Gag recruits host proteins Staufen1 and IMP1 while tethered to mRNA. HeLa cells were co-transfected with mRNA-reporter pGL3-basic/site, MS2-VN, MS2-Gag or MS2-Staufen1 and hGag-VC, Staufen1-VC or IMP-VC as indicated. TriFC analysis was performed at 24 and 40 hr post-transfection. MS2 RNA-tethered hGag recruits Gag (A), Staufen1 (B) and IMP1 (C) to generate TriFC signals in cells. MS2 RNA-tethered Staufen1 recruits hGag (D). Western blotting analysis for Staufen1, p24 (to identify Gag), and GFP (that recognizes C terminal part of Venus) and Luciferase (reporter mRNA, pGL3-basic/site expressed Luciferase) confirmed expression of DNA constructs. Negative controls included transfections that omitted either the VC fusion proteins (bottom left in panels (A)-(D)) or the bridging MS2 molecule (MS2-hGag, MS2-Gag(C36S) or MS2-Staufen1; data not shown). The size bars are equal to 10 μm.