Hypothesis-driven RNAi Screen for HSV1 Gene Expression

In addition to the primary and secondary DG screens, 207 genes (Fig. 16; Supplement Table 2) were selected that could play a potential role at different steps of HSV1 cell entry such as proteins involved in the actin-myosin cytoskeleton, endocytosis, autophagy, innate immunity, microtubule transport, or nuclear import and export. Furthermore, we included genes implicated in virus susceptibility (Fumagalli et al., 2010), in cell entry of vaccinia (Moser et al., 2010), and from the 50-kinase HSV1 screen (Snijder et al., 2012).

Figure 16: 207 host genes were chosen for a hypothesis-driven RNAi screen on HSV1 gene expression.

The pie chart displays the functional groups covered by the screen and their fraction in the list of 207 genes.

Figure 17: Outline of the hypothesis-driven RNAi screen for HSV1 gene expression.

The hypothesis-driven screen was performed in parallel with the secondary DG screen using exactly the same protocol and analysis tools (c.f. Chapter 5.1.4.). In total, 88 HITs were identified by the hypothesis-driven RNAi screen; 51 host factors were classified as DOWN (25%) and 37 as UP (18%) HITs (Table 10).

Genes

mean median # 2 of 3 mean median # 2 of 3

Total number of HITs 13 15 67 6 8 47

DOWN (≤ 60 %) 6 14 50 4 4 16

UP (≥ 140 %) 7 1 17 2 4 31

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Table 10: Results of the hypothesis-driven RNAi screen on HSV1 gene expression. The data of the hypothesis-driven screen were analyzed by six different HIT scoring methods: three for the “% infected cells” data set (MEAN, MEDIAN, 2 of 3) and three for the “GFP/cell” data set (MEAN, MEDIAN, 2 of 3). The number of host factors whose perturbation decreased HSV1 gene expression below 60% are shown in red fields and host factors whose perturbation increased HSV1 gene expression above 140% in green fields for the MEAN or the MEDIAN, or if at least one of the MEANS derived from the 3 siRNA pairs scored as a HIT.

Similar to the results of the primary and secondary DG screens, most HITs were scored by the “2 of 3” infection index. The analysis of the “% infected cells” showed three times more DOWN than UP HITs, whereas about two times more UP than DOWN HITs were scored by the “GFP/cell” infection index (Fig. 18).

Figure 18: Overlap between the primary DG and the hypothesis-driven screen for HSV1 gene expression based on “% infected cells”. All targeted host factors were sorted according to the MEAN or MEDIAN, or # 2 of 3 of % infected cells and plotted in a dot plot (Ai-iii) each data point representing one host factor. The number of host factors classified as HITs based on analyzing the MEAN, MEDIAN, and “2 of 3” and shown in Venn diagrams (Bi-iii). The numbers for the primary DG screen are showed in dark blue fields and the numbers for the hypothesis-driven screen in light purple fields. The numbers of host factors whose silencing reduced HSV1 gene expression are shown in red fields, host factors whose silencing resulted in increased HSV1 gene expression are shown in green fields. The overlapping regions between circles contain the number of HITs falling into overlapping categories.

According the MEAN and the MEDIAN infection indices, there were only a few HITs (Fig. 18 Ai-ii) compared to the 3.6 times more HITs when based on the “2 of 3” infection index. The HITs of the “% infected cells” infection indices were compared to the raw data sets of the primary DG. There was only an overlap of one gene if classified by the MEAN but of 11

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genes according to the “2 of 3” infection index. Similar results were obtained for the

“GFP7cell” infection index (Fig. 19). These results were to be expected as the library of the hypothesis-driven screen was designed to expand the number of genes covered already by the DG screen.

Figure 19: Overlap between the primary DG and the hypothesis-driven screen for HSV1 gene expression based on “GFP/cell”. All targeted host factors were sorted according to the MEAN, MEDIAN, or # 2 of 3 of the GFP grey value per cell (GFP/cell) and plotted in a dot plot (Ai-iii) each data point representing one host factor.

The number of host factors classified as HITs based on analyzing the MEAN, MEDIAN, and “2 of 3” and shown in Venn diagrams (Bi-iii). The numbers for the primary DG screen are shown in dark blue fields and the numbers for the hypothesis-driven screen in purple fields. The numbers of host factors whose silencing reduced HSV1 gene expression are shown in red fields, host factors whose silencing resulted in increased HSV1 gene expression are shown in green fields. The overlapping regions between circles contain the number of HITs falling into overlapping categories.

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Table 11: Host factors of the hypothesis-driven screen resulting in decreased HSV1 gene expression. The data of the hypothesis-driven RNAi screen were analyzed by six different scoring methods: 3 for the “% infected cells” (mean, median, 2 of 3) and 3 for “GFP/cell” (mean, median, 2 of 3). The host factors were ranked according to the MEAN of the “% infected cells” infection index. The degree of reduced gene expression is shown in red fields for the mean and the median, for the “2 of 3” index the number of MEAN pairs resulting in HIT scoring are listed.

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Table 12: Host factors of the hypothesis-driven RNAi screen resulting in increased HSV1 gene expression. The data of the hypothesis-driven RNAi screen were analyzed by six different scoring methods: 3 for the “% infected cells” (mean, median, 2 of 3) and 3 for “GFP/cell” (mean, median, 2 of 3). The host factors were ranked according to the MEAN of the “% infected cells” infection index. The degree of increased gene expression is shown in green fields for the mean and the median, for the “2 of 3” index the number of MEAN pairs resulting in HIT scoring are listed.

Gene % GFP expressing cells GFP/cell

mean median 2 of 3 mean median 2 of 3 functions and pathways using the annotation tool DAVID (Huang da et al., 2008) that clusters host genes to functional annotations derived from public databases (Fig. 20). Four of the clusters are part of or regulate the cytoskeleton dynamics of the actin-myosin cytoskeleton and transport along microtubules (cortical cytoskeleton, cytoskeleton organization, cytoskeleton and myosin). Two clusters were assigned to the functions of innate immunity

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(response to virus, inflammatory response). Another three clusters were involved in vesicle formation and transport (vesicle, RAS small GTPase, endocytosis). In addition, the three functional clusters ligases, nuclear pores and kinases were identified.

Figure 20: Functional clustering of 66 HSV1 host factors identified in the hypothesis-driven RNAi screen.

The host factors were bundled in functional clusters (grey boxes with dashed outlines; DAVID). The gene clusters were positioned on the scheme of a single cell at their approximate subcellular localization.

5.1.6 Comparison to other siRNA Screens of HSV1 and other Viral Infections