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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 interact with SAMD9 independently and in the absence of other viral proteins. (A) HeLa cells were infected with vK1L−C7L−/GFP+ and transfected with 3  µg of T7-C7L-V5 and increasing amounts of T7-K1L-FLAG or with 3 µg of T7-K1L-FLAG and increasing amounts of T7-C7L-V5. The amounts of T7-C7L-V5 and T7-K1L-FLAG (in micrograms) are given above the gels (−, none). After 16 h, 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 with antibodies to endogenous SAMD9 and V5 or FLAG epitope tag. The positions of mass markers (in kilodaltons) are shown to the left of the gels. The positions of tagged proteins are shown to the right of the gels. The position of the antibody heavy chain is indicated by an asterisk. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope. (B) Uninfected HeLa cells were transfected with plasmids that express C7L-HA, K1L-myc, or enhanced GFP (eGFP) regulated by CMV promoters. After 24 h, the cells were lysed and incubated with antibodies to the HA or myc epitope tag. Input and eluted proteins were analyzed by SDS-PAGE and Western blotting to detect C7L-HA, K1L-myc, and endogenous SAMD9.
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fig4: C7L and K1L interact with SAMD9 independently and in the absence of other viral proteins. (A) HeLa cells were infected with vK1L−C7L−/GFP+ and transfected with 3  µg of T7-C7L-V5 and increasing amounts of T7-K1L-FLAG or with 3 µg of T7-K1L-FLAG and increasing amounts of T7-C7L-V5. The amounts of T7-C7L-V5 and T7-K1L-FLAG (in micrograms) are given above the gels (−, none). After 16 h, 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 with antibodies to endogenous SAMD9 and V5 or FLAG epitope tag. The positions of mass markers (in kilodaltons) are shown to the left of the gels. The positions of tagged proteins are shown to the right of the gels. The position of the antibody heavy chain is indicated by an asterisk. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope. (B) Uninfected HeLa cells were transfected with plasmids that express C7L-HA, K1L-myc, or enhanced GFP (eGFP) regulated by CMV promoters. After 24 h, the cells were lysed and incubated with antibodies to the HA or myc epitope tag. Input and eluted proteins were analyzed by SDS-PAGE and Western blotting to detect C7L-HA, K1L-myc, and endogenous SAMD9.

Mentions: The above data could be explained by direct or indirect binding of C7 and K1 proteins to SAMD9. To eliminate the possibility of another viral protein mediating the association, we expressed C7 and K1 with different epitope tags using a cytomegalovirus (CMV) promoter in uninfected HeLa cells. SAMD9 was captured in association with hemagglutinin (HA) epitope-tagged C7 protein (C7-HA) and myc epitope-tagged K1 protein (K1-myc) and detected by Western blotting (Fig. 4A). These data eliminated the possibility that the binding of C7 and K1 to SAMD9 is mediated by another VACV protein but did not exclude the participation of another cellular protein.


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 interact with SAMD9 independently and in the absence of other viral proteins. (A) HeLa cells were infected with vK1L−C7L−/GFP+ and transfected with 3  µg of T7-C7L-V5 and increasing amounts of T7-K1L-FLAG or with 3 µg of T7-K1L-FLAG and increasing amounts of T7-C7L-V5. The amounts of T7-C7L-V5 and T7-K1L-FLAG (in micrograms) are given above the gels (−, none). After 16 h, 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 with antibodies to endogenous SAMD9 and V5 or FLAG epitope tag. The positions of mass markers (in kilodaltons) are shown to the left of the gels. The positions of tagged proteins are shown to the right of the gels. The position of the antibody heavy chain is indicated by an asterisk. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope. (B) Uninfected HeLa cells were transfected with plasmids that express C7L-HA, K1L-myc, or enhanced GFP (eGFP) regulated by CMV promoters. After 24 h, the cells were lysed and incubated with antibodies to the HA or myc epitope tag. Input and eluted proteins were analyzed by SDS-PAGE and Western blotting to detect C7L-HA, K1L-myc, and endogenous SAMD9.
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fig4: C7L and K1L interact with SAMD9 independently and in the absence of other viral proteins. (A) HeLa cells were infected with vK1L−C7L−/GFP+ and transfected with 3  µg of T7-C7L-V5 and increasing amounts of T7-K1L-FLAG or with 3 µg of T7-K1L-FLAG and increasing amounts of T7-C7L-V5. The amounts of T7-C7L-V5 and T7-K1L-FLAG (in micrograms) are given above the gels (−, none). After 16 h, 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 with antibodies to endogenous SAMD9 and V5 or FLAG epitope tag. The positions of mass markers (in kilodaltons) are shown to the left of the gels. The positions of tagged proteins are shown to the right of the gels. The position of the antibody heavy chain is indicated by an asterisk. Abbreviations: IP, immunopurification; αV5, antibody to V5 epitope; αFLAG, antibody to FLAG epitope. (B) Uninfected HeLa cells were transfected with plasmids that express C7L-HA, K1L-myc, or enhanced GFP (eGFP) regulated by CMV promoters. After 24 h, the cells were lysed and incubated with antibodies to the HA or myc epitope tag. Input and eluted proteins were analyzed by SDS-PAGE and Western blotting to detect C7L-HA, K1L-myc, and endogenous SAMD9.
Mentions: The above data could be explained by direct or indirect binding of C7 and K1 proteins to SAMD9. To eliminate the possibility of another viral protein mediating the association, we expressed C7 and K1 with different epitope tags using a cytomegalovirus (CMV) promoter in uninfected HeLa cells. SAMD9 was captured in association with hemagglutinin (HA) epitope-tagged C7 protein (C7-HA) and myc epitope-tagged K1 protein (K1-myc) and detected by Western blotting (Fig. 4A). These data eliminated the possibility that the binding of C7 and K1 to SAMD9 is mediated by another VACV protein but did not exclude the participation of another cellular protein.

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