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. 2021 Jan 7;296:100236. doi: 10.1074/jbc.RA120.016571

Figure 1.

Figure 1

Timestamping the viral replication state inside individual cells.A, schematic illustration of HSV-1 replication mechanism and the engineered reporter virus expression pattern. Fluorescently labeled Immediate early (ICP0) and late (gC) gene expression products mark different stages during lytic infection. B, structured illumination microscopy (SIM) was used to generate high-resolution images with low background. C, classification using SIM images of HFF cells infected with eYFP-ICP0/gC-mCherry HSV-1. Live cells were imaged at 3.5, 5.5, 7.5, and 9.5 h post infection (hpi). Appearance and localization of the two fluorescently labeled viral proteins can be correlated with four distinct stages in infection (stage 1–stage 4). D, representative images taken by SIM (top images show ICP0 and bottom images show gC) and (E) widefield microscopy display great variation of infection stages at each time point (hpi). Widefield images were taken at 6 hpi, both SIM and widefield images at MOI 3. Color of arrows indicates the stage of infection. Cells that do not show expression of viral proteins are counted as noninfected. F, progression of infection on the single cell level for different multiplicities of infection (MOIs) by classification of widefield images as shown in (E). Over 135 cells were analyzed for each time point (3–8 hpi) and MOI (MOI 0.3: n = 255, 245, 297, 240, 204, 264; MOI 3: n = 202, 223, 213, 190, 193, 161; MOI 30: n = 135, 194, 166, 137, 161, 169). Scale bar 10 μm.