Determination of the HTLV‐1 pro‐pol Frameshift Site Secondary Structure

Abstract

Expression of human t‐cell leukemia virus type I (HTLV‐I) enzymes requires two ‐1 programmed ribosomal frameshifts. These events occur between the gag‐pro and pro‐pol open reading frames. Each frameshift site includes a heptanucleotide slippery sequence followed by a downstream structure, which act in cis to produce specific frameshift efficiencies. While ‐1 PRF and the slippery sequences of these frameshift sites have been established in HTLV‐I, their frameshift efficiencies and the structures within these sites have not been determined. In pro‐pol frameshift site, an RNA pseudoknot is predicted to fold downstream of the UUUAAAC slippery sequence. However, no structural data exist for this RNA. Here, we report a preliminary structure of the HTLV‐1 pro‐pol frameshift site RNA. Nucleotide reactivity data acquired from selective 2′‐hydroxyl acylation experiments analyzed by primer extension and thermodynamics‐based secondary structure predictions are consistent with a pseudoknot secondary structure. Additionally, non‐denaturing PAGE analysis of the wild‐type structure and two mutant RNAs confirmed that the fold of the wild‐type RNA is significantly different from RNAs of identical length that cannot form a pseudoknot structure. These results establish the existence of a pseudoknot structure in the HTLV‐1 pro‐pol frameshift site.