Spatiotemporal resolution of suppression and recovery of protein synthesis at single cell level during cell-cell transmission.

<p>(A) Representation of a field of uninfected cells pulse-labeled using 0.5 mM of HPG for 30 min, showing homogenous levels of protein synthesis in all cells. A magnified view of a single cell is shown in the right hand panel. (B) Workflow of standard HPG pulse-labeling during single particle infection. Cells were infected with single particle and pulse-labeled with HPG from 24.5 to 25 hr p.i. before processing. (C) Cells were infected with HSV-2[186] according to the standard workflow, and stained for VP5 (red), followed by the click reaction. Three different zones of protein synthesis (green channel) are demarcated by the dotted lines. The central zone of active protein synthesis corresponds to the developing plaque and is co-stained with a viral antigen marker (red channel). This zone is surrounded by cells (see DAPI staining blue channel) which are severely suppressed for protein synthesis but appear morphologically normal and with no apparent virus antigen yet. Reference cells are numbered in this zone and corresponding panels for DAPI and phase. This translationally supressed zone is further surrounded by cells exhibiting normal levels of protein synthesis. The merged image is shown in the right hand panel. (D) Cells were infected as above but with HSV-1[KOS] and stained for ICP4 (red), followed by the click reaction. (E) Quantitative analysis of protein synthesis levels based on HPG intensity (green) using Image Pro Plus software (Media Cybernetics). Translation levels were colour coded with yellow representing a threshold of 30% or below of the maximum observed and pink above this threshold (panel iv).