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Thus, there is a need to identify antiretrovirals that act on viral life cycle stages not targeted by drugs in use, such as the events of HIV-1 Gag assembly. To address this gap, we developed a compound screen that recapitulates the intracellular events of HIV-1 assembly, including virus-host interactions that promote assembly. This effort led to the identification of a new chemotype that inhibits HIV-1 replication at nanomolar concentrations, likely by acting on assembly. This compound colocalized with Gag and two host enzymes that facilitate capsid assembly. However, resistance selection did not result in compound-specific mutations in\n                    <jats:italic>gag<\/jats:italic>\n                    , suggesting that the chemotype does not directly target Gag. 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