Author: Griffiths, Samantha J.; Koegl, Manfred; Boutell, Chris; Zenner, Helen L.; Crump, Colin M.; Pica, Francesca; Gonzalez, Orland; Friedel, Caroline C.; Barry, Gerald; Martin, Kim; Craigon, Marie H.; Chen, Rui; Kaza, Lakshmi N.; Fossum, Even; Fazakerley, John K.; Efstathiou, Stacey; Volpi, Antonio; Zimmer, Ralf; Ghazal, Peter; Haas, Jürgen
Title: A Systematic Analysis of Host Factors Reveals a Med23-Interferon-? Regulatory Axis against Herpes Simplex Virus Type 1 Replication Document date: 2013_8_8
ID: 0lyt8gfq_7
Snippet: Host factors (HFs) which positively or negatively regulate HSV-1 replication were identified by screening a druggable genome siRNA library (4 siRNAs per gene) targeting 7,237 human genes against a HSV-1 reporter virus expressing the enhanced green fluorescent protein (eGFP; HSV-1 strain C12) in the epithelial Hela cell line, due to their ease of transfection and susceptibility to HSV-1 infection [34] . To generate a robust and reliable dataset th.....
Document: Host factors (HFs) which positively or negatively regulate HSV-1 replication were identified by screening a druggable genome siRNA library (4 siRNAs per gene) targeting 7,237 human genes against a HSV-1 reporter virus expressing the enhanced green fluorescent protein (eGFP; HSV-1 strain C12) in the epithelial Hela cell line, due to their ease of transfection and susceptibility to HSV-1 infection [34] . To generate a robust and reliable dataset the screen was carried out three times in triplicate, with one replicate used in a cell viability assay to determine any cytotoxic effects of gene depletion and duplicates infected for the virus infection assay. The siRNA library was reversetransfected into Hela cells before infecting with HSV-1 and monitoring virus growth kinetics as a measure of GFP-fluorescence (Figure 1b). By following virus growth over multiple rounds of replication, host proteins involved in all stages of the virus life cycle can be identified. Replication slopes during linear growth were normalized to controls (mock-transfected cells, and cells transfected with a siRNA unable to be processed by the RNA Silencing Complex, RSCF) and the mean of six replicates was calculated. siRNAs found to be cytotoxic (81 in total) were excluded from further analyses, and a hitlist of 358 containing the top 2.5% inhibitory and the top 2.5% enhancing HFs was generated ( Table S1 in Text S2). The identified HSV-1 HFs were compared to datasets from published siRNA depletion screens aimed at identifying cellular factors affecting HIV-1 [7, 8, 9] , West Nile Virus (WNV) [14] , Hepatitis C Virus (HCV) [13] , Dengue virus [15] and Influenza A virus [10, 11, 12] . Of our 358 HFs, 54 cellular proteins (15.1%) overlapped with these other virus screens (Influenza A, 29; HIV-1, 24; HCV, 6; WNV, 2; Dengue virus, 1) ( Figure 1c ; Table S2 in Text S2).
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