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A virally encoded high resolution screen of cytomegalovirus host dependencies

ABSTRACT: Genetic screens have transformed our ability to interrogate cellular factor requirements in infection, yet current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information which is often based on infected cell survival. Here, by engineering human cytomegalovirus to express sgRNA libraries directly from the viral genome, we developed a sensitive, versatile, viral centric approach that allows profiling of different stages along viral infection in a pooled format. Using this approach, which we termed VECOS (Virus Encoded CRISPR-based direct readOut Screening system), we identified hundreds of novel dependency and restriction factors and we quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on viral-host interactions. These high resolution measurements reveal that perturbations that alter late stages in HCMV life cycle mostly regulate HCMV particle quality rather than quantity, defining correct virion assembly as a critical stage that is heavily reliant on viral-host interactions. Overall, VECOS facilitates systematic high resolution dissection of human proteins' role along the infection cycle, providing a roadmap for in-depth dissection of host–herpesvirus interactions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE246735 | GEO | 2024/04/09

REPOSITORIES: GEO

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Project description:We have established that human cytomegalovirus (HCMV) infection modulates the biology of target primary blood monocytes, allowing HCMV to use monocytes as 'vehicles' for its systemic spread. HCMV infection of monocytes results in rapid induction of PI(3)K and NF-kB activity. Integrins, which are upstream of the PI(3)K and NF-kB pathways, were shown to be involved in HCMV binding to and entry into fibroblasts, suggesting that receptor-ligand-mediated signaling following viral binding to integrins on monocytes could trigger the functional changes seen in infected monocytes. We now show that integrin engagement and the activation of the integrin/Src-signaling pathway is essential for the induction of HCMV-infected monocyte motility. To investigate how integrin engagement by HCMV triggers monocyte motility, we examined the infected monocyte transcriptome and found that the integrin/Src-signaling pathway regulates the expression of paxillin, which is an important signal transducer in the regulation of actin rearrangement during cell adhesion and movement. Functionally, we observed that paxillin is activated via the integrin/Src-signaling pathway and is required for monocyte motility. Because motility is intimately connected to cellular cytoskeletal organization, a process that is also important in viral entry, we investigated the role paxillin regulation plays in the process of viral entry of monocytes. New results confirmed that HCMV`s ability to enter target monocytes is significantly inhibited in cells deficient in paxillin expression or that had their integrin/Src/paxillin signaling pathway blocked. From our data, HCMV-cell interactions emerge as an essential trigger for the cellular changes that allow for HCMV entry and hematogenous dissemination. Monocytes were mock-infected, HCMV-infected, or pretreated with PP2 inhibitor prior to HCMV infection. There were three samples analyzed per individual replicate. Three replicates are included. comparative studies with a use of the specific Src kinase activity inhibitor

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A virally encoded high resolution screen of cytomegalovirus host dependencies

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