AbstractThe viral protein, Vif, is essential for the production of infectious progeny virions in natural target cells of human immunodeficiency virus type 1 (HIV‐1). Several recent reports indicate that Vif acts by antagonizing the activity of an endogenous human antiviral protein, APOBEC3G. To investigate this route to restrict HIV‐1 infection, we employed mutagenesis to assess APOBEC3G function during HIV‐1 infection including interaction with Vif, localization, and activity in virions. We found that APOBEC3G binds Vif in infected cells and the C′‐terminal region is required for this interaction. APOBEC3G was only incorporated into virions in the absence of Vif and deletion of either the N′‐terminal or C′‐terminal regions of APOBEC3G abrogated virion localization. Assaying endogenous reverse transcription we found that APOBEC3G and its C′‐terminal deletion mutant inhibited full‐length cDNA synthesis, possibly through binding to viral RNA, a function revealed through gel‐shift assays. Taken together, our studies suggest that APOBEC3G inhibits HIV‐1 infection through interference with reverse transcription and that Vif counteracts APOBEC3G by impeding its entry into virions. © 2004 Wiley‐Liss, Inc.