adjacent cells,Caco-2 monolayer was constructed,which is considered as the best in vitro model of human small intestinal epithelium containing brush borders.1.SCARB2 serves as a functional receptor of EV71 infection in Caco-2 cellsAs controls,RD cells were used to evaluate the level of SCARB2 expression,while THP-1 cells were utilized to evaluate the level of PSGL-1 expression.The levels of SCARB2 and PSGL-1 expression were tested by western blotting and q RT-PCR.The results showed that PSGL-1 is not expressed in Caco-2 cells,whereas SCARB2 is highly expressed in Caco-2 cells equal to in RD cells.Furthermore,silencing of SCARB2 with a specific si RNA significantly inhibited EV71 infection and entry.These data suggest that EV71 exploit SCARB2 as its receptor to infect Caco-2 cells.2.Identification of membrane-trafficking factors involved in EV71 infection using si RNA silencing screenA si RNA library was used to silence the expression of 140 human membrane trafficking genes,followed by infection with EV71.Viruses infection were detected by immunofluorescent and the results were obtained by image acquisition and data analysis.Among the 140 genes,silencing of 20 ones could decrease EV71 infectivity by more than 50%.These host factors involved in EV71 infection include DNM2,HGS,TSG101,VPS4 A,VPS36,PIK3 CG,BECN1,ARPC5,ENTH,EPN2,AP2B1,COPA,GAF1,GIT1,VAPB,VCP,NSF,RAB3 D,RAB7B,and RAB7L1.3.Infection of EV71 in Caco-2 cells does not depend on clathrin-mediated endocytosis and caveolae-mediated endocytosisSilencing of several key factors(CLTA,CLTB,CLTC and AP2A1)involved in clathrin-mediated pathway could not result in a reduction in EV71 infection in Caco-2 cells.Pretreatment of Caco-2 cells with chlorpromazine,which can specifically inhibit clathrin recycling and formation of clathrin coated-vesicle,did not exhibit an inhibitory effect on reduction in EV71 infection.Expression of EPS15 DN also did not result in a reduction of EV71 infection.Similarly,knockdown of several key factors(CAV1,CAV2,and CAV3)involved in caveolae-mediated pathway could not decrease EV71 infectivity.Filipin III pretreatment and expression of caveolin-1 DN(CAV1 DN)in Caco-2 cells successfully blocked caveolae-dependent endocytosis,whereas could not inhibit EV71 infection.Thus,these data suggest that EV71 infection in Caco-2 cells is clathrin-independent and caveolae-independent.4.EV71 infection is sensitive to chemical inhibitor EIPA but does not involve macropinocytosisEIPA is known as an important inhibitor of the sodium/hydrogen exchanger.Pretreatment of Caco-2 cells with EIPA not only effectively blocked micropinocytosis but also inhibited EV71 infection and cell entry.However,silencing of CDC42,RAC1,PKC,and PAK1 which are several key factors involved in micropinocytosis displayed no effect on EV71 infectivity.Pretreatment with NSC23766(a RAC1 inhibitor),IPA-3(a PAK1 inhibitor),and bisindolylmaleimide(a PKC inhibitor)also did not decrease EV71 infectivity.Taken together,these data indicate that while EV71 infection is EIPA-sensitive,it does not occur via micropinocytosis.5.Dynamin 2 is a prerequisite for EV71 entryTransfection of dynamin 2 si RNA in Caco-2 cells markedly inhibited EV71 infection and entry.Pretreatment with dynasore,a GTPase pharmacological inhibitor that can rapidly and reversibly inhibit dynamin,exhibited a significant inhibitory effect on EV71 infection and entry.Expression of dynamin 2 DN in Caco-2 cells also could result in a significant decrease in EV71 infectivity.Caco-2 cells were treated with dynasore at the indicated times during EV71 infection,and the results showed that dynasore could mainly inhibit EV71 infection in the early step of viral entry.Together,these data indicate that dynamin 2 is a prerequisite for EV71 internalization,and it mainly plays a role in the early stage of viral entry process.6.Cholesterol is required for EV71 infection and cell entryPretreatment of Caco-2 cells with M?CD,which is a cyclic oligosaccharide and has a function of extracting the cholesterol from the plasma membrane rapidly,resulted in a significant reduction of EV71 infection and entry.After recovery of physiological level of cellular cholesterol by adding cholesterol to M?CD-treated Caco-2 cells,EV71 infectivity and entry efficiency were also rolled back.EV71 infection was significantly inhibited when M?CD was added to Caco-2 cells at the early time points during infection.Therefore,the data indicate that cholesterol participant in the process of EV71 infection and cell entry,and it mainly plays a role in the early stage of EV71 entry.7.Cell entry of EV71 into Caco-2 cells involves the ESCRT components HGS,VPS36,VPS37 A,VPS37C,CHMP2 A and ESCRT-associated ATPase VPS4ATransfection of Caco-2 cells with specific si RNAs against several factors associated with ESCRT(endosomal sorting complex required for transport)such as HGS,VPS36,VPS37 A,VPS37C,CHMP2 A,and VPS4 A could significantly inhibit EV71 infection and entry,suggesting that ESCRT play a role in EV71 entry into Caco-2.The formation of a functional MVB requires the biosynthesis of the membrane lipid PI3 P by PIK3 CG,and PI3 P is crucial for endosomal localization of the ESCRT complexes.Inhibition of the function of PIK3 CG by si RNA or specific chemical inhibitor(wortmannin)significantly blocked EV71 infection and entry.Taken together,these data indicate that EV71 entry into Caco-2 cells depend on ESCRT,and with the participation of ESCRT machinery,virus particles might be delivered into MVB.8.PKA and SHP-2 are required for EV71 infection and entryBy screening a various of kinase inhibitors for their effect(s)on EV71 infection,H-89(PKA inhibitor)and NSC-87877(SHP-2 inhibitor)were identified to markedly reduce EV71 infectivity and entry.High concentration of NSC-87877 almost completely blocked EV71 infection and cell entry.To specifically identify which stage of EV71 entry was affected by NSC-87877 or H-89 treatment,Caco-2 cells were incubated with NSC-87877 or H-89 prior to the start of infection or at the indicated times after infection with EV71.The data showed that EV71 infection was significantly inhibited only when H-89 was added before or in very early stage of virus entry while NSC-87877 could inhibit EV71 infection only when it was added to cells in the late stage of virus entry.Therefore,these data suggest that both of PKA and SHP-2 are required for EV71 infection and cell entry,and PKA play a role in the initial stage of virus infection,while SHP-2 is involved in the late stage of viral entry.9.EV71 entry disrupts the tight junctions and increases the permeability of Caco-2 monolayer.Caco-2 cell monolayer was cultured in transwell inserts,and the status of the permeability barrier is validated by measuring measuring transepithelial electrical resistance(TEER)and immunostaining of tight junctions.Caco-2 cell monolayer was incubated with EV71 for 2 h,followed by measurement of TEER and staining of ZO-1.After this treatment,a decrease by more than 50% in TEER and rearrangement of tight junction were observed,indicating that the infectious entry of EV71 into the differentiated Caco-2 cell monolayer can change the arrangement of tight junction and increase permeability of monolayer.In conclusion,this work preliminarily elucidates the molecular and cellular mechanism of EV71 invading human intestine cells.The information obtained not only provides further insight into the life cycle of EV71 infection but also provides new targets for the design of antiviral drugs.