Abstract
Genetic screens have transformed our ability to interrogate cellular factor requirements for viral infections1,2, but most current approaches are limited in their sensitivity, biased towards early stages of infection and provide only simplistic phenotypic information that is often based on survival of infected cells2,3,4. Here, by engineering human cytomegalovirus to express single guide RNA libraries directly from the viral genome, we developed virus-encoded CRISPR-based direct readout screening (VECOS), a sensitive, versatile, viral-centric approach that enables profiling of different stages of viral infection in a pooled format. Using this approach, we identified hundreds of host dependency and restriction factors and quantified their direct effects on viral genome replication, viral particle secretion and infectiousness of secreted particles, providing a multi-dimensional perspective on virus–host interactions. These high-resolution measurements reveal that perturbations altering late stages in the life cycle of human cytomegalovirus (HCMV) mostly regulate viral particle quality rather than quantity, establishing correct virion assembly as a critical stage that is heavily reliant on virus–host interactions. Overall, VECOS facilitates systematic high-resolution dissection of the role of human proteins during the infection cycle, providing a roadmap for in-depth study of host–herpesvirus interactions.
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Data availability
All next-generation sequencing data files have been deposited in Gene Expression Omnibus under accession number GSE246735.
Code availability
All scripts required for screen analysis as well as input files have been deposited to Zenodo (https://doi.org/10.5281/zenodo.10944653 (ref. 48)).
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Acknowledgements
The authors thank I. Ulitsky, S. Schwartz and the members of the Stern-Ginossar laboratory for critical reading of the manuscript; H. Hezroni-Bravyi for guidance during early protocol establishment; and the Weizmann flow cytometry unit for technical assistance. This study was supported by MRC (MR/S00971X/1) and Wellcome Trust (226615/Z/22/Z) and European Research Council consolidator grant to N.S.-G (CoG-2019-864012).
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Y.F., M.S. and N.S.-G. conceptualized the study. Y.F., E.A., T.A., E.S., M.D., T.F. and A.G. performed experiments. Z.S. and R.J.S. generated reagents. Y.F. and A.N. undertook data analysis. Y.F., M.S. and N.S.-G. interpreted data. Y.F., M.S. and N.S.-G. wrote the manuscript with contributions from all other authors.
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Extended data figures and tables
Extended Data Fig. 1 Establishment of VECOS.
a, Schematic representation of the constructs used in a Gateway recombination reaction, the expected recombination product, and the features that support or do not support bacterial growth under the selection conditions (chloramphenicol and the absence of toxin resistance gene). Cm(R), chloramphenicol resistance gene; CcdB, toxin encoding gene; AttR1/2 and AttL1/2, Gateway recombination sites; UL1 and RL12, representative viral genes. b, Rate of indels at genomic loci (RRM2 and TRIP10) targeted by HCMV-encoded sgRNAs in Cas9-expressing fibroblasts infected for 10 h or uninfected. p-values were calculated using a chi-square test comparing the number of reads with and without indels at 10 hpi vs mock. n = 283,351(TRIP10 mock), n = 220,933(TRIP10 10 hpi), n = 304,913(RRM2 mock), n = 310,731(RRM2 10 hpi) reads per sample. c, Relative indel rate in the targeted genomic loci of the indicated genes (RRM2 and TRIP10) in Cas9-expressing fibroblasts at different times post infections with HCMV encoding the targeting sgRNAs. Indel values are normalized to levels measured in uninfected cells. d, Percentage of sgRNAs represented in each stage of library cloning out of the total number of sgRNA in the initial sgRNAs library. e, Graphical depiction of measurements of sgRNA abundance inside cells and in infectious progeny.
Extended Data Fig. 2 VECOS screen identifies host factors that affect HCMV propagation.
a, Percentage of sgRNAs represented in each stage of the screen out of the total number of sgRNA in the initial oligo library. Red bars, infectious progeny samples; blue bars, cell samples. Input samples represent three technical repeats of the input library. The number indicates selection round (1-3) and a letter indicates biological replicate (A-C). b, The relative abundance of the non-targeting sgRNAs at different stages of the screen, values of each sgRNA were normalized to the mean abundance in the input samples. Red, infectious progeny samples; blue, cell samples. c, Bubble plots showing the MAGeCK RRA score, comparing the sgRNA abundance in the infectious progeny from each selection round to the input library. Log10 MAGeCK RRA score is shown. Genes that increased and decreased in abundance are represented by positive and negative values, respectively. Point size reflects –log10 false discovery rate (FDR). Genes with log10 scores greater than 4 or lower than −4 are highlighted in red. d, Volcano plot of fitness score and –log10 FDR as calculated in the regression analysis. Genes highlighted in blue or red are those defined as significant dependency or restriction factors, respectively. e, Cumulative plot of FDR as calculated by the regression analysis. FDR was used as –log10 FDR for genes that increased in abundance and as +log10 FDR for gene that decreased in abundance. Genes defined as significant dependency or restriction factors are highlighted in red. Genes identified as restriction or dependency factors by the MAGeCK analysis but not by the regression analysis are highlighted in yellow, and their names are indicated.
Extended Data Fig. 3 Examples of identified cellular hits by VECOS.
a-b, Comparison between fitness score and viability score. Viability score was defined as the fold-change from input to 10 days in uninfected (a) or HCMV infected (b) fibroblasts (Hein and Weissman 2021), the average of Cas9 and dCas9 screens was used and for genes with multiple isoform the minimum value was used. Genes coding for 26 S proteasome subunits are highlighted in dark gray. c-d, Fluorescence microscopy of fibroblasts expressing a strep-tagged version of ARL6IP6 (red) either uninfected or infected with HCMV strain expressing GFP fused to the tegument protein UL32, which localizes to the viral assembly compartment, (green) at 24, 72 hpi and 6 dpi (c) and 96 hpi (d). Nuclei are stained with Hoechst (blue) and in (d) Calnexin staining is shown in cyan. Scale bar is 10 μm. Images are representative of two independent experiments.
Extended Data Fig. 4 VECOS facilitates the quantification of effects of perturbations on particles secretion.
a-b, Relative levels of sgRNAs (geometric mean) targeting the indicated genes, FLI1 (a) and SRSF7 (b), at different stages along the screen (cells, blue; infectious progeny, red). The geometric means of sgRNAs targeting the gene are shown. Gray lines connect the values of the same biological replicate. c, Relative viral genome levels as measured by qRT-PCR from DNA collected from cell samples (gray) or from viral supernatant samples (pink). d-g, Relative levels of sgRNAs (geometric means) targeting the indicated genes, FLI1 (d), SRSF7 (e), RRM2 (f) and ARL6IP6 (g), along the screen (cells, blue; secreted particles, black; infectious progeny, red). Gray lines connect the values of the same biological replicate. h, An image of VECOS HCMV library infected cells at 5 dpi, representative of 3 images taken from each of three independent replicates. i-j, Scatter plots showing the correlation between changes in secretion (i) or infectiousness (j) and changes in total infectious progeny at 5 dpi. Each point represents the change in one sgRNA in one replicate of one round of selection. Data is presented for significantly changing genes. Pearson’s R values are shown.
Extended Data Fig. 5 Effects of host factors on three stages in HCMV life cycle.
a and b, Heat maps of sgRNA abundance fold-change of the screen hits that had significant effects of individual selection stages (replication, secretion, or infectiousness). For each gene, the geometric means of all targeting sgRNAs (a) or the values of each individual sgRNA (b) is shown for each replicate of each selection round. Log2 transformed fold-change ratios without normalization are shown in (a), and normalized log2 transformed fold-change ratios are shown in (b). c, Ratio of viral DNA in the supernatant and viral DNA inside cells transfected with control siRNA or siRNA targeting a component of the 20 S proteasome (PSMB3), or of the 19 S proteasome (PSMD6) and infected with HCMV at 5 dpi, as measured by qRT-PCR. Values are presented relative to the control sample. p-values were calculated using a two-sided Student’s t-test. Bars represent the means of n = 4 biological replicates. d-e, Relative RNA levels measured by RT-qPCR of (d) two proteasome genes (e) three HCMV genes (UL123, UL44 and UL99) in cells treated with control siRNA or siRNA targeting a component of the 20 S proteasome (PSMB3), or of the 19 S proteasome (PSMD6) and infected with HCMV for 72 h. Values are normalized to a host gene and the control samples. p-values were calculated with linear regression of the dependence of normalized values on siRNA treatment and the viral gene tested. Bars represent the means of n = 3 (d) biological or (e) technical replicates.
Extended Data Fig. 6 Validation of host factors effects on different stages of HCMV infection.
a-h. Measurement of viral titers (a, d, red bars) and secreted viral DNA (b-c, e-h, dark gray bars). (a, d) Viral supernatants were collected at 5 dpi from knockout cells and control cells (a) or cells expressing inducible knockout cells in which CAS9 was induced 16hpi (d) and transferred to recipient wild-type fibroblasts. After 48 h, the recipient cells were analyzed by flow cytometry. PFU/ml were calculated from the percentages of GFP positive cells. (b-c, e-h). Viral supernatants collected from knockout (b-c, f-g) or inducible-CAS9 (e) and control cells were treated with proteinase K, boiled, and then viral DNA was quantified by qRT-PCR. n = 3 (a,h), n = 2 (b,c,f,g), n = 4 (d,e) independent biological replicates.
Supplementary information
Supplementary Figures
This file contains Supplementary Figs. 1 and 2
Supplementary Table 1
sgRNA library sequences and counts
Supplementary Table 2
Screen results for individual genes
Supplementary Table 3
Clustering based of effect size in each stage along the screen
Supplementary Table 4
Sequences primers and oligonucleotides sequences
Supplementary Table 5
Sequences of individually cloned sgRNA
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Finkel, Y., Nachshon, A., Aharon, E. et al. A virally encoded high-resolution screen of cytomegalovirus dependencies. Nature 630, 712–719 (2024). https://doi.org/10.1038/s41586-024-07503-z
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DOI: https://doi.org/10.1038/s41586-024-07503-z
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