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

The current model of the alphavirus structural polyprotein suggests the E2 and 6K proteins each contain two TM domains. 13, 28 However, there are two caveats to this model. First, cryo-EM structures reveal that the E2 protein only contains a single TM domain in the context of the viral envelope. 29, 30 Though it has been speculated that a second TM domain within E2 is somehow extruded from the membrane during processing, the marginal hydrophobicity of this segment also raises the possibility that it may fail to undergo translocon-mediated membrane integration in the first place. Second, the hydrophobic portion of the SINV 6K protein is only 35 residues in length, which is quite short for a segment containing two putative TM domains and a loop. These The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 ambiguous topological signals suggest this portion of the polyprotein is frustrated and could potentially form multiple topological isomers, 31 as has been suggested for the coronavirus E protein. 32 To survey the topological preferences of the E2-6K region, we scanned its sequence using a knowledge-based algorithm that predicts the energetics associated with the transfer of polypeptide segments from the translocon to the ER membrane (ΔG predictor). 33 Energetic predictions suggest only the regions corresponding to the first hydrophobic segments within the E2 (TM1) and 6K (TM3) proteins are sufficiently hydrophobic to undergo robust membrane integration (ΔG < 0 kcal/ mol, Fig. 1C ). In contrast, the translocon-mediated membrane integration of the second hydrophobic segment within E2 (TM2) is predicted to be inefficient (Fig. 1C) . To test these predictions, we measured the translocon-mediated membrane integration of each putative TM domain using a glycosylation-based translocation assay. 34 Briefly, the sequences of each putative TM domain were cloned into a chimeric leader peptidase (Lep) reporter protein. 34 Membrane integration of the putative TM segment (purple helix, Fig. 1D ) results in the modification of only a single glycosylation site in Lep, whereas the passage of the segment into the lumen results in the modification of two glycosylation sites (Fig. 1D ). Chimeric Lep proteins were produced by in vitro translation in the presence of canine rough microsomes, which contain native ER membranes and translocons. Consistent with predictions, Lep proteins containing TMs 1 & 3 acquire a single glycosyl modification, which suggests these segments undergo robust translocon-mediated membrane integration (Fig. 1E ). In contrast, the translocon-mediated membrane integration of the second putative TM domain of E2 (TM2) is significantly less efficient (Fig. 1E) Fig. 2A ). It should also be noted that -1PRF only modifies the sequence of the loops downstream from these TM domains (Fig. 1C , orange line), and is therefore unlikely to impact their topological preferences.