Elucidating structure-function relationships of viral tRNA-like structures

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Project Summary/Abstract Viruses have evolved diverse mechanisms to hijack the host’s cellular machinery for their own advantage. Ubiquitous in RNA viral genomes are discrete structured RNA elements that play an essential role in evading host cell immunity and promoting viral propagation. Therefore, viral RNA structure is intricately linked to viral infection and disease. Importantly, a major goal of biomedical research is to understand how viruses manipulate the host machinery. Thus, understanding the structure-function relationship of structured RNA elements found in viral genomes is fundamental for future development of vaccines and therapeutics. Many RNA viral genomes contain regions that structurally or functionally mimic transfer RNA (tRNA) as part of their strategy to interact with and manipulate the host cell machinery. These tRNA mimics are found throughout viral genomes and in viruses that infect diverse hosts. Important examples of tRNA mimicry that have served as a model system for this type of viral mechanism are tRNA Like Structures (TLS) found in the 3’ untranslated region of plant-infecting viruses. These TLSs were first identified decades ago and have been shown to mimic tRNAs in three ways and enhance translation in cell-free extracts. Despite decades of study and high-resolution structural information of a few TLSs, how they enhance translation remains unknown. This proposal aims to understand the contribution of the TLS in translation enhancement of the viral genome. The first step is to identify additional host cell components the TLS interacts with and how this compares to cognate tRNAs. Identifying the full set of interacting partners will allow for building testable models to elucidate the mechanism behind the translation enhancement function of the TLS. Furthermore, it is important to determine if the TLSs are recognized by additional tRNA-targeted enzymes, such as tRNA modification enzymes as recent evidence has shown chemical modifications to RNA are important for structure and function. Aim 2 proposes to investigate the hypothesized model of the TLS as a pseudo-poly(A) tail. Using fluorescence resonance energy transfer (FRET) followed by translation assays will determine if intrinsic folding of the RNA transcript is important for communication of the 3’ TLS to the 5’ cap. Identifying how tRNA mimics at the 3’ end of certain viruses can bypass the necessity of a poly(A) tail will enhance the understanding of the mandatory and potential alternative requirements of the host translation machinery. This work will yield insight into how viruses use tRNA mimicry for their benefit, how tRNA mimics are able to delude host cell machinery, and furthermore enhance the general understanding of tRNA mimicry and its influence on gene expression.