Fig. 4: Multiplexed protein maps link cellular changes to infection heterogeneity.

a Heatmap of 4i marker intensities in 44 MCUs from infected and uninfected HeLa cells at 1.5–12 hpi (n = 5500 cells per time point; n_total = 27,500 cells). Order of markers and MCUs was calculated by applying hierarchical clustering to the distance matrix created by Euclidean distance measure. b Stacked bar plots summarize abundance of MCUs in three cell subpopulations (1, uninfected cells without infected neighbors; 2, uninfected cells with infected neighbors; 3, infected cells). Markers enriched in the selected MCUs are indicated. Dashed yellow lines: 1/3 and 2/3 abundance. c UMAP of MCUs colored by their abundance in infected versus uninfected cells at 12 hpi. MCUs were clustered using their spatial interactions from 27,500 infected and uninfected cells (1.5–12 hpi). Markers dominant in the selected MCUs are indicated. Size of each MCU was normalized to the largest MCU. d UMAP of MCUs colored by their abundance in high versus low-ICP27 expressing cells at 12 hpi. MCUs were clustered using their spatial interactions from 1520 infected cells at 12 hpi. MCU size on UMAP as in c. Low-expressing cells: mean intensity of ICP27 <35^th percentile. High-expressing cells: mean intensity of ICP27 >65^th percentile. e Infected HeLa cells at 12 hpi (n = 1520) were clustered to 13 subpopulations using abundance of each MCU in each cell and Leiden algorithm. Left heatmap: median of MCU abundance in a subpopulation. Right heatmap: median of viral feature in a subpopulation. Medians of subpopulations were standardized and then normalized between 0 and 1. Order of objects was calculated using distance matrix created by Euclidean distance measure and hierarchical clustering. For HSV-1 proteins, sum intensities are shown. For UL29 and UL19, spot counts are shown. f Spatial projections of MCUs onto example cells of selected subpopulations. Cells are not drawn to the scale. g Cell morphology features of single cells in selected subpopulations. n = cells per subpopulation. Boxplots definitions as in Fig. 1c. Outliers are indicated by points.