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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

C7L and K1L proteins physically interact with SAMD9. (A) Expression of C7L-V5 and K1L-FLAG following transfection with bacteriophage T7 promoter plasmids. HeLa cells were infected with vK1L−C7L−/GFP+, which expresses the T7 RNA polymerase, and transfected 1 h later with plasmids T7-C7L-V5 and T7-K1L-FLAG encoding C7L-V5 (two clones) or K1L-FLAG regulated by T7 promoters, respectively. After 14 h, the cells were lysed, and their proteins were resolved by SDS-PAGE and Western blotting with antibodies to the V5 and FLAG epitope tags. The positions of molecular mass markers (M) (in kilodaltons) are indicated to the left of the gel. (B) Rescue of vK1L−C7L−/GFP+ by expression of C7 or K1 protein. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (upper panel) or transfected with T7-C7L-V5 or T7-K1L-FLAG for 16 h. Cells were imaged by GFP fluorescence microscopy and bright-field microscopy. (C) Association of C7 and K1 proteins with SAMD9. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (−) or transfected (+) with T7-C7L-V5 or T7-K1L-FLAG for 16 h. The cells were lysed and incubated with antibodies to the V5 or FLAG epitope tag and captured with magnetic beads conjugated to protein G. Input and eluted proteins were resolved by SDS-PAGE following Western blotting with antibodies to endogenous SAMD9 and to V5 and FLAG tags. The position of the heavy chain of the antibody is indicated by an asterisk to the right of the gel. The experiment was repeated three times with similar results. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope.
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fig3: C7L and K1L proteins physically interact with SAMD9. (A) Expression of C7L-V5 and K1L-FLAG following transfection with bacteriophage T7 promoter plasmids. HeLa cells were infected with vK1L−C7L−/GFP+, which expresses the T7 RNA polymerase, and transfected 1 h later with plasmids T7-C7L-V5 and T7-K1L-FLAG encoding C7L-V5 (two clones) or K1L-FLAG regulated by T7 promoters, respectively. After 14 h, the cells were lysed, and their proteins were resolved by SDS-PAGE and Western blotting with antibodies to the V5 and FLAG epitope tags. The positions of molecular mass markers (M) (in kilodaltons) are indicated to the left of the gel. (B) Rescue of vK1L−C7L−/GFP+ by expression of C7 or K1 protein. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (upper panel) or transfected with T7-C7L-V5 or T7-K1L-FLAG for 16 h. Cells were imaged by GFP fluorescence microscopy and bright-field microscopy. (C) Association of C7 and K1 proteins with SAMD9. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (−) or transfected (+) with T7-C7L-V5 or T7-K1L-FLAG for 16 h. The cells were lysed and incubated with antibodies to the V5 or FLAG epitope tag and captured with magnetic beads conjugated to protein G. Input and eluted proteins were resolved by SDS-PAGE following Western blotting with antibodies to endogenous SAMD9 and to V5 and FLAG tags. The position of the heavy chain of the antibody is indicated by an asterisk to the right of the gel. The experiment was repeated three times with similar results. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope.

Mentions: In view of the evidence that SAMD9 is a major restriction factor for replication of the K1L−C7L− mutant in HeLa cells, we designed an experiment to determine whether the two proteins interact directly or indirectly with SAMD9. The parental VACV used to construct vK1L−C7L−/GFP+ was vTF7-3 (25), which carries the gene encoding the bacteriophage T7 RNA polymerase regulated by a VACV early/late promoter and has been used extensively in transfection assays for expression of genes preceded by a T7 promoter. Accordingly, we constructed plasmids with genes encoding V5 and FLAG epitope-tagged C7 and K1 proteins, respectively, with T7 promoters. Both proteins were expressed following transfection of permissive and nonpermissive cells that had been infected with vK1L−C7L−/GFP+ (Fig. 3A). Furthermore, expression of either protein alone rescued replication of the host range mutant, demonstrating their biological activity (Fig. 3B). Next, we carried out binding experiments in HeLa cells infected with vK1L−C7L−/GFP+. Antibodies to V5 and FLAG were used to capture C7 and K1, respectively. Western blotting showed that SAMD9 was pulled down with either viral protein (Fig. 3C).


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)

C7L and K1L proteins physically interact with SAMD9. (A) Expression of C7L-V5 and K1L-FLAG following transfection with bacteriophage T7 promoter plasmids. HeLa cells were infected with vK1L−C7L−/GFP+, which expresses the T7 RNA polymerase, and transfected 1 h later with plasmids T7-C7L-V5 and T7-K1L-FLAG encoding C7L-V5 (two clones) or K1L-FLAG regulated by T7 promoters, respectively. After 14 h, the cells were lysed, and their proteins were resolved by SDS-PAGE and Western blotting with antibodies to the V5 and FLAG epitope tags. The positions of molecular mass markers (M) (in kilodaltons) are indicated to the left of the gel. (B) Rescue of vK1L−C7L−/GFP+ by expression of C7 or K1 protein. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (upper panel) or transfected with T7-C7L-V5 or T7-K1L-FLAG for 16 h. Cells were imaged by GFP fluorescence microscopy and bright-field microscopy. (C) Association of C7 and K1 proteins with SAMD9. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (−) or transfected (+) with T7-C7L-V5 or T7-K1L-FLAG for 16 h. The cells were lysed and incubated with antibodies to the V5 or FLAG epitope tag and captured with magnetic beads conjugated to protein G. Input and eluted proteins were resolved by SDS-PAGE following Western blotting with antibodies to endogenous SAMD9 and to V5 and FLAG tags. The position of the heavy chain of the antibody is indicated by an asterisk to the right of the gel. The experiment was repeated three times with similar results. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope.
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fig3: C7L and K1L proteins physically interact with SAMD9. (A) Expression of C7L-V5 and K1L-FLAG following transfection with bacteriophage T7 promoter plasmids. HeLa cells were infected with vK1L−C7L−/GFP+, which expresses the T7 RNA polymerase, and transfected 1 h later with plasmids T7-C7L-V5 and T7-K1L-FLAG encoding C7L-V5 (two clones) or K1L-FLAG regulated by T7 promoters, respectively. After 14 h, the cells were lysed, and their proteins were resolved by SDS-PAGE and Western blotting with antibodies to the V5 and FLAG epitope tags. The positions of molecular mass markers (M) (in kilodaltons) are indicated to the left of the gel. (B) Rescue of vK1L−C7L−/GFP+ by expression of C7 or K1 protein. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (upper panel) or transfected with T7-C7L-V5 or T7-K1L-FLAG for 16 h. Cells were imaged by GFP fluorescence microscopy and bright-field microscopy. (C) Association of C7 and K1 proteins with SAMD9. HeLa cells were infected with vK1L−C7L−/GFP+ and mock transfected (−) or transfected (+) with T7-C7L-V5 or T7-K1L-FLAG for 16 h. The cells were lysed and incubated with antibodies to the V5 or FLAG epitope tag and captured with magnetic beads conjugated to protein G. Input and eluted proteins were resolved by SDS-PAGE following Western blotting with antibodies to endogenous SAMD9 and to V5 and FLAG tags. The position of the heavy chain of the antibody is indicated by an asterisk to the right of the gel. The experiment was repeated three times with similar results. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope.
Mentions: In view of the evidence that SAMD9 is a major restriction factor for replication of the K1L−C7L− mutant in HeLa cells, we designed an experiment to determine whether the two proteins interact directly or indirectly with SAMD9. The parental VACV used to construct vK1L−C7L−/GFP+ was vTF7-3 (25), which carries the gene encoding the bacteriophage T7 RNA polymerase regulated by a VACV early/late promoter and has been used extensively in transfection assays for expression of genes preceded by a T7 promoter. Accordingly, we constructed plasmids with genes encoding V5 and FLAG epitope-tagged C7 and K1 proteins, respectively, with T7 promoters. Both proteins were expressed following transfection of permissive and nonpermissive cells that had been infected with vK1L−C7L−/GFP+ (Fig. 3A). Furthermore, expression of either protein alone rescued replication of the host range mutant, demonstrating their biological activity (Fig. 3B). Next, we carried out binding experiments in HeLa cells infected with vK1L−C7L−/GFP+. Antibodies to V5 and FLAG were used to capture C7 and K1, respectively. Western blotting showed that SAMD9 was pulled down with either viral protein (Fig. 3C).

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