Fig 1.
Relative fluorescence intensity of infected cells correlates with viral gene expression.
(A-B) Cells were infected with the barcode-viruses at MOI 10 (A) or 100 (B). At 3 HPI, the cells were sorted into three groups according to their fluorescence intensity from lowest (blue 30% of total), through middle (base line levels 40%) and highest (red 30%). Total RNA was collected from each group and RT-qPCR was performed for several viral genes as indicated. In each experiment, expression levels from highest and lowest groups were normalized to that in the middle group. Box plots of five biological repeats generated by SPSS are presented. Outliers (1.5<box length<3) are marked with circles, and extreme scores (3<box length) are marked by star shapes. (C, D) Vero cells infected with OK21 viral recombinants at MOI 10 (C) and MOI 100 (D) were visualized during the infection at the indicated time points. The graphs represent the relative yellow fluorescence (mVenus) vs. the relative red fluorescence (mCherry). Each graph represents 3 biological replicates, in each replicate three frames were analyzed. A total of 671 and 639 cells were plotted for MOI 10 and MOI 100, respectively. A trend line that was calculated using the ordinary least squares (OLS) method and the measured Pearson correlation, are presented in each graph.
Fig 2.
Relative fluorescence is maintained throughout infection.
(A-E) Cells infected with a mixture of the barcode-viruses at MOI 100 were monitored for 18 hours. (A) Snapshots of cells at different time points (as indicated) are presented. Scale bar 20μm. (B-E) The infected cells were divided according to the fluorescence intensity of the cells at 4 HPI, from lowest (blue 30% of total), through middle (purple 40%) and highest (red 30%). (B) The fluorescent profile of 97 individual cells from a representative single well (4 different frames) is presented as a function of time. (C) The mean of the cell fluorescent profiles, for each of the groups of fluorescence intensity, was calculated for each well. A mean of three wells (with the standard deviation between the wells-stripe lines) is presented. (D-E) At each time point indicated, cell profiles were sorted according to fluorescence levels. The 30% lowest population (D) and 30% highest population (E) were compared to the 4 HPI time point. Each bar represents the ratio of cells from the 4 HPI segregation (colored as above), as found at the indicated time point. An average of the ratios from three different wells is presented. (F-G) Cells were infected with three different fluorescence expressing viral recombinants (each expressing one of the following fluorescent genes: mCherry, EYFP or mTurq2) at MOI of 100. Ten representative cells were plotted according to their hues on triangular barymetric plot. Each vertex represents a different pure color (red, blue and green), edges represent a combination of two colors and the inside of the triangle represent mixture of three colors. (F) The ten cells (dots) are shown at 3 HPI as indicated. (G) A three dimensional plot of the changes in hue over time (z-axis) for the ten cells (lines) is presented. Each cell is plotted in a representative color.
Fig 3.
Experimental system to identify replicating HSV-1 barcodes in single cells.
A schematic illustration of the steps undertaken in a single cell experiment. (A) Genetically-tagged HSV-1 viruses were evenly mixed and used to inoculate Vero cells (B) The infected cells were incubated for 3 hours, then detached from the plate and transferred to a sorting flow cytometer. (C) The cells were sorted according to their fluorescence intensity into a 96-well plate. (D) Each well of the 96-well plate was pre-seeded with uninfected Vero cells, such that the sorted infected cell was dropped onto a layer of Vero cells. (E) At 24 and 48 HPI, each well was scanned with a fluorescence microscope to discover a single-focus plaque per well, which was further characterized. (F) At 144 HPI, the cells were lysed and analyzed with qPCR to discover the identity and number of barcode progeny.
Fig 4.
The number of barcodes replicating in individual Vero cells correlates with fluorescence.
(A) The relative proportion of each barcode of the total number of barcodes detected from all infectious centers analyzed. The results are presented according to MOI, and each barcode is represented by a different color. (B-E) The output infectious centers, each originated from one individual infected cell, were lysed and analyzed with qPCR. A total of 137 cells from three experiments are presented. (B, C) The distribution of the number of barcodes per cell is depicted at MOI 10 (B, open bars) and MOI 100 (C, solid bars). Each MOI is categorized according to the fluorescence intensity of the cells, from low (blue), through intermediate (purple) and high (red). (D, E) The number of replicated barcode progeny from individual cells, infected at MOI 10 (D) and MOI 100 (E), was plotted against expressed fluorescence, as measured by the cell sorter. Each point is color coded as above. A trend line that was calculated using the ordinary least squares (OLS) method and the measured Pearson correlation, are presented in each graph.
Fig 5.
The correlation between the number of barcodes replicating in cells and viral fluorescence is maintained in individual human fibroblasts.
(A) The relative proportion of each barcode of the total number of barcodes detected from all infectious centers analyzed. The results are presented according to MOI, and each barcode is represented by a different color. (B-E) The output infectious centers, each originated from one individual infected cell, were lysed and analyzed with qPCR. A total of 135 cells from three experiments are presented. (B, C) A distribution of the number of barcodes per cell is depicted at MOI 10 (B, open bars) and MOI 100 (C, solid bars). Each MOI is categorized according to the fluorescence intensity of the cells, from low (pink), through intermediate (light red) and high (red). (D, E) The number of replicated barcode progeny from individual cells, infected at MOI 10 (D) and MOI 100 (E), was plotted against expressed fluorescence, as measured by the cell sorter. Each point is color coded as above. A trend line that was calculated using the ordinary least squares (OLS) method and the measured Pearson correlation, are presented in each graph.
Fig 6.
Cells with higher expression of a housekeeping gene have a decreased expression of viral genes.
GFP expressing HeLa cells were infected with one of the barcoded viruses. (A). A representative image at 8 HPI is shown. Scale bar 10μm. (B). Fluorescence levels from GFP (shades of green) and mCherry (shade of red) were monitored during HeLa cells infection every 30 minutes for 10 HPI. Infection at MOI 100 (darkest color) or MOI 10 were compared to uninfected cells (lightest color). An average fluorescence of nine frames from cells infected with one of three viral recombinants (three from each recombinant) is shown. Standard deviation for each time point is represented. (C, D) A combination of 780 cells infected with one of three viral recombinants, (two frames from each recombinant, 130 cells per frame) were analyzed from images taken at 10 HPI. Cells were grouped according to the relative fluorescence levels in both GFP and mCherry as indicated for MOI 10 (C) and MOI 100 (D). Each number in the Heatmap represent the proportions of cells out of the total cells analyzed. (E, F) A combination of 1200 cells from 12 different LARCs infected with one viral recombinant, (two frames from each LARC, 50 cells per frame) were analyzed from images taken at 8 HPI. Cells were grouped according to the relative fluorescence levels in both GFP and mCherry as indicated for MOI 10 (E) and MOI 100 (F). Each number in the Heatmap represents the proportions of cells out of the total cells analyzed. The color scheme reflects the relative proportions as indicated in the side bar (log 2 scale).
Fig 7.
The correlation between the number of barcodes replicating in cells and viral fluorescence is maintained in gHeLa cells.
(A) The relative proportion of each barcode of the total number of barcodes detected from all infectious centers analyzed. The results are presented per MOI and each barcode is represented by a different color. (B-G) The output infectious centers, each originated from one individual infected gHela cell, were lysed and analyzed with qPCR. A total of 117 cells from four experiments are presented. For each MOI (10—open bars/diamonds and 100—solid bars/diamonds), the cells are divided according to fluorescence intensity, from low (yellow), through intermediate (orange) and high (red). (B, C) A distribution of the number of barcodes per cell is depicted at MOI 10 (B) and MOI 100 (C). (D, E) The number of replicated barcode progeny from individual cells, infected at MOI 10 (D) and 100 (E), was plotted against expressed green (cellular) fluorescence, as measured by the cell sorter. (F, G) The number of replicated barcode progeny from individual cells, infected at MOI 10 (F) and 100 (G), was plotted against expressed red (viral) fluorescence, as measured by the cell sorter. A trend line that was calculated using the ordinary least squares (OLS) method and the measured Pearson correlation is presented in each graph.
Fig 8.
A mathematical model predicts the number of barcodes replicated per cell.
Four linear models based on results from all 389 cells (A), Vero cells (B), HFF (C) and gHeLa cells (D) were constructed. The Vero (purple), HFF (light red) and HeLa (orange) cells infected in MOI 10 (open diamonds) and MOI 100 (solid diamonds) are plotted. The predicted number of barcodes per cell was plotted against the measured number of barcodes for all 389 cells. Linear trend lines for each experiment are marked (best fit in black and forced to intercept 0 in gray). (E, F) Results derived from all 389 cells (red), Vero cells (purple), HFF (light red) and gHeLa cells (yellow) are shown. (E) Pearson correlations from each model are plotted against each cell type separately and all together (as indicated). (F) The slopes of the linear trend lines (forced to intercept at 0; slopes of gray lines in A-D and S6 Fig) are plotted.