Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

Haley R. Harrington; Matthew H. Zimmer; Laura M. Chamness; Veronica Nash; Wesley D. Penn; Thomas F. Miller; Suchetana Mukhopadhyay; Jonathan P. Schlebach

To determine whether the cotranslational membrane integration of TM2 impacts translational recoding, we measured the effects of these substitutions on ribosomal frameshifting. PRF is most commonly measured using dual-luciferase reporters, which fuse luciferase domains to the 5' (renilla luciferase, 0-frame) and 3' (firefly luciferase, -1 frame) of the gene of interest. The activity of firefly luciferase serves as a reporter for -1PRF and is normalized relative to translational efficiency based on the activity of renilla luciferase. Current versions of these reporter constructs contain self-cleaving 2A segments that release these luciferase domains from the polypeptide of interest. 39 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/790444 doi: bioRxiv preprint preserve the integrity of the signal peptide, we generated a series of reporter constructs in which translation begins at the native E3 signal peptide and continues until the ribosome reaches a fluorescent mKate fusion domain that is encoded in the -1 reading frame downstream from the PRF site (Supplemental Fig. 4 ). To control for variations in transfection efficiency at the singlecell level, we included a downstream IRES cassette that drives the bicistronic expression of GFP from the reporter transcript. Reporter constructs encoding TM2 variants of the polyprotein were expressed in HEK293T cells, and cellular mKate intensities were quantitatively compared across cells within a discrete range of IRES-GFP intensities by flow cytometry (Supplemental Fig. 5 ).