Periodic Reporting for period 1 - Traitor-Viruses (Traitor-virus-guided discovery of antiviral factors)
Periodo di rendicontazione: 2022-07-01 al 2024-12-31
As outlined below, we successfully established and applied the CRISPR/Cas9-based HIV-guided approach for efficient discovery of novel antiviral factors and mechanisms. In brief, we equipped libraries of replication-competent HIV-1 and SIVcpz constructs with guide RNAs acting as genetic scissors that may inactivate human genes in the presence of Cas9. HIV-1 or SIVcpz variants expressing single gRNAs inactivating antiviral genes will be selected during cell-culture passage and reveal the identity of the targeted genes. We found that our approach directly indicates which cellular genes HIV-1 or SIVcpz have to eliminate to achieve increased replication fitness and identified a variety of antiviral factors and mechanisms as well as novel targets of the accessory viral proteins.
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.
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.
In the present project, we exploit the replication fitness of infectious HIV-1 or SIVcpz constructs expressing sgRNAs to decipher antiviral mechanisms. We named this technology “Traitor-virus” (TV) approach since populations of replication-competent viral constructs engineered to express sgRNAs not only allow the pathogen to inactivate antiviral genes (i.e. confer a selective advantage) but also reveal their identity (i.e. the targeted sequence). Each sgRNA represents a unique molecular barcode allowing the association of a selection advantage with a specific cellular gene. Unlike previous methods, this virus-driven technology is highly effective, robust and sensitive because the effect of selective advantages associated with specific sgRNAs is amplified at each round of viral replication. Notably, this closely reflects the impact of fitness advantages during HIV-1 or SIVcpz replication in vivo. Competition-based TV screens enable simultaneous evaluation of numerous cellular targets using complex HIV-1-U6-sgRNA-scaffold libraries. Currently, we work with libraries targeting all ~21.000 cellular genes. Since the readout relies on changes in viral replication fitness and hence changes in the relative frequencies of sgRNAs our approach is highly robust and barely affected by variations in the number of input sgRNA copies. Functional analyses confirmed that TVs identify physiologically relevant cellular factors that restrict HIV-1 replication in primary CD4+ T cells as well as Nef targets.
The combination of the revolutionary CRISPR/Cas9 technology with the enormous adaptive capacity of replication-competent HIV-1 or SIVcpz constructs to identify cellular factors restricting viral replication goes substantially beyond state-of-the-art and offers a novel, innovative and effective tool to elucidate virus-host interactions. Our approach is the first taking advantage of replication-competent viruses equipped with highly specific and effective genetic tools (i.e. the gRNA) to eliminate inhibitory cellular genes to elucidate antiviral defense mechanisms by natural selection of HIV-1 or SIVcpz gRNA variants counteracting antiviral mechanisms in various settings. Our results show that its highly robust and effective. It is also safe since any selective advantage for the virus is limited to cells artificially expressing the Cas9 endonuclease. The combination of molecular and evolutionary biology, virology and bioinformatics approaches and utilization of viruses themselves to identify the cellular opponents will fundamentally advance our understanding of intrinsic cellular antiviral mechanisms.
The combination of the revolutionary CRISPR/Cas9 technology with the enormous adaptive capacity of replication-competent HIV-1 or SIVcpz constructs to identify cellular factors restricting viral replication goes substantially beyond state-of-the-art and offers a novel, innovative and effective tool to elucidate virus-host interactions. Our approach is the first taking advantage of replication-competent viruses equipped with highly specific and effective genetic tools (i.e. the gRNA) to eliminate inhibitory cellular genes to elucidate antiviral defense mechanisms by natural selection of HIV-1 or SIVcpz gRNA variants counteracting antiviral mechanisms in various settings. Our results show that its highly robust and effective. It is also safe since any selective advantage for the virus is limited to cells artificially expressing the Cas9 endonuclease. The combination of molecular and evolutionary biology, virology and bioinformatics approaches and utilization of viruses themselves to identify the cellular opponents will fundamentally advance our understanding of intrinsic cellular antiviral mechanisms.