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Functional single-cell genomics of human cytomegalovirus infection

ABSTRACT: The complex life cycle of herpesviruses is orchestrated by the interplay of host factors and hundreds of viral genes. Understanding how they work together and how perturbations of viral and host factors impact infection represents both a fundamental problem in virology and the basis for designing antiviral interventions. Here, we use CRISPR screening to study infection of human cytomegalovirus (HCMV) in primary cells, comprehensively defining the functional contribution of each viral and host factor. We then record the transcriptomes of tens of thousands of single cells, and monitor how genetic perturbation of critical host and viral factors alters the timing, course, and progression of infection. We find that normally, the large majority of cells follow a stereotypical transcriptional trajectory. Perturbing critical host factors does not change this trajectory per se, but can either stall, delay or accelerate progression along the trajectory, allowing us to pinpoint systematically the stage of infection at which each host factor acts. Conversely, perturbation of viral factors can create distinct abortive trajectories. Our results reveal a dichotomy between the roles of host and viral factors and more generally provide a road map for functional dissection of host-pathogen interactions.

ORGANISM(S): Human herpesvirus 5 strain Merlin Homo sapiens

PROVIDER: GSE165291 | GEO | 2021/01/22

REPOSITORIES: GEO

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Project description:During HIV-1 infection, there is a massive perturbation of host gene expression, but as yet, genome-wide studies have not identified host genes affecting HIV-1 replication in lymphatic tissue, the primary site of virus-host interactions. In this study, we isolated RNA from the inguinal lymph nodes of 22 HIV-1-infected individuals and utilized a microarray approach to identify host genes critically important for viral replication in lymphatic tissue by examining gene expression associated with viral load. Strikingly, ~95% of the transcripts (558) in this data set (592 transcripts total) were negatively associated with HIV-1 replication. Genes in this subset (1) inhibit cellular activation/proliferation (ex.: TCFL5, SOCS5 and SCOS7, KLF10), (2) promote heterochromatin formation (ex.: HIC2, CREBZF, ZNF148/ZBP-89), (3) increase collagen synthesis (ex.: PLOD2, POSTN, CRTAP), and (4) reduce cellular transcription and translation. Potential anti-HIV-1 restriction factors were also identified (ex.: NR3C1, HNRNPU, PACT). Only ~5% of the transcripts (34) were positively associated with HIV-1 replication. Paradoxically, nearly all these genes function in innate and adaptive immunity, particularly highlighting a heightened interferon system. The predominance of negative correlations as well as the disconnect between host defenses and viral load point to the importance of genes that regulate target cell activation and genes that code for potentially new restriction factors as determinants of viral load rather than conventional host defenses. Total RNA was isolated from the inguinal lymph nodes of 22 HIV-1-infected subjects at different clinical stages (and varying viral loads) and prepared for RNA extraction and hybridization on Affymetrix Human Genome U133 Plus 2.0 microarrays. Replicate arrays were performed for lymph node samples to minimize assay noise and host genes critically important for viral replication in lymphatic tissue were identified by examining gene expression and its association with viral load. Replicates were not performed for samples WB91 and TS35 due to limited amounts of biomaterial.

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Functional single-cell genomics of human cytomegalovirus infection

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