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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

Validation of SAMD9 as a major host restriction factor for vK1L−C7L−/GFP+. (A) Comparison of positive-hit siRNAs. The indicated siRNAs were individually transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ per cell and incubated for 18 h. GFP-positive cells were scored by flow cytometry. Data from three experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001; *, P ≤ 0.05. (B) Replication of vK1L−C7L−/GFP+ in HeLa cells transfected with SAMD9 siRNA. Three different SAMD9 siRNAs and control siRNA were transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ and incubated for 24 h. Cells were lysed by freezing and thawing, and infectious virus titers were determined by plaque assay on permissive BS-C-1 cells. Data from two experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.005) from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated (**). (C) Synthesis of viral proteins in HeLa cells transfected with SAMD9 siRNA and infected with vK1L−C7L−/GFP+. HeLa cells were transfected with control (siCtr) or three different SAMD9-specific siRNAs for 48 h and then infected with 3 PFU of vK1L−C7L−/GFP+ per cell for the indicated hours postinfection (HPI). The cells were lysed, and the proteins were resolved by SDS-PAGE and Western blotting with broadly reactive antibodies to VACV proteins. The electrophoretic positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.
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fig2: Validation of SAMD9 as a major host restriction factor for vK1L−C7L−/GFP+. (A) Comparison of positive-hit siRNAs. The indicated siRNAs were individually transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ per cell and incubated for 18 h. GFP-positive cells were scored by flow cytometry. Data from three experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001; *, P ≤ 0.05. (B) Replication of vK1L−C7L−/GFP+ in HeLa cells transfected with SAMD9 siRNA. Three different SAMD9 siRNAs and control siRNA were transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ and incubated for 24 h. Cells were lysed by freezing and thawing, and infectious virus titers were determined by plaque assay on permissive BS-C-1 cells. Data from two experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.005) from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated (**). (C) Synthesis of viral proteins in HeLa cells transfected with SAMD9 siRNA and infected with vK1L−C7L−/GFP+. HeLa cells were transfected with control (siCtr) or three different SAMD9-specific siRNAs for 48 h and then infected with 3 PFU of vK1L−C7L−/GFP+ per cell for the indicated hours postinfection (HPI). The cells were lysed, and the proteins were resolved by SDS-PAGE and Western blotting with broadly reactive antibodies to VACV proteins. The electrophoretic positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.

Mentions: Ambion siRNAs targeting the selected genes and control siRNA were transfected individually into HeLa cells, which were subsequently infected with 0.01 PFU of vK1L−C7L−/GFP+ per cell and analyzed for GFP fluorescence by flow cytometry. The siRNAs to WDR6, although less effective than the siRNA to SAMD9, allowed the host range mutant to spread to about 15% of the cells (Fig. 2A). While still lower in efficacy, the siRNAs to FTJ1 and CDC37 increased spread above that of the control siRNA with a P value of <0.01 for the former (Fig. 2A). In the present study, we focused on SAMD9 and to a lesser extent on WDR6 as major human cell restriction factors for the K1L−C7L− host range mutant.


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)

Validation of SAMD9 as a major host restriction factor for vK1L−C7L−/GFP+. (A) Comparison of positive-hit siRNAs. The indicated siRNAs were individually transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ per cell and incubated for 18 h. GFP-positive cells were scored by flow cytometry. Data from three experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001; *, P ≤ 0.05. (B) Replication of vK1L−C7L−/GFP+ in HeLa cells transfected with SAMD9 siRNA. Three different SAMD9 siRNAs and control siRNA were transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ and incubated for 24 h. Cells were lysed by freezing and thawing, and infectious virus titers were determined by plaque assay on permissive BS-C-1 cells. Data from two experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.005) from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated (**). (C) Synthesis of viral proteins in HeLa cells transfected with SAMD9 siRNA and infected with vK1L−C7L−/GFP+. HeLa cells were transfected with control (siCtr) or three different SAMD9-specific siRNAs for 48 h and then infected with 3 PFU of vK1L−C7L−/GFP+ per cell for the indicated hours postinfection (HPI). The cells were lysed, and the proteins were resolved by SDS-PAGE and Western blotting with broadly reactive antibodies to VACV proteins. The electrophoretic positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig2: Validation of SAMD9 as a major host restriction factor for vK1L−C7L−/GFP+. (A) Comparison of positive-hit siRNAs. The indicated siRNAs were individually transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ per cell and incubated for 18 h. GFP-positive cells were scored by flow cytometry. Data from three experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001; *, P ≤ 0.05. (B) Replication of vK1L−C7L−/GFP+ in HeLa cells transfected with SAMD9 siRNA. Three different SAMD9 siRNAs and control siRNA were transfected into HeLa cells in a 24-well format. After 48 h, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+ and incubated for 24 h. Cells were lysed by freezing and thawing, and infectious virus titers were determined by plaque assay on permissive BS-C-1 cells. Data from two experiments each carried out in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.005) from the siControl value calculated by Bonferroni test after the one-way ANOVA test using PRISM GraphPad software are indicated (**). (C) Synthesis of viral proteins in HeLa cells transfected with SAMD9 siRNA and infected with vK1L−C7L−/GFP+. HeLa cells were transfected with control (siCtr) or three different SAMD9-specific siRNAs for 48 h and then infected with 3 PFU of vK1L−C7L−/GFP+ per cell for the indicated hours postinfection (HPI). The cells were lysed, and the proteins were resolved by SDS-PAGE and Western blotting with broadly reactive antibodies to VACV proteins. The electrophoretic positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel.
Mentions: Ambion siRNAs targeting the selected genes and control siRNA were transfected individually into HeLa cells, which were subsequently infected with 0.01 PFU of vK1L−C7L−/GFP+ per cell and analyzed for GFP fluorescence by flow cytometry. The siRNAs to WDR6, although less effective than the siRNA to SAMD9, allowed the host range mutant to spread to about 15% of the cells (Fig. 2A). While still lower in efficacy, the siRNAs to FTJ1 and CDC37 increased spread above that of the control siRNA with a P value of <0.01 for the former (Fig. 2A). In the present study, we focused on SAMD9 and to a lesser extent on WDR6 as major human cell restriction factors for the K1L−C7L− host range mutant.

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