Replication competent HIV-guided CRISPR screen identifies novel antiviral factors

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Date

2024-12-20

Authors

Prelli Bozzo, Caterina

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Dissertation

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Abstract

Innate defense mechanisms govern the outcome of pathogen exposure. A major part of these mechanisms are host-encoded proteins called restriction factors (RFs), which may inhibit viral pathogens, such as HIV-1, at essentially each step of their replication cycle. Although many antiviral factors have been identified, it has also become clear that some RFs that are limiting HIV-1 transmission and/or are targeted by the viral accessory proteins Vpu, Vpr or Nef remain to be discovered. CRISPR/Cas9 approaches are frequently used to identify novel RFs. However, previous KO screens are either based on transduction with lentiviral vectors co-expressing Cas9 with sgRNAs and subsequent infection with HIV-1 or on particles packaging Cas9 with the virus. Thus, they only allow to analyze single rounds of replication, and often focus on early steps of the viral replication cycle before integration. To overcome these limitations, I established a new method combining the CRISPR/Cas9 technology with the adaptive capacity of replication-competent HIV-1. To this end, I engineered pooled libraries of 1537 replication-competent HIV-1 each expressing a single sgRNAs to target >500 potential RFs thus equipping them with a defined selection advantage. Replication and selection over multiple rounds of replication thus allows enrichment of HIV-1 clones that target RFs, enabling their virus-driven identification. Screening of recombinant HIV-1 libraries in CD4+ T cells stably expressing Cas9 revealed that viruses expressing sgRNAs targeting several candidate RFs, including GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA were efficiently selected over multiple rounds of viral replication. The inhibitory impact of PGRN, CIITA and CEACAM3 was confirmed in primary CD4+ T cells. Functional analysis revealed that PGRN and CIITA suppress viral transcription whereas CC2D1B inhibits viral particle release. RhoA causes a cell cycle arrest in the G2/M phase which may be beneficial for the replication of the virus. To identify factors which are targeted by the accessory protein Nef, I disrupted the nef gene in the HIV-1 NL4-3 backbone. Lack of the accessory nef gene increased selection of sgRNAs targeting SERINC5, a well-known Nef target, confirming that the system works. Surprisingly, this approach also identified IFI16 as a new Nef target. Subsequent functional studies confirmed that Nef counteracts the inhibitory effects of IFI16. In summary, I established an innovative, robust, highly versatile and sensitive approach that allows the virus-guided identification of relevant antiviral factors by turning HIV-1 into “traitors” revealing their cellular opponents. This groundbreaking technique, allows the identification of new antiviral factors and opens a wide range of possibilities to dissect and understand virus-host interactions.

Description

Faculties

Medizinische Fakultät

Institutions

UKU. Institut für Molekulare Virologie
UKU. Institut für Virologie

Citation

DFG Project uulm

EU Project THU

Other projects THU

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CC BY 4.0 International

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DOI external

DOI external

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Periodical

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DFG Project THU

item.page.thu.projectEU

item.page.thu.projectOther

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Keywords

CRISPR/Cas9, Restriction factors, HIV, CRISPR/Cas-Methode, HIV-1, CRISPR-associated protein 9, CRISPR-Cas systems, HIV-1; Immunology, DDC 610 / Medicine & health