Human SCARB2-mediated entry and endocytosis of EV71

Abstract

Enterovirus (EV) 71 infection is known to cause hand-foot-and-mouth disease (HFMD) and in severe cases, induces neurological disorders culminating in fatality. An outbreak of EV71 in South East Asia in 1997 affected over 120,000 people and caused neurological disorders in a few individuals. The control of EV71 infection through public health interventions remains minimal and treatments are only symptomatic. Recently, human scavenger receptor class B, member 2 (SCARB2) has been reported to be a cellular receptor of EV71. We expressed human SCARB2 gene in NIH3T3 cells (3T3-SCARB2) to study the mechanisms of EV71 entry and infection. We demonstrated that human SCARB2 serves as a cellular receptor for EV71 entry. Disruption of expression of SCARB2 using siRNAs can interfere EV71 infection and subsequent inhibit the expression of viral capsid proteins in RD and 3T3-SCARB2 but not Vero cells. SiRNAs specific to clathrin or dynamin or chemical inhibitor of clathrin-mediated endocytosis were all capable of interfering with the entry of EV71 into 3T3-SCARB2 cells. On the other hand, caveolin specific siRNA or inhibitors of caveolae-mediated endocytosis had no effect, confirming that only clathrin-mediated pathway was involved in EV71 infection. Endocytosis of EV71 was also found to be pH-dependent requiring endosomal acidification and also required intact membrane cholesterol. In summary, the mechanism of EV71 entry through SCARB2 as the receptor for attachment, and its cellular entry is through a clathrin-mediated and pH-dependent endocytic pathway. This study on the receptor and endocytic mechanisms of EV71 infection is useful for the development of effective medications and prophylactic treatment against the enterovirus.

Figure 1. Expression of SCARB2 in RD, Vero, NIH3T3, and 3T3-SCARB2 cells.

(A) Lysates prepared from the tested cells were analyzed by immunoblotting using a polyclonal rabbit anti-SCARB2 antiserum. The SCARB2 protein has molecular size of 80 KDa. The internal cellular β-actin was detected by blotting the stripped membrane with monoclonal anti-β-actin antibody which corresponded to 44 KDa products. (B) The tested cells were incubated with biotinylated anti-SCARB2 antiserum (filled curve) or without antiserum (empty curve) prior fixed by methanol and then stained with FITC-conjugated avidin. Stained cells were run on a FACScan flow cytometer and analyzed by using CellQuest software (Becton Dickinson Immunocytometry System).

Figure 2. Susceptibility of SCARB2-expressing cells infected with EV71.

(A) Four different cells, NIH3T3, 3T3-SCARB2, RD, and Vero cells, were infected with two different MOI of EV71 and then cultured for 48 hours. After incubation, cell lysates were prepared for western blot using anti-capsid MAB979 antibody. (B) Immune plaque forming assay was performed in four different cells infected with 10-fold serial diluted 10⁸ pfu/ml stocks of EV71 as described in the Materials and Methods. The immuno-stained plaques were counted and photography was taken. (C) NIH3T3 or SCARB2-expressing NIH3T3, RD, and Vero cells cultured in 96-well plate were inoculated with various MOI of EV71 for one hour at room temperature. After incubation, cells were washed three times with PBS and then cultured for another 24 hours before fixed by methanol. EV71 capsid protein in the cells was detected by ELISA assay as described in the materials and methods.

Figure 3. Association of EV7 with SCARB2 in 3T3-SCARB2 cells.

Lysates prepared from the 3T3-SCARB2 and NIH3T3 cells were mixed individually with EV71 particles and then precipitated by protein G-conjugated anti-SCARB2 antibody as described in the Materials and Methods. Lysate-viruses mixtures without treatment of co-immunoprecipitation were also prepared. Following the co-precipitates and lysate-virus mixtures were individually subjected to western blot against associated EV71 capsid protein by blotting with MAB979 antibody (the top panel). The same membrane was stripped and re-blotted with ant-SCARB2 antibody.

Figure 4. Infection of EV71 in RD cells but not Vero cells is SCARB2-dependent.

(A) RD cells, and (B) Vero cells were transfected with two different amount of SCARB2 siRNA and then incubated for 24 or 48 hours before MOI = 0.04 of EV71 infection. Lysates prepared at 24 or 48 hours after siRNA transfection were subjected to western blot using anti-SCARB2 antibody (the tope panel). Cell lysates were extracted at 24 or 48 hours after EV71 infection and subjected to detect the synthetic viral capsid protein by western blot using MAB979 antibody (the lower panel). The internal control of cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Corresponding changes in SCARB2 protein levels compared to the expression levels in 48 hours transfection of the cells with 100 pmoles control siRNA (Mock) as 1.0 were quantified using Image-Pro Plus 6.0 software. Data represent one of two independent experiments.

Figure 5. EV71 infection is impacted by clathrin specific inhibitors.

(A) 3T3-SCARB2 cells were pretreated with different doses of CPZ for one hour prior to the addition of MOI = 0.04 of EV71. (B) 50 or 100 pmoles of siRNA specific to clathrin or 100 pmoles of control siRNA (MOCK) were transfected to 3T3-SCARB2 cells as following the treatment protocol as Figure 4 described above. After 24 or 48 hours of transfection, cells were following lysed and detected the content of clathrin light chain B (CLTB) by Western blot using anti-clathrin antibody. Relative expression of siRNA-targeting genes shown in the top panel represented the relative CLTB protein levels of the cells transfected with specific siRNA, compared to the expression in 48 hours transfection of the cells with 100 pmoles control siRNA (Mock) as 1.0 was shown. Lysates were also prepared after 24 or 48 hours of EV71 infection and subjected to analyze the expression of synthetic EV71 capsid protein by western blot using MAB979 antibody. The result was shown in the lower panel. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.

Figure 6. EV71 infection is blocked by dynamin-2 specific siRNA.

3T3-SCARB2 cells were treated with 100 and 200 pmoles of dynamin-2 siRNA or 200 pmole of control siRNA (MOCK) prior to the infection of EV71 as following the treatment protocol as Figure 4 described above. After 24 or 48 hours of transfection, the detection of dynamin-2 by western blot using anti-dynamin antibody was conducted, or following the transfectants were infected with MOI = 0.04 of EV71 and incubated for 24 or 48 hours before lysate preparation. Expression of dynamin-2 in the lysates was shown in the top panel. Relative dynamin-2 protein levels in specific siRNA-treated cells compared to the levels in 48 hours of cells transfected with 200 pmoles control siRNA (Mock) as 1.0 was shown. The lower panel showed the expression of EV71 capsid protein which was detected by western blotting of MAB979 antibody. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.

Figure 7. EV71 entry was not affected by a tyrosine kinase inhibitor treatment.

(A) Pretreatment of 3T3-SCARB2 cells with 20, 10, and 5 µg/mL of genestin for one hour before MOI = 0.04 of EV71 infection was conducted. After one hour infection, the cells were incubated for 24 hours. Cells lysate were further prepared and subjected to western blot using MAB979 antibody to detect EV71 capsid protein. The internal cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. (B) As a positive control of genistein inhibitory effect, A549 cells were pre-treated with vehicle or 20 µg/mL of genistein for one hour before infected with MOI = 0.1 of lentivirus-eGFP, and then following the cell culture until 72 hours incubation. Cells without lentivirus-eGFP infection as negative control were also included. The same field of fluorescent and visible pictures was taken under the UV-fluorescent microscopy and GFP-positive cells were counted.

Figure 8. Caveolae-independence of EV71 entry.

(A) Pretreatment of 3T3-SCARB2 cells with 2. 1, and 0.5 µg/mL of filipin was conducted as the same condition as the legend of Fig. 7A described above. Cells lysates were analyzed by western blot using MAB979 antibody to detect EV71 capsid protein. The internal cellular β-actin was also detected by blotting the same membrane. (B) Uptake of 5 µg/mL CT-B conjugated with alexa fluor 594 in 3T3-SCARB2 cells treated with 2 µg/mL of filipin or vehicle (0.1% DMSO) was conducted following the protocol described in the Materials and Methods. The same field of fluorescent and visible pictures was taken under confocal microscopy. (C) Transfection of 3T3-SCARB2 cells with 100 pmoles of caveolin (CAV-1) siRNA or control siRNA or control siRNA (Mock) prior to the infection of EV71 as following the treatment protocol as Figure 4 described above was conducted. Expression of CAV-1 in the lysates was shown in the top panel. Relative expression of CAV-1 in specific siRNA-treated cells compared to the protein levels in 48 hours of cells transfected with 100 pmoles control siRNA (Mock) as 1.0 was shown. The lower panel showed the expression of EV71 capsid protein which was detected by western blotting of MAB979 antibody. Cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody. Data represent one of two independent experiments.

Figure 9. EV71 endocytosis is endosomal pH dependent and the intact membrane cholesterol dependent.

3T3-SCARB2 cells were pretreated with various doses of (A) chloroquine and NH₄Cl, or (B) methyl β-cyclodextrin (MβCD), for one hour prior infection with MOI = 0.04 of EV71. After 24 hours incubation in the presence of drugs, cells lysate were prepared and subjected to western blot using MAB979 antibody to detect synthetic EV71 capsid protein. The internal cellular β-actin was detected by blotting the same membrane with monoclonal anti-β-actin antibody.