show Abstracthide AbstractInnate cellular defense mechanisms and viral countermeasures govern the outcome of pathogen exposure but the complex virus-host interplay remains poorly understood. Here, we developed a virus-guided technology platform where the pathogen itself reveals its cellular opponents. To accomplish this, we engineered replication-competent HIV-1 expressing single guide RNAs (sgRNAs) targeting potential antiviral genes in Cas9 expressing CD4+ T cells. Screening of HIV-1 constructs targeting >500 potential antiviral genes revealed that sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B, RHOA and HMOX1 provide significant advantages for viral replication. We verified that GRN and CIITA inhibit HIV-1 in primary CD4+ T cells by reducing viral transcription. Lack of the accessory nef gene increased selection for sgRNAs targeting SERINC5 and IFI16. Functional analyses demonstrated that Nef counteracts the inhibitory effects of IFI16. Altogether, we established a highly versatile, effective and robust approach that forces HIV-1 to reveal its cellular opponents. Overall design: RNA from virus was reverse transcribed and a cassette containing a sgRNA was amplified to sequence the sgRNA