Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef

• Published in: Nature communications Vol. 15; no. 1; p. 3813
• Main Authors: Prelli Bozzo, Caterina, Laliberté, Alexandre, De Luna, Aurora, Pastorio, Chiara, Regensburger, Kerstin, Krebs, Stefan, Graf, Alexander, Blum, Helmut, Volcic, Meta, Sparrer, Konstantin M. J., Kirchhoff, Frank
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 07.05.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/106; 13/31; 38/23; 38/47; 631/250/262; 631/326/596; 82/80; CD4 antigen; CD4-Positive T-Lymphocytes - immunology; CD4-Positive T-Lymphocytes - metabolism; CD4-Positive T-Lymphocytes - virology; Cell lines; CIITA protein; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CRISPR; CRISPR-Cas Systems; HEK293 Cells; HIV; HIV Infections - genetics; HIV Infections - immunology; HIV Infections - virology; HIV-1 - genetics; HIV-1 - physiology; Human immunodeficiency virus; Humanities and Social Sciences; Humans; Infectivity; Lymphocytes; Lymphocytes T; multidisciplinary; nef Gene Products, Human Immunodeficiency Virus - genetics; nef Gene Products, Human Immunodeficiency Virus - metabolism; Nef protein; Phosphoproteins - genetics; Phosphoproteins - metabolism; Proteins; Replication; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; RhoA protein; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Guide, CRISPR-Cas Systems - metabolism; Science; Science (multidisciplinary); Target recognition; Transcription factors; Virus Internalization; Virus Replication - genetics
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-48228-x

Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4 + T cells robustly enriched HIV-1 encoding sgRNAs against GRN , CIITA , EHMT2 , CEACAM3 , CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4 + T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle. Innate immune mechanisms are critical for antiviral defense. Here, the authors developed a CRISPR/Cas9-based HIV-driven approach to identify cellular factors compromising viral transcription, assembly, release or infectivity in human T cells. They identify targets of the Nef protein as antiviral factors.