A Virus-Packageable CRISPR Screen Identifies Host Factors Mediating Interferon Inhibition of HIV

Interferon (IFN) inhibits HIV replication by inducing an array of antiviral effectors. Here we describe a novel CRISPR knockout screening approach to identify the ensemble of these HIV restriction factors. We assembled a CRISPR sgRNA library specific for Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. We identify a small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5 which together explain the inhibitory effects of IFN on the HIV-1 LAI strain in THP-1 cells. Further, we identify novel HIV dependency factors, including SEC62 and TLR2. The ability of IFN-induced restriction factors to inhibit an HIV strain to replicate in human cells suggests that these human restriction factors are incompletely antagonized. A CRISPR screen targeting Interferon-Stimulated Genes in which enrichment of sgRNA sequences is compared in viral RNA to the library representation in genomic DNA. 2 biological replicates were performed in 3 indpendent cell lines (one wild type THP-1 and two independent ZAP Knockout THP-1 clonal cell lines). Cells were infected following treatment overnight with IFN and viral supernatants and cell pellets collected 3 days post-infection. sgRNA representation in viral RNA is compared to genomic DNA to determine which sgRNAs are over- or under-represented in the viral population.