5 Results
5.1 Identification of Host Factors Contributing to HSV1 Gene Expression
5.1.3 Optimization for Secondary Druggable and Hypothesis-driven Screens
optimized for the follow-up screens. The infection time was reduced to measure viral gene expression as early as possible prior to the HSV1 induced enlargement of the nuclei (Simpson-Holley et al., 2005), and two new analysis algorithms were developed to streamline image processing, and to evaluate the viral infection. The number of seeded cells used in the primary DG screen had resulted in very high cell densities in the individual wells that hampered the efficiency of HSV1 infection (Marozin et al., 2004; Schelhaas et al., 2003;
Yoon and Spear, 2002) and the characterization of the cell population context (Snijder et al., 2009; Snijder et al., 2012). The analysis of population context depends on the classification of cells within their microenvironment in edge and non-edge cells for which a cell density of about 70% confluence is most suitable (Pauli Rämö and Lucas Pelkmans, Zürich, personal communication).
Figure 9: Optimal cell densities differed among chosen cell lines. HeLa CNX, HeLa MZ, HeLa Kyoto, RPE or HEp-2 cells were seeded and reversely transfected with scrambled siRNA in 384 well plates. At 72 h post transfection, the cells were fixed, stained with DAPI, and documented with an automated fluorescence microscope. For each cell line 5 representative pictures of different numbers of seeded cells are shown.
Therefore, the number of cells to be seeded for the different HeLa cell lines was further optimized, and HEp-2 and RPE cells that have been used extensively in HSV1 research (Fuller and Spear, 1987; Rosenthal et al., 1989; Topp et al., 1994; Wittels and Spear, 1991) were also tested under screening conditions. Cells of each line were seeded in 384 well plates and transfected with scrambled siRNA. After three days, the cells were fixed and the morphology of the cell lawns analyzed. HeLa CNX and RPE cells adhered over a larger
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substrate area and did not grow in in cell patches or cell islets. HeLa MZ, HeLa Kyoto and HEp-2 cells grew in cell patches, and exposed only a small area of plasma membrane to the medium, especially those cells that had grown to a colony (Fig. 9). The HeLa Kyoto cells were therefore not further analyzed, since they were characterized by a high level of auto-fluorescence that seemed to increase upon transfection (data not shown).
Cell line HeLa CNX HeLa MZ HeLa Kyoto RPE HEp-2
# of seeded cells 1 to 1.1 x 103 1.2 to 1.3 x 103 1 to 1.1 x 103 0.9 to 1 x 103 1 to 1.1 x 103 Linear GFP response
at virus dose 5 to 100 x 103 PFU/well - 5 to 100 x 103 PFU/well
grey value threshold 300 50 - 600 300
Table 6: Cell densities and HSV1 dosages used in the secondary DG and the hypothesis-driven screens.
Although their cell densities appeared lower than for the other three cell lines, HeLa CNX and RPE cells grew to approximately 70% confluence at 1,000 to 1,100 or 900 to 1,000 seeded cells, respectively. Good cell densities were obtained by seeding 1,000 to 1,100 HEp-2 and HeLa Kyoto cells, and 1,200 to 1,300 HeLa MZ cells per well (Table 6).
Figure 10: Automated image analysis of the secondary DG and the hypothesis-driven RNAi screens.
HSV1 infection indices were evaluated at the single cell levels using the MetaXpress software to determine the % of cells expressing GFP (% infected cells), or the CellProfiler software to measure the average GFP intensity per cell (GFP/cell).
The optimization protocols for the primary DG RNAi screen in Hannover had relied on measuring GFP using a plate reader as an indicator for HSV1 infection, but it turned out later that these results could not be directly transferred to the results obtained by automated fluorescence microscopy. Therefore, I developed two image-based methods for quantifying HSV1 gene expression with our automated fluorescence microscope in Hannover. With the image based analysis, I quantified the fraction of infected cells (% infected cells) and the amount of GFP expressed per cell (GFP/cell). For both algorithm pipelines, the nuclei were segmented first (Fig. 10). An infection index (% infected cells) was determined by setting a
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threshold grey value for infected cells in the direct surrounding of the segmented nuclei. If the grey value reached the threshold, the cells were classified as infected cells (Fig. 10, top row).
Furthermore, the grey value of the GFP channel was measured in the nuclear area expanded by a 3 pixel ring surrounding the nucleus of each cell (Fig. 10, bottom row). For both methods, the MEAN of all cells in all pictures per well was used for further analysis.
Figure 11: Checker boards for HSV1 concentrations and definitions of infection thresholds. HeLa CNX cells were seeded at 1,000 cells/well (Ai-Ci), HEp-2 at 1,000 cells/well, (Aii-Cii), RPE at 900 cells/well (Aiii-Ciii), and HeLa MZ at 1,200 cells/well (Aiv-Civ) and reversely transfected with 50 nM scrambled siRNA. After 72 h, the cells were infected with a range of HSV1 dosages for 8 h, fixed and the nuclei stained with DAPI. Images of each well were obtained using an automated fluorescence microscope. Image based analysis was performed to measure the amount of GFP expressed per cell (GFP/cell; Ai-Aiv, Bi-Biv). Different thresholds for a cut-off grey value (50, 150, 300 or 600) were used to determine an infection index based on the % of infected cells (Ci-Civ).
The data points show the MEANs from three independent experiments each performed in one well.
A good infection ratio for siRNA screens appears to be 20 to 30% for cells transfected with a scrambled, non-targeting siRNA (Mercer et al., 2012; Pelkmans et al., 2005; Snijder et al., 2009; Snijder et al., 2012; Wippich et al., 2013). The different cell lines were infected with a range of HSV1 concentrations to obtain such conditions at which perturbations that increase or decrease viral infection could be identified. The amount of GFP expressed varied substantially between different cell lines. While HeLa CNX (Fig. 11, Ai) and HEp-2 cells (Fig.
11, Aii) synthesized GFP to similar levels, RPE cells showed a ~2.5fold higher GFP expression (Fig. 11, Aiii), while infection of HeLa MZ cells was rather restricted so that
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infected cells were only identified unequivocally at higher HSV1 concentrations (Fig. 11, Aiv).
For each cell line, the GFP expression was set to 100% for the highest virus concentration used. Thereby, a range of HSV1 doses could be identified that resulted in a linear relationship to the amount of GFP synthesized (Fig. 11, Bi-Biv; Tab. 6 below). To define an infection index (% infected cells), different thresholds ranging from grey values of 50 to 600 were then tested to identify GFP expressing cells (Fig. 11 Ci-Civ). At a low threshold of 50, nearly all HeLa CNX, HEp-2 and RPE cells (Fig. 11 Ci-iii) were infected at 1 x 105 PFU/well (2.5 PFU/ mL). However, even at a high virus concentrations of 1 x 106 PFU/well (2.5 x 107 PFU/mL), only 28% of the HeLa MZ cells became infected (Fig. 11 Civ). In summary, these optimization experiments identified conditions at which HSV1 infection in different cell lines can be analyzed.