Defining the host dependencies and the transcriptional landscape of RSV infection and bystander activation

Figure 1:. Experimental design and infection characterization. A. Experimental approach for characterizing the transcriptional response to RSV infection in single cells. B. We quantified the percent infection for each time point and condition. In the bar plot, the number of cells per time point and condition is displayed. C. Single-cell transcriptomes for all conditions were projected into UMAP space and cells treated with heat inactivated RSV or live RSV are colored by viral transcript fraction per cell and overlaid onto vehicle control treated cells (gray).

Figure 2:. Single-cell transcriptome characterization 12 hours after RSV infection. A-C. Single-cell transcriptomes were projected into UMAP space and the density of cells for each condition were overlaid onto all cells (gray). The RSV infected condition (C) was further broken down by classification as infected or bystander cells. D. Transcriptional analysis of infected and bystander cells revealed distinct patterns in UMAP space, including differential expression for interferon-stimulated genes (ISG15, IFIT1) and genes associated with the unfolded protein response (HERPUD1, DDIT3). E. In the dot plot, mean expression for a subset of differentially expressed genes is plotted by condition. Dot size indicates fraction of cells in a group and color indicates mean gene expression.

Figure 3:. Genome-wide CRISPR/Cas9 knockout screen in human A549 cells for identification of host factors important for RSV infection. A. Experimental design for CRISPR/Cas9 screens. B-C. Gene enrichment scores for the “Viability Screen” (B) and the “Early Infection Screen” (C). Enrichment scores were determined using the MAGeCK algorithm [23] and gene hits are colored by function. The top hit for both screens, TMEM165, is highlighted in brown. A complete list of enrichment scores and genes can be found in Table S2.

Figure 4:. Comparison of RSV screening results with 29 published CRISPR/Cas9 screens for viral dependency factors. Host factor genes that were identified in at least one CRISPR screen using the MAGeCK analysis method (FDR < 0.01) were visualized on a heatmap for 29 published screens in addition to the two CRISPR screens from this study. Rows represent genes and columns represent CRISPR screen datasets. For genes that passed a threshold of −log10(FDR) > 1.3, heatmap blocks are colored by the −log10(FDR).