Ribosomal frameshifting is defined as a shift in reading frame. During translation, a ribosomal frameshift (FS) can occur when the ribosome encounters a slippery sequence followed by a stable secondary structure. A slippery sequence specifically allows for the opportunity for tRNA repainring with the codons in the A- and P- sites of the ribosome. Retroviruses include programmed ribosomal frameshift (PRF) sites within their RNA, which increase viral genomic coding capacity by allowing translation of multiple reading frames from a single RNA. Human T-cell leukemia virus type-II (HTLV-II) uses PRF sites to translate its enzymatic proteins. These proteins are encoded in the pro and pol open reading frames. The first HTLV-II PRF site is located at the end of the gag open reading frame and is described as the gag-pro PRF site. This FS site contains a heptanucleotide slippery sequence (AAAAAAC) followed by a stem-loop structure. Recently, a study showed that local stability of the secondary structure was correlated to frameshift efficiency in HIV-1. We hypothesize that local stability has greater control of frameshift efficiency than global stability in the gap-pro PRF site in HTLV-II. To test this hypothesis, five variant stem-loops were designed to decouple local and global stability. There variant frameshift sites were inserted into a dual-luciferase reporter plasmid using molecular cloning techniques. Successful cloning was verified by DNA sequencing. These plasmid DNAs will be used for in vitro transcription of RNA. The resulting RNAs will be purified and utilized in an in vitro translation assay to determine the frameshift efficiencies for each variant frameshift site. Finally, the frameshift efficiencies will be compared to each other to determine if local or global stability has a stronger effect on frameshift efficiency.
