Traditional approaches to discover innate antiviral factors typically focus on the initial stages of viral replication, are confined to a single round of infection and thus not very sensitive. Since cells exert numerous antiviral mechanisms the contribution of individual factors to the control of viral pathogens may seem small. However, a 2-fold growth advantage in a single round may result in >1000-fold higher virus yields after just 10 rounds of replication. Thus, effects that may be missed in single round of infection screens can have a major impact on viral spread in vivo. To address the limitations of available screening methods, we initially engineered libraries consisting of over 1,500 replication-competent HIV-1 constructs, each expressing a single gRNA altogether targeting more than 500 cellular genes. About 200 target genes were chosen based on previous analyses of protein-coding genes that share features of known antiviral factors, such as the in vivo response to HIV-1 infection and/or IFNs, codon-specific positive selection, burden of synonymous, missense and non-sense variation, as well as the number of paralogs. The remaining factors were selected because of their putative roles in pathogen sensing or in the various steps in the HIV-1 replication cycle. This novel approach allowed efficient virus-driven discovery of antiviral factors. Through passaging in engineered Cas9-expressing CD4+ T cells, we achieved robust enrichment of HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B, and RHOA by more than 50-fold. Additionally, we used an HIV-1 library lacking the accessory nef gene to identify IFI16 as a novel Nef target. Functional analyses in cell lines and primary CD4+ T cells confirmed that the HIV-1-driven CRISPR screen successfully identified restriction factors targeting various stages of the viral life cycle, including virus entry, transcription, release, and infectivity. These results demonstrated that the HIV-guided CRISPR technique represents a sensitive and comprehensive method for discovering physiologically relevant cellular defense mechanisms throughout the entire viral replication cycle (Prelli Bozzo, Laliberte et al., Nat. Com. 2024).
More recently, we generated libraries of replication-competent HIV-1 expressing more than 75.000 sgRNAs targeting all ~21.000 human genes for unbiased identification of antiviral mechanisms. To ensure the relevance of the analyses, we generated the libraries in two different HIV-1 backbones. Amongst others, we found that variants of the primary transmitted-founder HIV-1 CH077 clone carrying sgRNA targeting RHOA are enriched by several orders of magnitude especially in the presence of interferon. Functional analysis showed that depletion of RHOA during HIV-1 replication was beneficial for the virus and caused cell cycle arrest in the G2/M phase. In addition, lack of the viral vpr gene significantly enhanced the selection advantage mediated by sgRNA targeting RHOA. Altogether, we identified RHOA as a novel antiviral factor and potential Vpr target that modulates the cell cycle.
Pandemic HIV-1 strains are well adapted to humans and largely resistant to innate restriction factors representing a first-line of defense against viral zoonoses. HIV-1 originates from SIVcpz infecting chimpanzees and had to overcome numerous defense mechanisms after zoonotic transmission to spread and ultimately cause the AIDS pandemic. To identify these hurdles, we generated derivatives of the infectious molecular clone SIVcpz MB897 containing sgRNA expression cassettes between the nef gene and the 3’LTR. SIVcpz MB897 is one of the closest non-human relatives of HIV-1 M (major). SIVcpz containing ~1500 sgRNAs was passaged in the human T cell line SupT1 CCR5 expressing Cas9. These analyses showed that IFITM2 restricts SIVcpz more efficiently than HIV-1 group M strains. Recently, we generated SIVcpz gRNA libraries targeting all human genes for unbiased discovery and obtained interesting hits.
