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

Using an array of biochemical, cellular, and computational methods we show that the nascent SINV structural polyprotein forms a spectrum of topological intermediates during biosynthesis, and that -1PRF is primarily driven by the translocon-mediated membrane integration of a marginally hydrophobic TM domain within the E2 protein. We also provide evidence to suggest this mechanism is likely to be conserved across the alphavirus genus. To date, the mechanistic basis of -1PRF has been generally attributed to the kinetic effects of mechanochemical forces that are generated by structural elements within the mRNA. Indeed, we do find that PRF in the SINV structural polyprotein is dependent upon the RNA stem loop downstream of the slip-site (Supplemental Fig. 6 ). Nevertheless, it is clear that the forces generated by the translocon-mediated membrane integration of TM2 dramatically enhance the frameshifting efficiency. To our knowledge, the findings reported herein constitute the first instance in which forces generated by conformational transitions in the nascent polypeptide chain have been implicated in the efficiency . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 of PRF. Though additional investigations are needed to elucidate how pulling forces in the nascent chain physically stimulate -1PRF, a causative role for tension in both the transcript and nascent chain seems plausible given that ribosomal frameshifting fundamentally arises from the movement of the tRNA with respect to the mRNA. It seems likely that cotranslational folding is one of many regulators, which include both host and viral effectors, that tune the net efficiency of PRF. This creates the potential for mechanistic diversity that could provide an evolutionary benefit for alphaviruses, as -1PRF is rendered tunable by either downstream or upstream mutations that impact the stability of the mRNA hairpin or the conformational ensemble of the nascent chain, respectively. This flexibility could also potentially provide the virus with a means of maintaining desired -1PRF levels in the presence host factors that globally regulate -1PRF through mRNA interactions. 12 It should be noted that the implications of these findings potentially extend beyond the realm of viral proteins. A wide variety of molecular transitions have been found to generate tension within the nascent chain including the folding of soluble domains near the ribosomal exit tunnel 43 and the translocon-mediated membrane integration of nascent TM domains. 40, 41 These observations suggest the tension in the nascent chain should fluctuate as the structural features emerge from the ribosome (Fig. 6A) , which may therefore provide the ribosome with a readout for the progress of cotranslational folding. In the case of the SINV structural polyprotein, the topological frustration within the nascent chain leads to the production of two competing topomers that generate distinct pulling forces on the ribosome in a manner that ultimately impacts the fidelity and processivity of translation ( Fig. 6 B & C) . Additional investigations are needed to explore the potential relevance of this type of cotranslational feedback to protein homeostasis. Indeed, interactions between the . CC-BY 4.0 International license is made available under a The copyright holder for this preprint