Limits...
Global Reprogramming of Host SUMOylation during Influenza Virus Infection.

Domingues P, Golebiowski F, Tatham MH, Lopes AM, Taggart A, Hay RT, Hale BG - Cell Rep (2015)

Bottom Line: This is paralleled by widespread host deSUMOylation.Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes.Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression.

View Article: PubMed Central - PubMed

Affiliation: MRC-University of Glasgow Centre for Virus Research, Garscube Campus, 464 Bearsden Road, Glasgow G61 1QH, UK.

No MeSH data available.


Related in: MedlinePlus

SUMO Modification Promotes the Function of CDC73 in Mediating an ISG Response(A) Impact of CDC73 knockdown on ISG expression. A549s were transfected for 48 hr with four independent siRNAs targeting CDC73 (or scrambled) before stimulation with 100 IU/ml IFNα for 8 hr. The mRNA levels of CDC73, ISG15, and GAPDH were quantified. (Left) IFNα-mediated induction of ISG15 mRNA relative to mock is shown.(B) Induction of an ISRE-containing promoter by overexpressed CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (12.5–200 ng) together with pGL3-Mx1P-FFluc and pRL-SV40. After 36 hr, FF luciferase activity was determined and normalized to Renilla. Parallel samples were harvested for western blot, probing for the indicated proteins.(C) Impact of CDC73 overexpression on NF-κB (left) and IFNβ (right) promoters. 293Ts were co-transfected with FLAG-tagged mCherry or CDC73 (200 ng) together with pNF-κB-FFLuc (or p125-FFLuc) and pRL-SV40. Control for NF-κB promoter activation was 10 ng/ml TNF-α for 12 hr (+ve); control for IFNβ promoter activation was co-transfection of 20 ng RIG-I2CARD (+ve). After 36 hr, relative activity was determined as in (B).(D) The siRNAs targeting CDC73 abrogate the effect of CDC73 overexpression on inducible gene expression. 293Ts were transfected/processed as in (B) except four independent siRNAs targeting CDC73 (or scrambled) also were transfected.(E) CDC73-mediated induction of an ISRE-containing promoter is independent of STAT1 function. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (100 ng) together with plasmids encoding GST or PIV5-V (100 ng) and pGL3-Mx1P-FFluc and pRL-SV40. Control cells were stimulated with 100 IU/ml IFNα. Then, 36 hr post-transfection, relative activity was determined as in (B). Data represent fold induction in promoter activation relative to mCherry-expressing cells.(F) CDC73-mediated induction of an ISRE-containing promoter is dependent on SUMOylation. Experiment was as in (E) except plasmids encoding GST or SENP2 (100 ng) were co-transfected.(G) The K136 SUMOylation site in CDC73 is essential for stimulating inducible gene expression. Experiment was as in (B) but included a panel of FLAG-tagged CDC73 lysine mutants (wild-type [WT]; K136R; K301R; K385R; or a triple mutant, 3KR).(H) K136 is a major SUMOylation site in CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged CDC73-WT or CDC73-K136R, together with 6His-tagged SUMO2-GG or SUMO2-AA. Following denaturing Ni2+ pull-down, purified proteins were detected by western blot with anti-6His or anti-FLAG. For all graphs, bars represent mean values from triplicates (±SD) and are derived from three independent experiments. Statistical significance was determined using the Student’s t test (∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p < 0.0001; ns, non-significant). See also Figure S6.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660286&req=5

fig7: SUMO Modification Promotes the Function of CDC73 in Mediating an ISG Response(A) Impact of CDC73 knockdown on ISG expression. A549s were transfected for 48 hr with four independent siRNAs targeting CDC73 (or scrambled) before stimulation with 100 IU/ml IFNα for 8 hr. The mRNA levels of CDC73, ISG15, and GAPDH were quantified. (Left) IFNα-mediated induction of ISG15 mRNA relative to mock is shown.(B) Induction of an ISRE-containing promoter by overexpressed CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (12.5–200 ng) together with pGL3-Mx1P-FFluc and pRL-SV40. After 36 hr, FF luciferase activity was determined and normalized to Renilla. Parallel samples were harvested for western blot, probing for the indicated proteins.(C) Impact of CDC73 overexpression on NF-κB (left) and IFNβ (right) promoters. 293Ts were co-transfected with FLAG-tagged mCherry or CDC73 (200 ng) together with pNF-κB-FFLuc (or p125-FFLuc) and pRL-SV40. Control for NF-κB promoter activation was 10 ng/ml TNF-α for 12 hr (+ve); control for IFNβ promoter activation was co-transfection of 20 ng RIG-I2CARD (+ve). After 36 hr, relative activity was determined as in (B).(D) The siRNAs targeting CDC73 abrogate the effect of CDC73 overexpression on inducible gene expression. 293Ts were transfected/processed as in (B) except four independent siRNAs targeting CDC73 (or scrambled) also were transfected.(E) CDC73-mediated induction of an ISRE-containing promoter is independent of STAT1 function. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (100 ng) together with plasmids encoding GST or PIV5-V (100 ng) and pGL3-Mx1P-FFluc and pRL-SV40. Control cells were stimulated with 100 IU/ml IFNα. Then, 36 hr post-transfection, relative activity was determined as in (B). Data represent fold induction in promoter activation relative to mCherry-expressing cells.(F) CDC73-mediated induction of an ISRE-containing promoter is dependent on SUMOylation. Experiment was as in (E) except plasmids encoding GST or SENP2 (100 ng) were co-transfected.(G) The K136 SUMOylation site in CDC73 is essential for stimulating inducible gene expression. Experiment was as in (B) but included a panel of FLAG-tagged CDC73 lysine mutants (wild-type [WT]; K136R; K301R; K385R; or a triple mutant, 3KR).(H) K136 is a major SUMOylation site in CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged CDC73-WT or CDC73-K136R, together with 6His-tagged SUMO2-GG or SUMO2-AA. Following denaturing Ni2+ pull-down, purified proteins were detected by western blot with anti-6His or anti-FLAG. For all graphs, bars represent mean values from triplicates (±SD) and are derived from three independent experiments. Statistical significance was determined using the Student’s t test (∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p < 0.0001; ns, non-significant). See also Figure S6.

Mentions: In agreement with the antiviral role of PAF1 in mediating RNA polymerase II transcription elongation of ISGs (Marazzi et al., 2012), we found that siRNA-mediated depletion of endogenous CDC73 resulted in defective induction of ISG15 mRNA following IFNα treatment (Figure 7A). In addition, overexpression of CDC73 alone was able to stimulate expression from a promoter containing an interferon-stimulated response element (ISRE) in a dose-dependent manner (Figure 7B). The effect of CDC73 overexpression was not limited to ISRE-containing promoters, as a similar enhancing effect was observed for an NF-κB promoter, although minimally for the IFNβ promoter reporter, indicating a degree of specificity in CDC73’s capacity to regulate inducible gene expression (Figure 7C). Promoter stimulation in these assays was specific to CDC73 overexpression, as co-transfection of siRNAs targeting CDC73 mRNA ablated protein production downstream of the ISRE promoter (Figure 7D). Furthermore, consistent with a model for CDC73 acting in RNA polymerase II-mediated transcription elongation, we found that the effect of CDC73 on ISRE promoter-driven expression was insensitive to depletion of the STAT1 transcription factor, which is otherwise essential for IFNα-stimulated activation of the ISRE (Figure 7E). These data suggest that CDC73 may act as an antiviral factor by potentiating inducible antiviral gene expression at a level subsequent to transcription factor activation.


Global Reprogramming of Host SUMOylation during Influenza Virus Infection.

Domingues P, Golebiowski F, Tatham MH, Lopes AM, Taggart A, Hay RT, Hale BG - Cell Rep (2015)

SUMO Modification Promotes the Function of CDC73 in Mediating an ISG Response(A) Impact of CDC73 knockdown on ISG expression. A549s were transfected for 48 hr with four independent siRNAs targeting CDC73 (or scrambled) before stimulation with 100 IU/ml IFNα for 8 hr. The mRNA levels of CDC73, ISG15, and GAPDH were quantified. (Left) IFNα-mediated induction of ISG15 mRNA relative to mock is shown.(B) Induction of an ISRE-containing promoter by overexpressed CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (12.5–200 ng) together with pGL3-Mx1P-FFluc and pRL-SV40. After 36 hr, FF luciferase activity was determined and normalized to Renilla. Parallel samples were harvested for western blot, probing for the indicated proteins.(C) Impact of CDC73 overexpression on NF-κB (left) and IFNβ (right) promoters. 293Ts were co-transfected with FLAG-tagged mCherry or CDC73 (200 ng) together with pNF-κB-FFLuc (or p125-FFLuc) and pRL-SV40. Control for NF-κB promoter activation was 10 ng/ml TNF-α for 12 hr (+ve); control for IFNβ promoter activation was co-transfection of 20 ng RIG-I2CARD (+ve). After 36 hr, relative activity was determined as in (B).(D) The siRNAs targeting CDC73 abrogate the effect of CDC73 overexpression on inducible gene expression. 293Ts were transfected/processed as in (B) except four independent siRNAs targeting CDC73 (or scrambled) also were transfected.(E) CDC73-mediated induction of an ISRE-containing promoter is independent of STAT1 function. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (100 ng) together with plasmids encoding GST or PIV5-V (100 ng) and pGL3-Mx1P-FFluc and pRL-SV40. Control cells were stimulated with 100 IU/ml IFNα. Then, 36 hr post-transfection, relative activity was determined as in (B). Data represent fold induction in promoter activation relative to mCherry-expressing cells.(F) CDC73-mediated induction of an ISRE-containing promoter is dependent on SUMOylation. Experiment was as in (E) except plasmids encoding GST or SENP2 (100 ng) were co-transfected.(G) The K136 SUMOylation site in CDC73 is essential for stimulating inducible gene expression. Experiment was as in (B) but included a panel of FLAG-tagged CDC73 lysine mutants (wild-type [WT]; K136R; K301R; K385R; or a triple mutant, 3KR).(H) K136 is a major SUMOylation site in CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged CDC73-WT or CDC73-K136R, together with 6His-tagged SUMO2-GG or SUMO2-AA. Following denaturing Ni2+ pull-down, purified proteins were detected by western blot with anti-6His or anti-FLAG. For all graphs, bars represent mean values from triplicates (±SD) and are derived from three independent experiments. Statistical significance was determined using the Student’s t test (∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p < 0.0001; ns, non-significant). See also Figure S6.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660286&req=5

fig7: SUMO Modification Promotes the Function of CDC73 in Mediating an ISG Response(A) Impact of CDC73 knockdown on ISG expression. A549s were transfected for 48 hr with four independent siRNAs targeting CDC73 (or scrambled) before stimulation with 100 IU/ml IFNα for 8 hr. The mRNA levels of CDC73, ISG15, and GAPDH were quantified. (Left) IFNα-mediated induction of ISG15 mRNA relative to mock is shown.(B) Induction of an ISRE-containing promoter by overexpressed CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (12.5–200 ng) together with pGL3-Mx1P-FFluc and pRL-SV40. After 36 hr, FF luciferase activity was determined and normalized to Renilla. Parallel samples were harvested for western blot, probing for the indicated proteins.(C) Impact of CDC73 overexpression on NF-κB (left) and IFNβ (right) promoters. 293Ts were co-transfected with FLAG-tagged mCherry or CDC73 (200 ng) together with pNF-κB-FFLuc (or p125-FFLuc) and pRL-SV40. Control for NF-κB promoter activation was 10 ng/ml TNF-α for 12 hr (+ve); control for IFNβ promoter activation was co-transfection of 20 ng RIG-I2CARD (+ve). After 36 hr, relative activity was determined as in (B).(D) The siRNAs targeting CDC73 abrogate the effect of CDC73 overexpression on inducible gene expression. 293Ts were transfected/processed as in (B) except four independent siRNAs targeting CDC73 (or scrambled) also were transfected.(E) CDC73-mediated induction of an ISRE-containing promoter is independent of STAT1 function. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged mCherry or CDC73 (100 ng) together with plasmids encoding GST or PIV5-V (100 ng) and pGL3-Mx1P-FFluc and pRL-SV40. Control cells were stimulated with 100 IU/ml IFNα. Then, 36 hr post-transfection, relative activity was determined as in (B). Data represent fold induction in promoter activation relative to mCherry-expressing cells.(F) CDC73-mediated induction of an ISRE-containing promoter is dependent on SUMOylation. Experiment was as in (E) except plasmids encoding GST or SENP2 (100 ng) were co-transfected.(G) The K136 SUMOylation site in CDC73 is essential for stimulating inducible gene expression. Experiment was as in (B) but included a panel of FLAG-tagged CDC73 lysine mutants (wild-type [WT]; K136R; K301R; K385R; or a triple mutant, 3KR).(H) K136 is a major SUMOylation site in CDC73. 293Ts were co-transfected with expression plasmids encoding FLAG-tagged CDC73-WT or CDC73-K136R, together with 6His-tagged SUMO2-GG or SUMO2-AA. Following denaturing Ni2+ pull-down, purified proteins were detected by western blot with anti-6His or anti-FLAG. For all graphs, bars represent mean values from triplicates (±SD) and are derived from three independent experiments. Statistical significance was determined using the Student’s t test (∗p < 0.01, ∗∗p < 0.001, and ∗∗∗p < 0.0001; ns, non-significant). See also Figure S6.
Mentions: In agreement with the antiviral role of PAF1 in mediating RNA polymerase II transcription elongation of ISGs (Marazzi et al., 2012), we found that siRNA-mediated depletion of endogenous CDC73 resulted in defective induction of ISG15 mRNA following IFNα treatment (Figure 7A). In addition, overexpression of CDC73 alone was able to stimulate expression from a promoter containing an interferon-stimulated response element (ISRE) in a dose-dependent manner (Figure 7B). The effect of CDC73 overexpression was not limited to ISRE-containing promoters, as a similar enhancing effect was observed for an NF-κB promoter, although minimally for the IFNβ promoter reporter, indicating a degree of specificity in CDC73’s capacity to regulate inducible gene expression (Figure 7C). Promoter stimulation in these assays was specific to CDC73 overexpression, as co-transfection of siRNAs targeting CDC73 mRNA ablated protein production downstream of the ISRE promoter (Figure 7D). Furthermore, consistent with a model for CDC73 acting in RNA polymerase II-mediated transcription elongation, we found that the effect of CDC73 on ISRE promoter-driven expression was insensitive to depletion of the STAT1 transcription factor, which is otherwise essential for IFNα-stimulated activation of the ISRE (Figure 7E). These data suggest that CDC73 may act as an antiviral factor by potentiating inducible antiviral gene expression at a level subsequent to transcription factor activation.

Bottom Line: This is paralleled by widespread host deSUMOylation.Depletion screening identified ten virus-induced SUMO targets as potential antiviral factors, including C18orf25 and the SMC5/6 and PAF1 complexes.Mechanistic studies further uncovered a role for SUMOylation of the PAF1 complex component, parafibromin (CDC73), in potentiating antiviral gene expression.

View Article: PubMed Central - PubMed

Affiliation: MRC-University of Glasgow Centre for Virus Research, Garscube Campus, 464 Bearsden Road, Glasgow G61 1QH, UK.

No MeSH data available.


Related in: MedlinePlus