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

Based on these observations, we hypothesize that the translocon-mediated membrane integration of TM2 is mechanistically linked to -1PRF and the translation of the TF protein. Our model . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 suggests mutations that alter the translocon-mediated membrane integration of TM2 should have a direct impact on -1PRF (Fig. 3) . To test this hypothesis, we assessed whether mutations that alter the hydrophobicity of TM2 also influence -1PRF. Both energetic predictions and in vitro translation measurements suggest the introduction of two non-native leucine residues into TM2 (T738L & S739L, LL mutant) enhances the translocon-mediated membrane integration of TM2 (predicted ΔΔG = -1.7 kcal/ mol, Supplemental Fig. 3 ). In contrast, the introduction of two glutamate residues into TM2 (V735E & I736E, EE mutant) is predicted to increase its transfer free energy by 3.3 kcal/ mol, which should significantly reduce its membrane integration efficiency (Supplemental Fig. 3A ). The effects of the EE substitutions appear to be subtle in the context of the Lep protein (Supplemental Fig. 3B ), though this likely reflects the limited dynamic range of this translocation assay. 34, 38 Nevertheless, these results clearly show that the LL and EE mutations alter the translocon-mediated membrane integration of TM2.