Capsid (CA)-specific restrictions are determinants of retroviral tropism in mammalian cells. One such restriction, human Ref1, targets strains of murine leukemia virus bearing an arginine at CA residue 110 (N-MLV), resulting in decreased accumulation of viral cDNA. The cellular factors accounting for Ref1 activity are unknown. As(2)O(3) increases N-MLV titer in Ref1-positive cells, possibly by counteracting Ref1. Restriction factor saturation experiments suggest that Ref1 may also target human immunodeficiency virus type 1 (HIV-1), but only if its CA is not bound to the cellular protein cyclophilin A (CypA). As a step towards understanding the genetic determinants of Ref1, we subjected Ref1-positive TE671 cells to three sequential rounds of selection with N-MLV reporter viruses. We isolated a subclone, 17H1, that was permissive for N-MLV infection and therefore deficient in Ref1 activity. Stimulation of N-MLV replication by As(2)O(3) was attenuated in 17H1, confirming that the drug acts by overcoming Ref1 activity. HIV-1 infection of 17H1 cells was resistant to disruption of the CA-CypA interaction, demonstrating that Ref1 restricts CypA-free HIV-1. Our results suggest that interaction with CypA evolved to protect HIV-1 from this human antiviral activity.