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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

WDR6 is a restriction factor for vK1L−C7L−/GFP+. (A) Generation of WDR6-depleted cells by CRISPR/Cas9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Cells from individual colonies 1 and 2 were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous WDR6, SAMD9, and actin. (B) HeLa cells and two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ (0.01 PFU per cell) and incubated for 18 h. GFP-positive cells were quantified using flow cytometry. Data from three experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that are significantly different (P ≤ 0.001) relative to the value for HeLa cells calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***). (C) HeLa cells and cells of two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ at 3 PFU per cell and mock transfected or transfected with C7L-V5 or K1L-FLAG regulated by the T7 promoter. Eighteen hours later, the cells were lysed and incubated with antibodies for the V5 or FLAG epitope tag. Input and proteins captured by magnetic beads conjugated to protein G were resolved by SDS-PAGE and Western blotting for endogenous SAMD9 and V5 or FLAG epitope tag.
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fig7: WDR6 is a restriction factor for vK1L−C7L−/GFP+. (A) Generation of WDR6-depleted cells by CRISPR/Cas9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Cells from individual colonies 1 and 2 were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous WDR6, SAMD9, and actin. (B) HeLa cells and two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ (0.01 PFU per cell) and incubated for 18 h. GFP-positive cells were quantified using flow cytometry. Data from three experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that are significantly different (P ≤ 0.001) relative to the value for HeLa cells calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***). (C) HeLa cells and cells of two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ at 3 PFU per cell and mock transfected or transfected with C7L-V5 or K1L-FLAG regulated by the T7 promoter. Eighteen hours later, the cells were lysed and incubated with antibodies for the V5 or FLAG epitope tag. Input and proteins captured by magnetic beads conjugated to protein G were resolved by SDS-PAGE and Western blotting for endogenous SAMD9 and V5 or FLAG epitope tag.

Mentions: WDR6 was the second strongest hit in the genome-wide screen. In order to confirm the siRNA data, we used CRISPR/Cas9 technology to inactivate the WDR6 gene in HeLa cells. There was a partial reduction of WDR6 expression in cell line 1 and a more complete inactivation in cell line 2, suggesting knockout of one and two alleles, respectively (Fig. 7A). In neither cell line, however, was a reduction in SAMD9 noted (Fig. 7A). Moreover, SAMD9 retained the ability to interact with C7 and K1 proteins in the absence of WDR6. Replication of vK1L−C7L− corresponded inversely to the WDR6 level: WDR6#2 > WDR6#1 > HeLa cells (Fig. 7B).


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)

WDR6 is a restriction factor for vK1L−C7L−/GFP+. (A) Generation of WDR6-depleted cells by CRISPR/Cas9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Cells from individual colonies 1 and 2 were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous WDR6, SAMD9, and actin. (B) HeLa cells and two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ (0.01 PFU per cell) and incubated for 18 h. GFP-positive cells were quantified using flow cytometry. Data from three experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that are significantly different (P ≤ 0.001) relative to the value for HeLa cells calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***). (C) HeLa cells and cells of two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ at 3 PFU per cell and mock transfected or transfected with C7L-V5 or K1L-FLAG regulated by the T7 promoter. Eighteen hours later, the cells were lysed and incubated with antibodies for the V5 or FLAG epitope tag. Input and proteins captured by magnetic beads conjugated to protein G were resolved by SDS-PAGE and Western blotting for endogenous SAMD9 and V5 or FLAG epitope tag.
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fig7: WDR6 is a restriction factor for vK1L−C7L−/GFP+. (A) Generation of WDR6-depleted cells by CRISPR/Cas9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Cells from individual colonies 1 and 2 were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous WDR6, SAMD9, and actin. (B) HeLa cells and two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ (0.01 PFU per cell) and incubated for 18 h. GFP-positive cells were quantified using flow cytometry. Data from three experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that are significantly different (P ≤ 0.001) relative to the value for HeLa cells calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***). (C) HeLa cells and cells of two WDR6 depleted colonies were infected with vK1L−C7L−/GFP+ at 3 PFU per cell and mock transfected or transfected with C7L-V5 or K1L-FLAG regulated by the T7 promoter. Eighteen hours later, the cells were lysed and incubated with antibodies for the V5 or FLAG epitope tag. Input and proteins captured by magnetic beads conjugated to protein G were resolved by SDS-PAGE and Western blotting for endogenous SAMD9 and V5 or FLAG epitope tag.
Mentions: WDR6 was the second strongest hit in the genome-wide screen. In order to confirm the siRNA data, we used CRISPR/Cas9 technology to inactivate the WDR6 gene in HeLa cells. There was a partial reduction of WDR6 expression in cell line 1 and a more complete inactivation in cell line 2, suggesting knockout of one and two alleles, respectively (Fig. 7A). In neither cell line, however, was a reduction in SAMD9 noted (Fig. 7A). Moreover, SAMD9 retained the ability to interact with C7 and K1 proteins in the absence of WDR6. Replication of vK1L−C7L− corresponded inversely to the WDR6 level: WDR6#2 > WDR6#1 > HeLa cells (Fig. 7B).

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