FIG. 3.

Effect of hnRNP A1 and hnRNP A2 on EV71 replication. (A) Western blot to examine the expression of hnRNP A1 and hnRNP A2. hnRNP A1, hnRNP A2, or both were depleted by the siRNA knockdown technique as described in Materials and Methods. HeLa cell lysates were prepared 3 days after transfection of siRNA. Western blotting was carried out using antibodies against hnRNP A1 and hnRNP A2. The expression of actin was examined by Western blotting as a control. (B) Diagram of the bicistronic construct used in the dual-luciferase assay. (C) Effect of knockdown of hnRNP A1 and hnRNP A2 on EV71 IRES activity. HeLa cells were transfected with no siRNA, siRNA targeting hnRNP A1, siRNA targeting hnRNP A2, or siRNA targeting both. Three days after transfection, the CMV-RLuc-EV71 5′ UTR-FLuc RNA transcript was then transfected into the cells. Luciferase activity was measured 2 days after transfection. Mean values and standard errors from triplicate samples are shown in the bar graph. (D) Effect of knockdown of hnRNP A1 and hnRNP A2 on EV71 RNA synthesis. hnRNP A1 and hnRNP A2 were knocked down as described in Materials and Methods. HeLa cells were then infected with EV71 at an MOI of 2 PFU/cell and labeled from 20 to 24 h p.i. with [³H]uridine. Cell lysates were collected and resuspended in a solution of 0.6% NP-40 in PBS. After incubation at 4°C for 30 min, the nuclear fraction was removed by centrifugation, and the acid-insoluble radioactivity was determined in the supernatant (cytoplasmic) fraction. Mean values and standard errors from duplicate samples are shown. (E) Effect of knockdown of hnRNP A1 and hnRNP A2 on EV71 protein synthesis. HeLa cells were transfected with no siRNA or siRNA targeting hnRNP A1, hnRNP A2, or both. Cells were mock infected or infected with EV71 3 days after transfection. Lysates from mock-infected or EV71-infected cells were subjected to 12% SDS-PAGE. Western blotting analysis was carried out using an anti-3C antibody. (F) Replication of EV71 in hnRNP A1- and A2-depleted cells. HeLa cells treated with siRNA targeting hnRNP A1, hnRNP A2, or both were infected with EV71 at an MOI of 2 PFU/cell and incubated at 37°C. Medium was harvested 24 h p.i. and assayed for infectious virus by plaque formation on Vero cells. Mean values and standard errors from duplicate samples are shown.