Determining How Stem‐loop Structure Thermodynamic Stability Influences Frameshift Efficiency at the HTLV‐1 gag‐proFrameshift Site

Abstract

The human T‐lymphotropic virus type 1 (HTLV‐1) RNA genome includes two programmed ‐1 ribosomal frameshift (‐1 PRF) sites. These sites allow ribosomes access to alternate reading frames encoding critical viral enzymes. The gag‐proframeshift site includes a slippery sequence, spacer, and stem‐loop structure. How the stem‐loop acts to promote frameshifting is unclear. Previous HTLV‐2 research showed that changes to the gag‐pro frameshift site stem‐loop thermodynamic stability influenced its frameshift efficiency to a modest degree. There is substantial conservation between the HTLV‐1 and HTLV‐2 gag‐pro frameshift site sequences (86%) and structures. We hypothesized that the HTLV‐1 gag‐pro frameshift efficiency would be similarly influenced by its stem‐loop thermodynamic stability. To test this hypothesis, we designed 15 stem‐loop mutants (SLMs) with varied base‐pair composition. These mutations decoupled changes in overall thermodynamic stability from those localized to the stem‐loop base. The SLM thermodynamic stabilities were calculated using nearest neighbor parameters and the in vitro frameshift efficiencies were measured with a dual‐luciferase assay. Correlations between frameshift efficiency and thermodynamic stability were subsequently assessed. Preliminarily results reveal a moderate correlation between the SLM stem‐loop overall thermodynamic stability and frameshifting efficiency. No correlation was observed between the thermodynamic stability of the stem‐loop base and frameshifting efficiency. While the overall thermodynamic stability does impact the frameshift efficiency, it cannot be used exclusively to predict it. This reflects a complex interplay between the frameshift site elements. Overall, our preliminary results suggest a conserved function for the gag‐pro frameshift site stem‐loop between the HTLV‐1 and HTLV‐2 retroviruses.