SUMMARY: Acquiring extracellular genetic and functional novelty can forever change the evolutionary trajectory of organisms, from mitochondria to antibiotic resistance to placental formation. Retroviruses heritable through the germline, endogenous retroviruses (ERVs), comprise eight percent of the human genome (four times more than protein coding sequence). The vast majority of ERVs lack all protein coding potential. A select few ERV genes have been preserved over evolutionary timescales, a strong indication that these specific genes are beneficial. env is the most frequently preserved ERV gene. In retroviruses, env encodes a glycoprotein that interacts with specific cellular receptors and is the virus-cell membrane fusogen. These activities have been repurposed by a pair of ERV envs that form the syncytiotrophoblast, an essential placental structure of fused cells at the maternal- fetal interface. Divergent envs have been coopted by different mammalian lineages for the same function. Knowledge of how ERV genes are acquired and repurposed is largely restricted to DNA and mRNA, while it is typically the proteins that carry out their functions. A dearth of quality reagents for ERV proteins has impeded our ability to define when, where, and why these proteins are expressed. We will leverage our expertise as evolutionary biologists, protein biochemists, and B cell immunologists to move ERV research into the proteome by generating, quality controlling, and disseminating the reagents required for this transition. Our primary focus is EnvP(b)1, an env preserved for >40 million years with no known function. Evolution has maintained its membrane fusion activity across human, ape and monkey orthologs. Expression in cultured cells results in fused monolayers of multinucleated cells and rapid cell death. Expression in vivo must therefore be tightly regulated, and its activity tailored to its function. We find genetic and functional evidence of evolutionary refinement of EnvP(b)1 over the past >40 million years. We will, in vitro, define the tissue and species specificities of membrane fusion by human and non-human primate (NHP) EnvP(b)1 orthologs. Using ancestral reconstruction, we will determine how EnvP(b)1 function evolved to its present form across primate lineages. Given its cytotoxicity and ability to produce cells with variable chromosomal content, the preservation of EnvP(b)1 indicates it must perform critical functions. We will define the anatomical sites and context of EnvP(b)1 protein expression in humans and NHPs, and if cell-cell fusion occurs at these sites. This work will ultimately establish NHP models required to define the function and explain why EnvP(b)1 is maintained. Using a carefully controlled antibody reagent, we find EnvP(b)1 protein expression in healthy human and NHP tissues, including placenta. The placenta, a rapidly evolving structure, is a hotbed of functional ERV Env expression. We will develop the reagents needed to dissect ...