Viruses rely on the host cellular machinery for the production of their proteins and, ultimately, for the assembly of infectious virions. In most cases, viral proteins are recognized and processed in the same manner as host proteins. However, some viral sequence elements have evolved to distort the activity of the host machinery in a manner that increases the structural and/ or functional diversity among translation products. Like many other RNA viruses, alphaviruses leverage ribosomal errors to facilitate the production of multiple proteins from a single open reading frame. This process, which is known as -1 programmed ribosomal frameshifting (-1PRF), is often mediated by structural elements within the polyprotein mRNA. However, it was recently found that the protein downstream of the -1PRF site exhibits a distinct topology with respect to the endoplasmic reticulum (ER) membrane. This finding suggests the formation of distinct topological states in the nascent polypeptide is coupled to ribosomal frameshifting. In this proposal, we seek to explore the mechanistic basis of this phenomenon and to determine whether sequence elements in the viral polyprotein have evolved to exploit topological errors for functional gain. Sequence-based topology predictors reveal several ambiguous topological signals within the alphavirus polyprotein, which could potentially facilitate the formation of a distinct topology in the frameshifted translation product. Notably, we have identified a TM domain in a position to partition into the membrane and impose a tension on the nascent chain at the precise moment the frameshift site passes through the ribosome. Given that mechanical tension on the nascent chain is a hallmark of-1PRF, this observation is suggestive of a novel mechanistic link between the translocon and -1PRF. Moreover, this TM domain is somewhat polar and partitions into the membrane with a marginal efficiency that is comparable to the frequency of -1PRF. Based on this preliminary evidence, we hypothesize that the membrane integration of this TM domain serves as a trigger for -1PRF. In the following, we propose a series of experiments aimed at testing this hypothesis and elucidating the topological determinants within the polyprotein. More generally, we also seek to determine whether the ambiguous signals in the polyprotein have evolved to promote the formation of multiple topologies during translation. Using energetic predictors and multiple sequence alignments, we outline a novel protein engineering approach to trap the nascent polyprotein within discrete topologies during translation. We will then evaluate the manner in which these modifications to the topological energetics influence viral fitness in the context of both host and vector cells. Finally, to determine whether topological heterogeneity represents a selected trait, we will screen for revertant strains of these engineered viruses, and assess whether their sequences regenerate the topological energetics of the wild-type strain. Together these investigations will provide fundamental insights into the mechanisms of viral biogenesis that could have far reaching implications for the evolution of RNA viruses.
Proper folding and processing of viral polyproteins by host machinery is essential for the replication of infectious virions. Based on emerging evidence, we propose a series of experiments to explore potential mechanisms by which viruses may exploit the activity of the Sec61 translocon to expand the functionality of viral proteins. The results will highlight new modes of crosstalk between the ribosome and translocon, elucidate novel strategies for viral evolution, and provide new targets for the design of anti-viral therapeutics.
