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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

Interferon induces SAMD9 expression in Huh-7.5.1 cells and inhibits replication of vK1L−C7L−/GFP+. (A) Induction of SAMD9 in Huh-7.5.1 cells. Huh-7.5.1 and HeLa cells were transfected with control siRNA or SAMD9 siRNA, and 24 h later, the cells were treated with 200 U/ml of IFN-β or left untreated. After an additional 24 h, the cells were lysed and analyzed by Western blotting with antibodies to SAMD9 and β-actin as a loading control. (B) Quantification of SAMD9. The bands in panel A were quantified using Image Studio software from LI-COR. The intensities of SAMD9 bands were normalized to the intensities of the β-actin bands. (C) Inhibition of vK1L−C7L−/GFP+ replication in Huh-7.5.1 cells treated with IFN-β and partial reversal with SAMD9 siRNA. Huh-7.5.1 cells were transfected with control siRNA or siRNA to SAMD9 for 24 h and then were left untreated or treated with 200 U/ml of IFN-β for 24 h. After infection with 0.01 PFU of vK1L−C7L−/GFP+ per cell for 18 h, GFP was measured by flow cytometry. Data from two experiments each performed in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.001) calculated as Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***).
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fig6: Interferon induces SAMD9 expression in Huh-7.5.1 cells and inhibits replication of vK1L−C7L−/GFP+. (A) Induction of SAMD9 in Huh-7.5.1 cells. Huh-7.5.1 and HeLa cells were transfected with control siRNA or SAMD9 siRNA, and 24 h later, the cells were treated with 200 U/ml of IFN-β or left untreated. After an additional 24 h, the cells were lysed and analyzed by Western blotting with antibodies to SAMD9 and β-actin as a loading control. (B) Quantification of SAMD9. The bands in panel A were quantified using Image Studio software from LI-COR. The intensities of SAMD9 bands were normalized to the intensities of the β-actin bands. (C) Inhibition of vK1L−C7L−/GFP+ replication in Huh-7.5.1 cells treated with IFN-β and partial reversal with SAMD9 siRNA. Huh-7.5.1 cells were transfected with control siRNA or siRNA to SAMD9 for 24 h and then were left untreated or treated with 200 U/ml of IFN-β for 24 h. After infection with 0.01 PFU of vK1L−C7L−/GFP+ per cell for 18 h, GFP was measured by flow cytometry. Data from two experiments each performed in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.001) calculated as Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***).

Mentions: Meng and coworkers (21) had reported that the replication of the VACV K1L−C7L− mutant was restricted by type 1 interferons in permissive Huh-7 cells. Therefore, we wanted to determine whether these cells normally expressed SAMD9 and whether SAMD9 was induced by beta interferon (IFN-β). In contrast to HeLa cells, SAMD9 was not detected in Huh7.5.1 cells by Western blotting (Fig. 6A and B). However, IFN-β, but not IRF1, increased SAMD9 expression in Huh-7.5.1 cells (Fig. 6A and B). SAMD9 siRNA partially reduced SAMD9 expression induced by interferon (Fig. 6A and B) and enhanced vK1L−C7L−/GFP+ spread in interferon-treated Huh-7.5.1 cells (Fig. 6C). Taken together, these data suggest that insufficient SAMD9 could explain the permissiveness of Huh-7.5.1 cells in the absence of interferon.


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)

Interferon induces SAMD9 expression in Huh-7.5.1 cells and inhibits replication of vK1L−C7L−/GFP+. (A) Induction of SAMD9 in Huh-7.5.1 cells. Huh-7.5.1 and HeLa cells were transfected with control siRNA or SAMD9 siRNA, and 24 h later, the cells were treated with 200 U/ml of IFN-β or left untreated. After an additional 24 h, the cells were lysed and analyzed by Western blotting with antibodies to SAMD9 and β-actin as a loading control. (B) Quantification of SAMD9. The bands in panel A were quantified using Image Studio software from LI-COR. The intensities of SAMD9 bands were normalized to the intensities of the β-actin bands. (C) Inhibition of vK1L−C7L−/GFP+ replication in Huh-7.5.1 cells treated with IFN-β and partial reversal with SAMD9 siRNA. Huh-7.5.1 cells were transfected with control siRNA or siRNA to SAMD9 for 24 h and then were left untreated or treated with 200 U/ml of IFN-β for 24 h. After infection with 0.01 PFU of vK1L−C7L−/GFP+ per cell for 18 h, GFP was measured by flow cytometry. Data from two experiments each performed in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.001) calculated as Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***).
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fig6: Interferon induces SAMD9 expression in Huh-7.5.1 cells and inhibits replication of vK1L−C7L−/GFP+. (A) Induction of SAMD9 in Huh-7.5.1 cells. Huh-7.5.1 and HeLa cells were transfected with control siRNA or SAMD9 siRNA, and 24 h later, the cells were treated with 200 U/ml of IFN-β or left untreated. After an additional 24 h, the cells were lysed and analyzed by Western blotting with antibodies to SAMD9 and β-actin as a loading control. (B) Quantification of SAMD9. The bands in panel A were quantified using Image Studio software from LI-COR. The intensities of SAMD9 bands were normalized to the intensities of the β-actin bands. (C) Inhibition of vK1L−C7L−/GFP+ replication in Huh-7.5.1 cells treated with IFN-β and partial reversal with SAMD9 siRNA. Huh-7.5.1 cells were transfected with control siRNA or siRNA to SAMD9 for 24 h and then were left untreated or treated with 200 U/ml of IFN-β for 24 h. After infection with 0.01 PFU of vK1L−C7L−/GFP+ per cell for 18 h, GFP was measured by flow cytometry. Data from two experiments each performed in triplicate were combined. Values are means plus standard deviation (error bars). Values that are significantly different (P ≤ 0.001) calculated as Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated (***).
Mentions: Meng and coworkers (21) had reported that the replication of the VACV K1L−C7L− mutant was restricted by type 1 interferons in permissive Huh-7 cells. Therefore, we wanted to determine whether these cells normally expressed SAMD9 and whether SAMD9 was induced by beta interferon (IFN-β). In contrast to HeLa cells, SAMD9 was not detected in Huh7.5.1 cells by Western blotting (Fig. 6A and B). However, IFN-β, but not IRF1, increased SAMD9 expression in Huh-7.5.1 cells (Fig. 6A and B). SAMD9 siRNA partially reduced SAMD9 expression induced by interferon (Fig. 6A and B) and enhanced vK1L−C7L−/GFP+ spread in interferon-treated Huh-7.5.1 cells (Fig. 6C). Taken together, these data suggest that insufficient SAMD9 could explain the permissiveness of Huh-7.5.1 cells in the absence of interferon.

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