Visualization of viral FL RNA translation by dual RNA-Gag labeling. (A) Schematic representation of the MLV construct. MLV-ST-MS2 contains an in-frame insertion of the 24XST in Gag and the 24XMS2 in Pol. Ѱ refers to the RNA encapsidation signal, splice donor (SD, SD’) and acceptor (SA) sites are shown, as well as the start codons for Gag (ATG) and for gGag (CTG). (B) Representative images of a GFP/RFP cell expressing WT MLV-ST-MS2 acquired on a widefield microscope. Merge and maximum intensity projection (MIP) of several z-stacks are presented on the left. A cell treated with puromycin is shown in the lower panels. In the zoom insets, red arrows indicate non-translating RNA, yellow arrows indicate translating RNA, and green arrows indicate mature Gag. Scale bars are 10 and 1 µm for zoom insets. (C) Quantification of MS2 and ST spots per cell detected with Imaris in the presence or absence of puromycin. (D) Proportion of MS2 spots colocated with ST spots per cell. For all graphs, the mean and SEM are shown with n = 38 cells. The significance of differences was assessed using a nonparametric test (Mann–Whitney) (ns = nonsignificant, ****P ≤ 0.0001).