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

Viruses have evolved numerous mechanisms to exploit the host machinery in order to increase the coding capacity of their highly constrained genomes. There are at least 27 viral genera that utilize programmed ribosomal frameshifting (PRF) in order to produce multiple proteins from a single transcript (https://viralzone.expasy.org/860). PRF is genetically encoded, and minimally requires a portion of the transcript that contains a repetitive "slippery" heptanucleotide sequence (slip-site) followed by a region that forms stimulatory RNA secondary structures (an ensemble of stem loops and/ or pseudoknots). 1, 2 A collision between the translating ribosome and the stimulatory secondary structure increases the kinetic barrier to translocation, which causes the ribosome to dwell on the slip-site. [3] [4] [5] [6] [7] During this pause, the t-RNA that is annealed within the ribosomal P-site (and most often also the t-RNA in the A-site) 8 begin to sample alternative base pairing interactions that shift the reading frame of the ribosme. 9 Based on these mechanistic considerations, PRF is typically believed to be mediated at the level of RNA structure. Nevertheless, recent reports have also found that the efficiency of -1PRF can be tuned by a variety of regulatory proteins and/ or miRNA. [10] [11] [12] -1PRF is utilized to temporally and stoichiometrically regulate protein production during viral replication and assembly. For instance, the alphavirus structural proteins are most often produced from a single polyprotein that is cleaved into the capsid (CP), E3, E2, 6K, and E1 proteins ( Figure 1A ). 13 The E2 and E1 proteins are membrane glycoproteins that heterodimerize early in the assembly pathway. These dimeric units then form trimeric spike complexes, traffic to the plasma membrane, and initiate viral budding. 14-16 -1PRF during the translation of the 6K protein gives rise to a secondary form of the polyprotein containing the TransFrame (TF) protein, 13 ,17 a known . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 virulence factor, [18] [19] [20] [21] in place of the 6K and E1 proteins ( Figure 1B ). Because -1PRF precludes the translation of E1, the efficiency of ribosomal frameshifting (1-48% in alphaviruses) 22 influences the stoichiometric ratio of the E1 and E2 glycoproteins and the net accumulation of spike complexes. Current evidence suggests -1PRF is stimulated by a canonical poly-U slip site and a downstream RNA hairpin. 23 However, an effort to map the stimulatory RNA structures within alphavirus polyproteins revealed that deletions within the predicted hairpin region are capable of reducing the efficiency of -1PRF, but appear to be insufficient to knock out frameshifting completely. 22 This observation suggests there may be multiple regulatory mechanisms that mediate -1PRF within the alphavirus structural polyprotein.