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

Localization of these segments within the lumen is essential to ensure that the E3, E2, and E1 ectodomains form their native disulfides and undergo glycosylation. 15, 16, 24 Post-translational modifications are also critical for TF, which must be palmitoylated in order to reach the plasma membrane and incorporate into the viral envelope. 25 The palmitoylated cysteines in TF are positioned near the edge of a putative transmembrane (TM) domain that is found in both TF and 6K. 25, 26 Though these residues are present in both proteins, they are only palmitoylated in the context of the frameshifted polyprotein. 25 Considering palmitoylation only occurs on the cytosolic face of cellular membranes, 27 the distinct modification state of the two forms of the polyprotein is therefore suggestive of an underlying difference in their topologies. In this study, we set out to . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 gain insight into the interplay between -1PRF and the topology of the structural polyprotein. We first mapped the topology of the Sindbis virus (SINV) structural polyprotein. Our results demonstrate that the structural polyprotein forms two topological isomers. The predominant topology features two TM domains upstream of the -1PRF site, and its formation coincides with production of the 6K protein. Alternatively, the minor topology contains an additional TM domain upstream from the -1PRF site that is linked to the production of TF. Using protein engineering in conjunction with coarse-grained molecular dynamics (CGMD) simulations, we demonstrate that the efficiency of -1PRF is dependent upon the force generated by the translocon-mediated membrane integration of the extra TM domain within the minor topomer. Together, our observations highlight novel connections between the cotranslational folding, biosynthesis, and processing of the alphavirus structural polyprotein. Moreover, our findings reveal a novel mechanism that regulates the overall efficiency of -1PRF.