Structural Basis of -1 Programmed Ribosomal Frameshifting by the Human T-cell Lymphotropic Virus Type I RNA

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Programmed ribosomal frameshifting (PRF) is a common viral mechanism used to regulate the relative levels of various gene products. How RNA structures induce PRF is a fundamental question of relevance to human health, due to its prevalence in retroviruses that infect and cause human diseases. Human T-cell lymphotropic virus type I (HTLV-I) replication depends on two -1 PRF events, which occur at the gag-pro and pro-pol open reading frame junctions. How the cis-acting RNA elements at these genomic locations function to induce frameshifting is unknown. The long-term goal of this research is to improve understanding of how viral RNA structures manipulate host-translational machinery to ensure successful viral replication. The overall objective of this application is to determine the structural basis of -1 PRF in the HTLV-I retrovirus. Our central hypothesis is that specific regions of thermodynamic stability within each frameshift site structure are fundamental to frameshift stimulation. The rationale that underlies the proposed research is that once the structural basis of -1 PRF is understood, a significant gap in the knowledge base about the HTLV-I frameshift mechanisms would be filled. We propose two specific aims: 1) Define the HTLV-I frameshift site RNA structures, and 2) Investigate the relationship between each structure's local thermodynamic stability and -1 PRF efficiency. In this proposal, local thermodynamic stability is defined by the stability of base-pairs positioned directly outside of the mRNA entry channel at the time of frameshifting. To accomplish these aims, RNA chemical probing experiments will be combined with computational methods to define the RNA secondary structures at each frameshift site. Mutagenesis and in vitro frameshift assays will be used to evaluate the importance of each structure to -1 PRF and to investigate the relationship between local thermodynamic stability and frameshift efficiency. The results of the proposed research are significant because they will substantially increase what is known about HTLV-I structure-stimulated programmed ribosomal frameshifting. These studies promise to open new research horizons, particularly in targeting HTLV-I frameshift sites as a means of disrupting HTLV-I replication.