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 the computational and biochemical results in Figure 1 , we generated a topological model of the SINV structural polyprotein ( Fig. 2A) . This model correctly places the E2 and E1 ectodomains within the ER lumen, and places the two palmitoylated cysteine residues in E2 (C716 and C718) within the cytosol. 35, 36 To probe the topological preferences of the SINV polyprotein in the cell, we produced and characterized a series of reporter constructs that begin with the E3 protein and end at the C-terminal edge of each of the three putative TM domains within E2 and 6K ( Fig. 2A, Supplemental Fig. 1 ). Each of these fragments was genetically fused to a C-terminal cassette containing a short GS linker and glycosylatable GFP (gGFP) gene, which contains two glycosylation sites within the core of the eGFP protein. 37 Topological signals that direct the gGFP protein into the cytosol will produce a fluorescent gGFP, whereas the glycosylation of gGFP within the ER lumen renders the protein non-fluorescent (Fig. 2B ). Each construct was then expressed in HEK293T cells, and flow cytometry was used to quantify the fluorescence intensity of the gGFP reporter at a consistent expression level as judged by the intensity of a bicistronic reporter protein (Supplemental Fig. 2) . Expression of the reporter constructs containing gGFP downstream from Fig. 2A ) generates fluorescent gGFP (Fig. 2C) , which suggests the C-termini of these TM domains reside within the cytosol. In contrast, the reporter construct with gGFP downstream from TM3 (after R785) exhibits an attenuated GFP signal at an equivalent expression level (Fig. 2C) , which suggests the gGFP fused to the C-terminus of TM3 is projected into the ER lumen. Placement of the gGFP after the full stretch of hydrophobic amino acids in the 6K protein The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 (after Y807, see Fig. 2A ) also results in an attenuated gGFP signal (TM3+ in Fig. 2C ), which confirms the full stretch of hydrophobic residues near TM3 only spans the membrane once. Thus, results from this cellular reporter assay (Fig. 2C ) are consistent with predictions ( Fig. 1C) , in vitro translation data (Fig. 1E) , and the model shown in Figure 2A . These observations together confirm that the E1 and 6K proteins each contain a single TM domain in the most abundant form of the polyprotein.