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Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1L-C7L- Mutant.

Sivan G, Ormanoglu P, Buehler EC, Martin SE, Moss B - MBio (2015)

Bottom Line: Knockout of WDR6 did not reduce the levels of SAMD9 and interactions of WDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway.The coevolution of microbial pathogens with cells has led to an arms race in which the invader and host continuously struggle to gain the advantage.However, host-restricted viral mutants have lost one or more defense genes needed for their replication in nonpermissive cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus

Rescue of vK1L−C7L−/GFP+ by siRNAs. (A) Replication of vK1L−C7L−/GFP+ in monkey BS-C-1 cells, but not in human HeLa cells. Cells were infected with wild-type VACV expressing GFP (vWT) or vK1L−C7L−/GFP+ and incubated for 48 h at 37°C with a methylcellulose overlay. The images were taken with a fluorescence microscope. (B) Effect of virus multiplicity on virus spread. HeLa and BS-C-1 cells were seeded into 384-well plates, and 48 wells of each cell type were infected with each serial dilution of vK1L−C7L−/GFP+. After 18 h, the cells were fixed with 2% paraformaldehyde. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and imaged by automated fluorescence microscopy. GFP-positive cells were scored and plotted against a logarithmic scale of the multiplicity of infection (MOI). Values are means ± standard deviations (error bars). Values that are significantly different (P ≤ 0.0001) as calculated by two-way ANOVA and Bonferroni test after ANOVA comparing each dilution in permissive versus nonpermissive cells are indicated by an asterisk. Calculations were made using PRISM by GraphPad. (C) SAMD9 siRNAs restore replication of vK1L−C7L−/GFP+ in HeLa cells. Images of the GFP channel (top panels) and DAPI stain (bottom panels) were taken from the genome-wide siRNA screens. Three separate SAMD9 siRNAs and a control siRNA (siControl) were tested. The percentages of GFP-positive cells are indicated.
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fig1: Rescue of vK1L−C7L−/GFP+ by siRNAs. (A) Replication of vK1L−C7L−/GFP+ in monkey BS-C-1 cells, but not in human HeLa cells. Cells were infected with wild-type VACV expressing GFP (vWT) or vK1L−C7L−/GFP+ and incubated for 48 h at 37°C with a methylcellulose overlay. The images were taken with a fluorescence microscope. (B) Effect of virus multiplicity on virus spread. HeLa and BS-C-1 cells were seeded into 384-well plates, and 48 wells of each cell type were infected with each serial dilution of vK1L−C7L−/GFP+. After 18 h, the cells were fixed with 2% paraformaldehyde. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and imaged by automated fluorescence microscopy. GFP-positive cells were scored and plotted against a logarithmic scale of the multiplicity of infection (MOI). Values are means ± standard deviations (error bars). Values that are significantly different (P ≤ 0.0001) as calculated by two-way ANOVA and Bonferroni test after ANOVA comparing each dilution in permissive versus nonpermissive cells are indicated by an asterisk. Calculations were made using PRISM by GraphPad. (C) SAMD9 siRNAs restore replication of vK1L−C7L−/GFP+ in HeLa cells. Images of the GFP channel (top panels) and DAPI stain (bottom panels) were taken from the genome-wide siRNA screens. Three separate SAMD9 siRNAs and a control siRNA (siControl) were tested. The percentages of GFP-positive cells are indicated.

Mentions: We employed the VACV mutant vK1L−C7L−/GFP+ with a deletion of the K1L gene and replacement of the C7L gene with an open reading frame (ORF) encoding the green fluorescent protein (GFP) regulated by a VACV late promoter (7). GFP fluorescence was a sensitive and appropriate readout, since late genes are not expressed in nonpermissive cells. The ability of vK1L−C7L−/GFP+ to replicate in African green monkey BS-C-1 cells, but not in human HeLa cells, is shown by plaque formation in Fig. 1A. To prepare for the siRNA screen, we calibrated conditions by parallel infections of permissive BS-C-1 and restrictive HeLa cell lines in a 384-well plate with serial dilution of the mutant virus. GFP-positive (GFP+) cells were scored using automated fluorescence microscopy. Even at the highest multiplicity of infection, there were few GFP+ HeLa cells (Fig. 1B). We empirically chose to use a multiplicity of 0.1 PFU of the mutant virus per cell, which resulted in the spread of virus to ~50% of the permissive cells after 18 h but close to zero in the nonpermissive cells (Fig. 1B). These conditions provided an extremely robust screen capable of detecting even minute changes in virus replication.


Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1L-C7L- Mutant.

Sivan G, Ormanoglu P, Buehler EC, Martin SE, Moss B - MBio (2015)

Rescue of vK1L−C7L−/GFP+ by siRNAs. (A) Replication of vK1L−C7L−/GFP+ in monkey BS-C-1 cells, but not in human HeLa cells. Cells were infected with wild-type VACV expressing GFP (vWT) or vK1L−C7L−/GFP+ and incubated for 48 h at 37°C with a methylcellulose overlay. The images were taken with a fluorescence microscope. (B) Effect of virus multiplicity on virus spread. HeLa and BS-C-1 cells were seeded into 384-well plates, and 48 wells of each cell type were infected with each serial dilution of vK1L−C7L−/GFP+. After 18 h, the cells were fixed with 2% paraformaldehyde. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and imaged by automated fluorescence microscopy. GFP-positive cells were scored and plotted against a logarithmic scale of the multiplicity of infection (MOI). Values are means ± standard deviations (error bars). Values that are significantly different (P ≤ 0.0001) as calculated by two-way ANOVA and Bonferroni test after ANOVA comparing each dilution in permissive versus nonpermissive cells are indicated by an asterisk. Calculations were made using PRISM by GraphPad. (C) SAMD9 siRNAs restore replication of vK1L−C7L−/GFP+ in HeLa cells. Images of the GFP channel (top panels) and DAPI stain (bottom panels) were taken from the genome-wide siRNA screens. Three separate SAMD9 siRNAs and a control siRNA (siControl) were tested. The percentages of GFP-positive cells are indicated.
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fig1: Rescue of vK1L−C7L−/GFP+ by siRNAs. (A) Replication of vK1L−C7L−/GFP+ in monkey BS-C-1 cells, but not in human HeLa cells. Cells were infected with wild-type VACV expressing GFP (vWT) or vK1L−C7L−/GFP+ and incubated for 48 h at 37°C with a methylcellulose overlay. The images were taken with a fluorescence microscope. (B) Effect of virus multiplicity on virus spread. HeLa and BS-C-1 cells were seeded into 384-well plates, and 48 wells of each cell type were infected with each serial dilution of vK1L−C7L−/GFP+. After 18 h, the cells were fixed with 2% paraformaldehyde. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and imaged by automated fluorescence microscopy. GFP-positive cells were scored and plotted against a logarithmic scale of the multiplicity of infection (MOI). Values are means ± standard deviations (error bars). Values that are significantly different (P ≤ 0.0001) as calculated by two-way ANOVA and Bonferroni test after ANOVA comparing each dilution in permissive versus nonpermissive cells are indicated by an asterisk. Calculations were made using PRISM by GraphPad. (C) SAMD9 siRNAs restore replication of vK1L−C7L−/GFP+ in HeLa cells. Images of the GFP channel (top panels) and DAPI stain (bottom panels) were taken from the genome-wide siRNA screens. Three separate SAMD9 siRNAs and a control siRNA (siControl) were tested. The percentages of GFP-positive cells are indicated.
Mentions: We employed the VACV mutant vK1L−C7L−/GFP+ with a deletion of the K1L gene and replacement of the C7L gene with an open reading frame (ORF) encoding the green fluorescent protein (GFP) regulated by a VACV late promoter (7). GFP fluorescence was a sensitive and appropriate readout, since late genes are not expressed in nonpermissive cells. The ability of vK1L−C7L−/GFP+ to replicate in African green monkey BS-C-1 cells, but not in human HeLa cells, is shown by plaque formation in Fig. 1A. To prepare for the siRNA screen, we calibrated conditions by parallel infections of permissive BS-C-1 and restrictive HeLa cell lines in a 384-well plate with serial dilution of the mutant virus. GFP-positive (GFP+) cells were scored using automated fluorescence microscopy. Even at the highest multiplicity of infection, there were few GFP+ HeLa cells (Fig. 1B). We empirically chose to use a multiplicity of 0.1 PFU of the mutant virus per cell, which resulted in the spread of virus to ~50% of the permissive cells after 18 h but close to zero in the nonpermissive cells (Fig. 1B). These conditions provided an extremely robust screen capable of detecting even minute changes in virus replication.

Bottom Line: Knockout of WDR6 did not reduce the levels of SAMD9 and interactions of WDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway.The coevolution of microbial pathogens with cells has led to an arms race in which the invader and host continuously struggle to gain the advantage.However, host-restricted viral mutants have lost one or more defense genes needed for their replication in nonpermissive cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

No MeSH data available.


Related in: MedlinePlus