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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 inactivation of SAMD9 and inhibition of replication by IRF1. (A) Generation of SAMD9-deficient cells by CRISPR/CAS9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Colonies were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous SAMD9 and β-actin as a loading control. Colony 3 was chosen for further experiments and labeled as SAMD9−/− HeLa cells. (B) Functional validation of SAMD9−/− HeLa cells. Normal HeLa cells and SAMD9−/− HeLa cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. One set of infected SAMD9−/− cells were transfected with T7-SAMD9-HA. After 18 h, GFP-positive cells were scored by flow cytometry. (Inset) Western blot demonstrating expression of SAMD9 by T7-SAMD9-HA. Data from two separate experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The value that was significantly different (P ≤ 0.001) from the value for the untransfected control, calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software is indicated (***). (C) Overexpression of IRF1 prevents spread of vK1L−C7L−/GFP+ in SAMD9−/− HeLa cells. SAMD9−/− cells were mock transfected or transfected with plasmid expressing IRF1 regulated by the CMV promoter. At 30 h after transfection, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. After an additional 18-h incubation, GFP-positive cells were scored by flow cytometry. (Inset) Western blot showing IRF1 expression. Data from three separate experiments performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that were significantly different calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001 relative to the value for C7L−K1L−/GFP+; *, P ≤ 0.05 relative to the value for iFire.
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fig5: Rescue of vK1L−C7L−/GFP+ by inactivation of SAMD9 and inhibition of replication by IRF1. (A) Generation of SAMD9-deficient cells by CRISPR/CAS9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Colonies were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous SAMD9 and β-actin as a loading control. Colony 3 was chosen for further experiments and labeled as SAMD9−/− HeLa cells. (B) Functional validation of SAMD9−/− HeLa cells. Normal HeLa cells and SAMD9−/− HeLa cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. One set of infected SAMD9−/− cells were transfected with T7-SAMD9-HA. After 18 h, GFP-positive cells were scored by flow cytometry. (Inset) Western blot demonstrating expression of SAMD9 by T7-SAMD9-HA. Data from two separate experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The value that was significantly different (P ≤ 0.001) from the value for the untransfected control, calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software is indicated (***). (C) Overexpression of IRF1 prevents spread of vK1L−C7L−/GFP+ in SAMD9−/− HeLa cells. SAMD9−/− cells were mock transfected or transfected with plasmid expressing IRF1 regulated by the CMV promoter. At 30 h after transfection, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. After an additional 18-h incubation, GFP-positive cells were scored by flow cytometry. (Inset) Western blot showing IRF1 expression. Data from three separate experiments performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that were significantly different calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001 relative to the value for C7L−K1L−/GFP+; *, P ≤ 0.05 relative to the value for iFire.

Mentions: Our evidence for the major role of SAMD9 in restricting replication of vK1L−C7L−/GFP+ was derived using siRNAs. To further investigate the host range defect of K1L−C7L− mutants, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology was used to disrupt the SAMD9 ORF in HeLa cells. Only a trace of SAMD9 was detected by Western blotting in one of the three clonally derived cell lines tested (Fig. 5A). Using the latter, the replication of vK1L−C7L−/GFP+ was evident from the significant virus spread (Fig. 5B). Moreover, transfection of a SAMD9-expressing plasmid into the SAMD9 knockout cells prevented replication of the host range mutant (Fig. 5B).


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 inactivation of SAMD9 and inhibition of replication by IRF1. (A) Generation of SAMD9-deficient cells by CRISPR/CAS9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Colonies were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous SAMD9 and β-actin as a loading control. Colony 3 was chosen for further experiments and labeled as SAMD9−/− HeLa cells. (B) Functional validation of SAMD9−/− HeLa cells. Normal HeLa cells and SAMD9−/− HeLa cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. One set of infected SAMD9−/− cells were transfected with T7-SAMD9-HA. After 18 h, GFP-positive cells were scored by flow cytometry. (Inset) Western blot demonstrating expression of SAMD9 by T7-SAMD9-HA. Data from two separate experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The value that was significantly different (P ≤ 0.001) from the value for the untransfected control, calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software is indicated (***). (C) Overexpression of IRF1 prevents spread of vK1L−C7L−/GFP+ in SAMD9−/− HeLa cells. SAMD9−/− cells were mock transfected or transfected with plasmid expressing IRF1 regulated by the CMV promoter. At 30 h after transfection, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. After an additional 18-h incubation, GFP-positive cells were scored by flow cytometry. (Inset) Western blot showing IRF1 expression. Data from three separate experiments performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that were significantly different calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001 relative to the value for C7L−K1L−/GFP+; *, P ≤ 0.05 relative to the value for iFire.
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fig5: Rescue of vK1L−C7L−/GFP+ by inactivation of SAMD9 and inhibition of replication by IRF1. (A) Generation of SAMD9-deficient cells by CRISPR/CAS9 technology. HeLa cells were transfected with the CRISPR/Cas9 components as described in Materials and Methods. Colonies were lysed, and their proteins were resolved by SDS-PAGE and analyzed by Western blotting to detect endogenous SAMD9 and β-actin as a loading control. Colony 3 was chosen for further experiments and labeled as SAMD9−/− HeLa cells. (B) Functional validation of SAMD9−/− HeLa cells. Normal HeLa cells and SAMD9−/− HeLa cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. One set of infected SAMD9−/− cells were transfected with T7-SAMD9-HA. After 18 h, GFP-positive cells were scored by flow cytometry. (Inset) Western blot demonstrating expression of SAMD9 by T7-SAMD9-HA. Data from two separate experiments each performed in triplicate were combined. Values are means plus standard deviations (error bars). The value that was significantly different (P ≤ 0.001) from the value for the untransfected control, calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software is indicated (***). (C) Overexpression of IRF1 prevents spread of vK1L−C7L−/GFP+ in SAMD9−/− HeLa cells. SAMD9−/− cells were mock transfected or transfected with plasmid expressing IRF1 regulated by the CMV promoter. At 30 h after transfection, the cells were infected with 0.01 PFU of vK1L−C7L−/GFP+. After an additional 18-h incubation, GFP-positive cells were scored by flow cytometry. (Inset) Western blot showing IRF1 expression. Data from three separate experiments performed in triplicate were combined. Values are means plus standard deviations (error bars). The values that were significantly different calculated by Bonferroni test after one-way ANOVA using PRISM GraphPad software are indicated by asterisks as follows: ***, P ≤ 0.001 relative to the value for C7L−K1L−/GFP+; *, P ≤ 0.05 relative to the value for iFire.
Mentions: Our evidence for the major role of SAMD9 in restricting replication of vK1L−C7L−/GFP+ was derived using siRNAs. To further investigate the host range defect of K1L−C7L− mutants, clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology was used to disrupt the SAMD9 ORF in HeLa cells. Only a trace of SAMD9 was detected by Western blotting in one of the three clonally derived cell lines tested (Fig. 5A). Using the latter, the replication of vK1L−C7L−/GFP+ was evident from the significant virus spread (Fig. 5B). Moreover, transfection of a SAMD9-expressing plasmid into the SAMD9 knockout cells prevented replication of the host range mutant (Fig. 5B).

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