Abstract
Most antiviral proteins recognize specific features of viruses. In contrast, the recently described antiviral factor retroCHMP3 interferes with the "host endosomal complexes required for transport" (ESCRT) pathway to inhibit the budding of enveloped viruses. RetroCHMP3 arose independently on multiple occasions via duplication and truncation of the gene encoding the ESCRT-III factor CHMP3. However, since the ESCRT pathway is essential for cellular membrane fission reactions, ESCRT inhibition is potentially cytotoxic. This raises fundamental questions about how hosts can repurpose core cellular functions into antiviral functions without incurring a fitness cost due to excess cellular toxicity. We reveal the evolutionary process of detoxification for retroCHMP3 in New World monkeys using a combination of ancestral reconstructions, cytotoxicity, and virus release assays. A duplicated, full-length copy of retroCHMP3 in the ancestors of New World monkeys provides modest inhibition of virus budding while exhibiting subtle cytotoxicity. Ancient retroCHMP3 then accumulated mutations that reduced cytotoxicity but preserved virus inhibition before a truncating stop codon arose in the more recent ancestors of squirrel monkeys, resulting in potent inhibition. In species where full-length copies of retroCHMP3 still exist, their artificial truncation generated potent virus-budding inhibitors with little cytotoxicity, revealing the potential for future antiviral defenses in modern species. In addition, we discovered that retroCHMP3 restricts LINE-1 retrotransposition, revealing how different challenges to genome integrity might explain multiple independent origins of retroCHMP3 in different species to converge on new immune functions.