Secretory Carrier Membrane Protein 3 Interacts with 3A Viral Protein of Enterovirus and Participates in Viral Replication

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  FIG 1. Interaction of proteins with tagged protein 3A from EV-A71-infected cells. (A) Genome organization of EV-A71 and 3A protein. Arrows indicate sites of insertion. Amino acids that encode the FLAG tag are underlined. (B) Cytopathic effect of EV-A71-3A4-FLAG recombinant virus at an MOI of 20 at 12 h postinfection (magnification, 200x). (C) Effects of EV-A71-3A4-FLAG recombinant virus on viral RNA replication and viral protein synthesis. RD cells were infected with EV-A71-3A4-FLAG virus at an MOI of 20 or were mock infected. (i) Cells were collected to detect positive-strand viral RNA or negative-strand viral RNA by slot blot at different times following viral infection. (ii and iii) Cells were collected to detect viral protein levels using Western blotting at various times points after infection. (D) Experimental strategy for the immunoaffinity assay. RD cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20. Infected-cell lysates were immunoprecipitated with control IgG or anti-FLAG antibodies, washed, and eluted with sample buffer. The sample that contained eluted proteins was then boiled, subjected to 8 to 16% SDS-PAGE, and visualized by silver staining.…

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  FIG 2. 3A protein interacts and colocalizes with SCAMP3 in EV-A71-infected cells. (A) A co-immunoprecipitation assay was carried out. RD cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20 for 12 h. Infected-cell lysates were immunoprecipitated with control IgG or anti-FLAG antibodies, and proteins that bound to the resin were analyzed by 12% SDS-PAGE, followed by immunoblotting with anti-SCAMP3 antibody. (B) Colocalization of SCAMP3 with 3A protein. RD cells were mock infected or infected with EV-A71 at an MOI of 20. At different time points postinfection, cells were fixed with formaldehyde, washed, and immunostained with antibody against SCAMP3 or 3A-FLAG. DAPI was used to stain the nucleus. Images were captured by confocal laser scanning microscopy. Scale bar, 10 µm. (C) A co-immunoprecipitation assay was carried out. SF268 cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20 for 12 h. Infected-cell lysates were immunoprecipitated with control IgG or anti-FLAG antibodies, and proteins that bound to the resin were analyzed by 12% SDS-PAGE, followed by immunoblotting with anti-SCAMP3 antibody. (D) Colocalization of SCAMP3 with 3A protein.…

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  FIG 3. Effects of SCAMP3 knockdown or knockout on EV-A71 replication. (A) Effect of SCAMP3 knockdown on EV-A71/4643 RNA levels. SF268 cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with EV-A71/4643 at an MOI of 10 2 days after transfection. Total RNA was extracted and viral RNA levels were determined by quantitative RT-qPCR. (B) Effect of SCAMP3 knockdown on EV-A71/4643 viral 3D, 3CD, and P3 protein levels. SF268 cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with EV-A71/4643 at an MOI of 10 2 days after transfection. Total cell lysates were examined by Western blotting. (C) Effect of SCAMP3 knockdown on EV-A71/4643 viral growth. SF268 cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with EV-A71 at an MOI of 10 2 days after transfection. Viruses were harvested at different time points postinfection and assayed by plaque formation with RD cells. (D) Effect of SCAMP3 knockdown on EV-A71-3A4-FLAG RNA levels. RD cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with EV-A71-3A4-FLAG at an MOI…

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  FIG 4. EV-A71 3A protein, SCAMP3, and PI4KIIIβ associate and colocalize in EV-A71-infected cells. (A) A co-immunoprecipitation assay was carried out. RD cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20 for 6 h. Infected-cell lysates were immunoprecipitated with control IgG or anti-FLAG antibodies, and proteins that bound to the resin were analyzed by 12% SDS-PAGE, followed by immunoblotting with anti-SCAMP3, anti-FLAG, anti-3D, anti-ACBD3, and anti-PI4KIIIβ antibodies. (B) RD cells were infected with EV-A71-3A4-FLAG recombinant virus at an MOI of 20, and cell lysates were collected to isolate cytosol and membrane fractions at different time points postinfection. The fractions were analyzed for viral and host proteins levels by Western blotting. (C) SCAMP3, PI4KIIIβ, and viral 3A protein were localized to RNA replication complexes in EV-A71-infected RD cells. RD cells were transfected with pEGFP-C3-SCAMP3 for 48 h. Transfected RD cells were mock infected or infected with EV-A71-3A4-FLAG at an MOI of 20. At 6 h postinfection, cells were fixed with formaldehyde, washed, and immunostained with antibody against 3A-FLAG and PI4KIIIβ. DAPI was used to stain the…

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  FIG 5. SCAMP3 affects PI4KIIIβ and PI4P recruitment for viral replication during EV-A71 infection. (A) Scramble or SCAMP3 KO RD cells were mock infected or infected with EV-A71-3A4-FLAG at an MOI of 20. At 6 h postinfection, cells were fixed with formaldehyde, washed, and immunostained with antibody against 3A-FLAG, PI4KIIIβ, and PI4P. DAPI was used to stain the nucleus. Images were captured by confocal laser scanning microscopy. (B) Quantification of the FLAG fluorescence intensity of EV-A71-infected scramble and SCAMP3 KO cells. (C) Quantification of the PI4KIIIβ fluorescence intensity of EV-A71-infected cells. PI4KIIIβ level was determined by fluorescence intensity overlap of the 3A-FLAG area of the whole cells. (D) Quantification fluorescence intensity of PI4P of EV-A71-infected cells. PI4P level was determined by fluorescence intensity overlap of the 3A-FLAG area of the whole cells. Bars represent the means from 40 cells from the experiment. Significant differences compared to EV-A71-infected scramble cells are indicated as follows: **, P < 0.01, ***, P < 0.001. (E) Western blot analysis of viral protein and SCAMP3 levels of total cell lysates. (F) Total viral RNA level was…

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  FIG 6. SCAMP3-depleted cells indicate the ERK pathway, but not AKT pathway, is involved in EV-A71 replication. SF268 cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with EV-A71 at an MOI of 20 2 days after transfection. Total cell lysates were examined by Western blotting with anti-ERK, anti-pERK, anti-AKT, anti-pATK, anti-3Dpol, anti-SCAMP3, and anti-actin antibodies.

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  FIG 7. SCAMP3 associates with 3A protein of CVB3 and enhances viral RNA replication. (A) Sequence alignment of EV-A71 and CVB3 3A proteins. (B) A co-immunoprecipitation assay was carried out. RD cells were transfected with CVB3-3A-FLAG for 2 days. Transfected cell lysates were immunoprecipitated with control IgG or anti-FLAG antibodies, and proteins bound to the resin were analyzed by 12% SDS-PAGE, followed by immunoblotting with anti-SCAMP3, anti-PI4KIIIβ, anti-ACBD3, and anti-FLAG antibodies. (C) Colocalization of SCAMP3 with EGFP-C3-CVB3 3A protein. RD cells were transfected with pEGFP-C3 or pEGFP-C3-CVB3-3A. At 48 h posttransfection, cells were fixed with formaldehyde, washed, and immunostained with antibody against SCAMP3. DAPI was used to stain the nucleus. Images were captured by confocal laser scanning microscopy. (D) Effect of SCAMP3 knockdown on CVB3 RNA levels. RD cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with CVB3 at an MOI of 0.1 2 days after transfection. Total RNA was extracted and viral RNA levels were determined by RT-qPCR. (E) Effect of SCAMP3 knockdown on CVB3 viral 3Dpol protein levels. RD cells were…

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  FIG 8. SCAMP3 does not regulate DENV2 replication. (A) Effect of SCAMP3 knockdown on DENV2 RNA levels. A549 cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with DENV2 at an MOI of 1 2 days after transfection. Total RNA was extracted at 24 h postinfection, and viral RNA levels were determined by RT-qPCR. (B) Effect of SCAMP3 knockdown on DENV2 viral NS3 protein levels. A549 cells were transfected with NC siRNA or siRNA against SCAMP3. Cells were mock infected or infected with DENV2 at an MOI of 1 2 days after transfection. Total cell lysates were examined by Western blotting. (C) Effect of SCAMP3 knockdown on DENV2 viral growth. Viruses were harvested at 24 h postinfection and assayed by plaque formation with BHK-21 cells.