Retroviruses enter host cells through the interaction of their envelope (Env) protein with a cell surface receptor, which triggers the fusion of viral and cellular membranes. The sodium-dependent neutral amino acid transporter ASCT2 is the common receptor of the large RD114 retrovirus interference group, whose members display frequent
envrecombination events. Germ line retrovirus infections have led to numerous inherited endogenous retroviruses (ERVs) in vertebrate genomes, which provide useful insights into the coevolutionary history of retroviruses and their hosts. Rare ERV-derived genes display conserved viral functions, as illustrated by the fusogenic syncytin envgenes involved in placentation. Here, we searched for functional envgenes in the nine-banded armadillo ( Dasypus novemcinctus) genome and identified dasy-env1.1, which clusters with RD114 interference group envgenes and with two syncytingenes sharing ASCT2 receptor usage. Using ex vivopseudotyping and cell-cell fusion assays, we demonstrated that the Dasy-Env1.1 protein is fusogenic and can use both human and armadillo ASCT2s as receptors. This gammaretroviral envgene belongs to a provirus with betaretrovirus-like features, suggesting acquisition through recombination. Provirus insertion was found in several Dasypusspecies, where it has not reached fixation, whereas related family members integrated before diversification of the genus Dasypus>12 million years ago (Mya). This newly described ERV lineage is potentially useful as a population genetic marker. Our results extend the usage of ASCT2 as a retrovirus receptor to the mammalian clade Xenarthra and suggest that the acquisition of an ASCT2-interacting envgene is a major selective force driving the emergence of numerous chimeric viruses in vertebrates. IMPORTANCERetroviral infection is initiated by the binding of the viral envelope glycoprotein to a host cell receptor(s), triggering membrane fusion. Ancient germ line infections have generated numerous endogenous retroviruses (ERVs) in nearly all vertebrate genomes. Here, we report a previously uncharacterized ERV lineage from the genome of a xenarthran species, the nine-banded armadillo ( Dasypus novemcinctus). It entered the Dasypusgenus >12 Mya, with one element being inserted more recently in some Dasypusspecies, where it could serve as a useful marker for population genetics. This element exhibits an envgene, acquired by recombination events, with conserved viral fusogenic properties through binding to ASCT2, a receptor used by a wide range of recombinant retroviruses infecting other vertebrate orders. This specifies the ASCT2 transporter as a successful receptor for ERV endogenization and suggests that ASCT2-binding envacquisition events have favored the emergence of numerous chimeric viruses in a wide range of species.