Indels allow antiviral proteins to evolve functional novelty inaccessible by missense mutations.

Antiviral proteins often evolve rapidly at virus-binding interfaces to defend against new viruses. We investigated whether antiviral adaptation via missense mutations might face limits, which insertion or deletion mutations (indels) could overcome. Using high-throughput saturation missense mutagenesis, we identify one such case of a nearly insurmountable evolutionary challenge: the human anti-retroviral protein TRIM5α requires more than five missense mutations in its specificity-determining v1 loop to restrict a divergent simian immunodeficiency virus (SIV). However, through a novel saturating indel scanning methodology, we find that duplicating just one amino acid in v1 enables human TRIM5α to potently restrict SIV in a single evolutionary step. Moreover, natural primate TRIM5α v1 loops have evolved indels that confer novel antiviral specificities. Thus, indels enable antiviral proteins to overcome viral challenges otherwise inaccessible by missense mutations. Our findings reveal the potential of often-overlooked indel mutations in driving protein innovation.