Picornavirus 2A protease regulates stress granule formation to facilitate viral translation
Fig 2
2A induces aSGs by cleaving eIF4GI.
(A-C) Effects of protease activity of 2A on aSG formation. HeLa cells were transiently transfected with empty vector, Myc-tagged 2A, or 2AC110S for 24 h and then subjected to WB; GAPDH was the sample loading control. Arrow indicates the N-terminal cleavage products of eIF4G (A). IF assay of aSG formation in 2AC110S-expressing cells (B) and quantitative analysis of the 2A- (in S2B Fig) or 2AC110S-expressing cells with aSGs in B. N.D., not detected. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001 (C). (D-F) Effects of cleavage of eIF4GI on aSG formation. HeLa cells were transfected with HA-tagged eIF4GI or eIF4GI cleavage-resistant mutant (eIF4GIG689E) for 24 h, followed by EV71 infection or 2A transfection. The cleavage of eIF4GI and eIF4GIG689E was analyzed by WB assay, arrow indicates the C-terminal cleavage products of eIF4GI (D), and the formation of aSGs was viewed by IF assay (E). Quantitation analysis and comparison of eIF4GI- or eIF4GIG689E-expressing cells with aSGs (TIA-1 foci) among EV71-infected or 2A-expressing cells in E. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001 (F). “+” indicates the 2A/2AC110S-expressing or EV71-infected cells, and yellow arrows indicate the HA-tagged eIF4GI/eIF4GIG689E-expressing cells. Scale bars, 10 μm. See also S3 Fig.