Zinc mediates the allosteric activation of Shiftless to suppress programmed -1 ribosomal frameshifting.

Many medically important viruses, including HIV-1 and severe acute respiratory syndrome coronavirus 2, utilize programmed -1 ribosomal frameshifting (-1 PRF) to synthesize certain proteins essential for the viral life cycle. Shiftless (SHFL) is an interferon-simulated, zinc finger-containing protein that restricts -1 PRF and viral infection. However, its regulatory spectrum and mechanism remain largely unclear. Here, we showed that SHFL orthologs potently suppress a wide variety of viral and cellular -1 PRF, and that this activity is evolutionarily conserved in mammals. Notably, mammalian SHFL proteins contain three zinc finger domains and inhibit -1 PRF in a zinc ion- and zinc finger-dependent manner. Structural and biochemical analyses revealed that SHFL is an autoinhibited protein, in which the C-terminal domain (CTD) serves as the principal functional module through mediating RNA binding, homotypic oligomerization, and ribosomal interaction, whereas the N-terminal domain (NTD) negatively regulates CTD activity through intramolecular interaction. Either zinc binding to the three zinc fingers or genetical disruption of the NTD-CTD interaction by point mutagenesis likely drives conformational rearrangement that relieves autoinhibition and converts SHFL into an active conformation. Together, these findings uncover a previously unrecognized mechanism of zinc ion- and zinc finger-mediated allosteric activation of SHFL, providing a molecular framework for broad-spectrum suppression of viral and cellular -1 PRF.