Translating FL RNAs at the ER produced gGag proteins. (A) Schematic representation of the MLV mutants. MLV-ΔgGag-ΔNC construct contains a 4-nt deletion (∆364–367) that prevents the translation of gGag and the deletion of the NC domain. MLV-gGag-∆NC construct lacks the AUG_Gag, preventing the translation of Gag and deletion of the NC domain. (B) Representative image of cell expressing the MLV-∆NC, MLV-ΔgGag-ΔNC, and MLV-gGag-∆NC constructs. Images were acquired on confocal microscope. In zooms, the yellow and white arrows show the translating and non-translating RNA associated with the ER, respectively. A single z-plane is presented, and scale bars are 10 and 2 µm for insets. (C) Percentage of ER-associated RNA expressed as a fold change from the mean value of ∆NC. The graph shows the mean ± SEM, n = 40 cells for MLV-ΔNC and NV-MS2, and n ≥ 26 for MLV-ΔgGag-ΔNC and MLV-gGag-∆NC. *P ≤ 0.05 and ****P ≤ 0.0001 (Mann–Whitney test).