Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Sunshine, Sara; Puschnik, Andreas S.; Replogle, Joseph M.; Laurie, Matthew T; Liu, Jamin; Zha, Beth Shoshana; Nuñez, James K.; Byrum, Janie R.; McMorrow, Aidan H.; Frieman, Matthew B.; Winkler, Juliane; Qiu, Xiaojie; Rosenberg, Oren S.; Leonetti, Manuel D.; Ye, Chun Jimmie; Weissman, Jonathan S.; DeRisi, Joseph L.; Hein, Marco Y.

doi:10.1101/2022.07.15.500120

Cited by 7 publications

(9 citation statements)

References 85 publications

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“…However, aside from this study and a previous OPS of Sendai virus infection (Carlson et al, 2023), high-content profiling screens of host-pathogen interactions have been limited. Notable recent examples include targeted transcriptome screens that revealed perturbation-induced changes in transcriptional trajectories upon HCMV (Hein & Weissman, 2022) and SARS-CoV-2 (Sunshine et al, 2022) infection. Importantly, high-content perturbation screens like these and that which we report here did not require a priori specification of phenotypes of interest but rather resulted in rich data resources that can be mined to rapidly generate and investigate hypotheses based on primary screening data alone (Bock et al, 2022), provided the achievement of adequate quantitative performance of the perturbation and phenotyping steps, and adequate cell sampling.…”

Section: Discussionmentioning

confidence: 99%

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Carlson,

Patten,

Stefanakis

et al. 2024

Preprint

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Ebola virus (EBOV) is a high-consequence filovirus that gives rise to frequent epidemics with high case fatality rates and few therapeutic options. Here, we applied image-based screening of a genome-wide CRISPR library to systematically identify host cell regulators of Ebola virus infection in 39,085,093 million single cells. Measuring viral RNA and protein levels together with their localization in cells identified over 998 related host factors and provided detailed information about the role of each gene across the virus replication cycle. We trained a deep learning model on single-cell images to associate each host factor with predicted replication steps, and confirmed the predicted relationship for select host factors. Among the findings, we showed that the mitochondrial complex III subunit UQCRB is a post-entry regulator of Ebola virus RNA replication, and demonstrated that UQCRB inhibition with a small molecule reduced overall Ebola virus infection with an IC50 of 5 μM. Using a random forest model, we also identified perturbations that reduced infection by disrupting the equilibrium between viral RNA and protein. One such protein, STRAP, is a spliceosome-associated factor that was found to be closely associated with VP35, a viral protein required for RNA processing. Loss of STRAP expression resulted in a reduction in full-length viral genome production and subsequent production of non-infectious virus particles. Overall, the data produced in this genome-wide high-content single-cell screen and secondary screens in additional cell lines and related filoviruses (MARV and SUDV) revealed new insights about the role of host factors in virus replication and potential new targets for therapeutic intervention.

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Section: Discussionmentioning

confidence: 99%

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Carlson,

Patten,

Stefanakis

et al. 2024

Preprint

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“…This is corroborated by studies by Sunshine et al, who identified NFKBIA in a Perturb-Seq study as a host protein supporting viral replication. 36 …”

Section: Discussionmentioning

confidence: 99%

“…This is corroborated by studies by Sunshine et al, who identified NFKBIA in a Perturb-Seq study as a host protein supporting viral replication. 36 It remains to be determined what the relevant upstream and downstream signals of this proviral phenotype are and whether the continuous presence of NF-κB in the cell nucleus provides benefits for the SARS-CoV-2 lifecycle. Our findings indicate that after overexpression of the mutant IκBα protein, nuclear p65 levels were lower in infected cells, supporting a model in which IκBα promotes viral infection by constraining, albeit not completely, the well-known antiviral activities of NF-κB.…”

Section: Discussionmentioning

confidence: 99%

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids

Simoneau¹,

Chen²,

Xing³

et al. 2022

Preprint

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As SARS-CoV-2 continues to spread worldwide, simple and tractable primary airway cell models that accurately recapitulate the cell-intrinsic response to arising viral variants are needed. Here we describe an adult stem cell-derived human airway organoid model overexpressing the ACE2 receptor that supports robust viral replication while maintaining 3D architecture and cellular diversity of the airway epithelium. ACE2-OE organoids were infected with SARS-CoV-2 variants and subjected to single-cell RNA-sequencing. NF-kB inhibitor alpha was consistently upregulated in infected epithelial cells, and its mRNA expression positively correlated with infection levels. Single-cell imaging showed more IkBa expression in infected cells than uninfected bystander cells, but found concurrent nuclear translocation of NF-kB that IkBa usually prevents. Overexpressing a non-degradable IkBa mutant reduced NF-kB translocation and increased viral infection. These data identify IkBa as a cellular rheostat that controls viral replication by tuning NF-kB. Incomplete NF-kB control in infected cells may promote inflammation and severe disease.

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“…However, we did not detect the overexpression of interferon (IFN) and IFN-stimulated genes (ISGs) in these populations, likely a consequence of virus-mediated repression at the protein level 6 . Previous studies have shown that cytokine production in virus-infected populations is often confined to small subpopulations of abortively infected cells [33][34][35] . Clustering cells purely based on their transcriptome patterns indeed revealed that stress-related and cytokine transcripts are concentrated in a small cluster of infected cells (Fig.…”

Section: Single-cell Resolution Of the Transcriptional Landscape In V...mentioning

confidence: 99%

Spatio-temporal analysis of Vaccinia virus infection and host response dynamics using single-cell transcriptomics and proteomics

Matía,

McCarthy,

Woosley

et al. 2024

Preprint

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Poxviruses are a large group of DNA viruses with exclusively cytoplasmic life cycles and complex gene expression programs. A number of systems-level studies have analyzed bulk transcriptome and proteome changes upon poxvirus infection, but the cell-to-cell heterogeneity of the transcriptomic response, and the subcellular resolution of proteomic changes have remained unexplored. Here, we measured single-cell transcriptomes of Vaccinia virus-infected populations of HeLa cells and immortalized human fibroblasts, resolving the cell-to-cell heterogeneity of infection dynamics and host responses within those cell populations. We further integrated our transcriptomic data with changes in the levels and subcellular localization of the host and viral proteome throughout the course of Vaccinia virus infection. Our findings from single-cell RNA sequencing indicate conserved transcriptome changes independent of the cellular context, including widespread host shutoff, heightened expression of cellular transcripts implicated in stress responses, the rapid accumulation of viral transcripts, and the robust activation of antiviral pathways in bystander cells. While most host factors were co-regulated at the RNA and protein level, we identified a subset of factors where transcript and protein levels were discordant in infected cells; predominantly factors involved in transcriptional and post-transcriptional mRNA regulation. In addition, we detected the relocalization of several host proteins such as TENT4A, NLRC5, and TRIM5, to different cellular compartments in infected cells. Collectively, our comprehensive data provide spatial and temporal resolution of the cellular and viral transcriptomes and proteomes and offer a robust foundation for in-depth exploration of virus-host interactions in poxvirus-infected cells.

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Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Cited by 7 publications

References 85 publications

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids

Spatio-temporal analysis of Vaccinia virus infection and host response dynamics using single-cell transcriptomics and proteomics

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