Phosphorylation of shiftless is important for inhibiting the programmed −1 ribosomal frameshift

Shiftless (SFL) is a broad-spectrum inhibitor of programmed -1 ribosomal frameshift (-1 PRF) and exhibits various antiviral activities. Here, we characterized human SFL structurally and biochemically. The 2.0-angstrom resolution crystal structure of SFL reveals a boat-like module comprising an N-terminal helical bundle and three zinc fingers at the C terminus. A hyperphosphorylation loop (HPL) buried between the helical bundle and the zinc finger 1 harbors four phosphorylated residues (p-S249, p-T250 p-T253, and p-S256), which are important to protein folding. SFL forms monomers in solution and binds the HIV-1 -1 PRF sequence with nanomolar affinity (<i>K</i>_D = 5.7 nanomolar). Disruption of HPL phosphorylation decreased the RNA binding affinity and undermined the SFL-mediated -1 PRF inhibition of various viruses. Proximity-dependent biotinylation identified three cellular Ser/Thr kinases-EEF2K, NEK9, and PBK-that phosphorylate SFL in cells. These findings shed light on the mechanisms underlying -1 PRF regulation by SFL and provide insights into the role of SFL in virus inhibition.