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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

IAV Polymerase Activity Contributes to SUMO Remodeling(A) Western blot of lysates from IAV-infected A549 cells treated with different inhibitors. Cells were infected with IAV or UV-inactivated IAV (UV) at 5 PFU/cell, followed by incubation with 50 μg/ml cycloheximide (CHX), 11 nM leptomycin B (LMB), or 10 μM zanamivir (Zan) for 12 hr. SUMO1, SUMO2/3, NS1, NP, and actin were detected. (Bottom) Immunofluorescence shows NP staining at 12 hr post-infection in A549s ± LMB. DAPI was used to stain DNA.(B and C) Immunofluorescent analyses of MRC5s transiently expressing PB1, PB2, PA, NP, and a negative-sense viral-like mini-replicon mCherry reporter construct (+vRNA), or PB1, PB2, PA, NP, and mCherry (no viral-like reporter; −vRNA).(D) Immunofluorescent analysis of MRC5s individually expressing PB1, PB2, PA, or NP.(E) Immunofluorescent analysis of MRC5s transiently transfected with pDZ-NP, which expresses both NP protein from a pol-II promoter and NP vRNA from a pol-I promoter.(F) (Top) Luciferase-based mini-replicon assays in 293Ts to assess polymerase activity. (Left) (WSN, H1N1) Cells transiently expressing PB1 (or not), PB2, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. (Right) (KAN-1, H5N1) Cells transiently expressing PB1 (or an E445A/E446A inactive mutant), AvianPr-PB2-E627K, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. Bars represent mean values from triplicates (±SD). (Bottom) Quantification of SUMO1 nuclear foci for the conditions indicated above as determined by the mCherry-based mini-replicon reporter assay in MRC5s. For (B)–(F), cells were transfected for 36 hr prior to processing or fixation and immunostaining. Representative images are shown. Scale bars represent 5 μm. Statistical significance in panels (B), (C), (E), and (F) was determined using the Student’s t test (∗∗∗p < 0.0001; ns, non-significant). See also Figure S2.
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fig2: IAV Polymerase Activity Contributes to SUMO Remodeling(A) Western blot of lysates from IAV-infected A549 cells treated with different inhibitors. Cells were infected with IAV or UV-inactivated IAV (UV) at 5 PFU/cell, followed by incubation with 50 μg/ml cycloheximide (CHX), 11 nM leptomycin B (LMB), or 10 μM zanamivir (Zan) for 12 hr. SUMO1, SUMO2/3, NS1, NP, and actin were detected. (Bottom) Immunofluorescence shows NP staining at 12 hr post-infection in A549s ± LMB. DAPI was used to stain DNA.(B and C) Immunofluorescent analyses of MRC5s transiently expressing PB1, PB2, PA, NP, and a negative-sense viral-like mini-replicon mCherry reporter construct (+vRNA), or PB1, PB2, PA, NP, and mCherry (no viral-like reporter; −vRNA).(D) Immunofluorescent analysis of MRC5s individually expressing PB1, PB2, PA, or NP.(E) Immunofluorescent analysis of MRC5s transiently transfected with pDZ-NP, which expresses both NP protein from a pol-II promoter and NP vRNA from a pol-I promoter.(F) (Top) Luciferase-based mini-replicon assays in 293Ts to assess polymerase activity. (Left) (WSN, H1N1) Cells transiently expressing PB1 (or not), PB2, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. (Right) (KAN-1, H5N1) Cells transiently expressing PB1 (or an E445A/E446A inactive mutant), AvianPr-PB2-E627K, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. Bars represent mean values from triplicates (±SD). (Bottom) Quantification of SUMO1 nuclear foci for the conditions indicated above as determined by the mCherry-based mini-replicon reporter assay in MRC5s. For (B)–(F), cells were transfected for 36 hr prior to processing or fixation and immunostaining. Representative images are shown. Scale bars represent 5 μm. Statistical significance in panels (B), (C), (E), and (F) was determined using the Student’s t test (∗∗∗p < 0.0001; ns, non-significant). See also Figure S2.

Mentions: We used small-molecule inhibitors and UV-inactivation methods to map IAV-triggered SUMOylation to a process requiring viral genome replication and protein synthesis, but not genome nuclear export or later stages of the virus replication cycle, such as virion budding (Figure 2A). Given the tight association of active influenza virus replication complexes with nuclear processes (a distinguishing feature from cytoplasmic-replicating RNA viruses), we speculated that the stress of nuclear IAV polymerase activity may contribute to host SUMO remodeling. To test this hypothesis, we used a transfection-based mini-replicon reporter system, whereby viral ribonucleoprotein complexes (vRNPs) consisting of viral NP, PB1, PB2, and PA are assembled together in the nucleus following plasmid expression along with a negative-sense viral-like RNA genomic segment encoding mCherry. As the viral-like RNA cannot be transcribed into mRNA by cellular polymerases, mCherry protein is only produced in cells expressing all five viral components. Furthermore, the mCherry construct is unspliced such that this assay recapitulates IAV RNA transcription and replication, but not splicing.


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)

IAV Polymerase Activity Contributes to SUMO Remodeling(A) Western blot of lysates from IAV-infected A549 cells treated with different inhibitors. Cells were infected with IAV or UV-inactivated IAV (UV) at 5 PFU/cell, followed by incubation with 50 μg/ml cycloheximide (CHX), 11 nM leptomycin B (LMB), or 10 μM zanamivir (Zan) for 12 hr. SUMO1, SUMO2/3, NS1, NP, and actin were detected. (Bottom) Immunofluorescence shows NP staining at 12 hr post-infection in A549s ± LMB. DAPI was used to stain DNA.(B and C) Immunofluorescent analyses of MRC5s transiently expressing PB1, PB2, PA, NP, and a negative-sense viral-like mini-replicon mCherry reporter construct (+vRNA), or PB1, PB2, PA, NP, and mCherry (no viral-like reporter; −vRNA).(D) Immunofluorescent analysis of MRC5s individually expressing PB1, PB2, PA, or NP.(E) Immunofluorescent analysis of MRC5s transiently transfected with pDZ-NP, which expresses both NP protein from a pol-II promoter and NP vRNA from a pol-I promoter.(F) (Top) Luciferase-based mini-replicon assays in 293Ts to assess polymerase activity. (Left) (WSN, H1N1) Cells transiently expressing PB1 (or not), PB2, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. (Right) (KAN-1, H5N1) Cells transiently expressing PB1 (or an E445A/E446A inactive mutant), AvianPr-PB2-E627K, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. Bars represent mean values from triplicates (±SD). (Bottom) Quantification of SUMO1 nuclear foci for the conditions indicated above as determined by the mCherry-based mini-replicon reporter assay in MRC5s. For (B)–(F), cells were transfected for 36 hr prior to processing or fixation and immunostaining. Representative images are shown. Scale bars represent 5 μm. Statistical significance in panels (B), (C), (E), and (F) was determined using the Student’s t test (∗∗∗p < 0.0001; ns, non-significant). See also Figure S2.
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fig2: IAV Polymerase Activity Contributes to SUMO Remodeling(A) Western blot of lysates from IAV-infected A549 cells treated with different inhibitors. Cells were infected with IAV or UV-inactivated IAV (UV) at 5 PFU/cell, followed by incubation with 50 μg/ml cycloheximide (CHX), 11 nM leptomycin B (LMB), or 10 μM zanamivir (Zan) for 12 hr. SUMO1, SUMO2/3, NS1, NP, and actin were detected. (Bottom) Immunofluorescence shows NP staining at 12 hr post-infection in A549s ± LMB. DAPI was used to stain DNA.(B and C) Immunofluorescent analyses of MRC5s transiently expressing PB1, PB2, PA, NP, and a negative-sense viral-like mini-replicon mCherry reporter construct (+vRNA), or PB1, PB2, PA, NP, and mCherry (no viral-like reporter; −vRNA).(D) Immunofluorescent analysis of MRC5s individually expressing PB1, PB2, PA, or NP.(E) Immunofluorescent analysis of MRC5s transiently transfected with pDZ-NP, which expresses both NP protein from a pol-II promoter and NP vRNA from a pol-I promoter.(F) (Top) Luciferase-based mini-replicon assays in 293Ts to assess polymerase activity. (Left) (WSN, H1N1) Cells transiently expressing PB1 (or not), PB2, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. (Right) (KAN-1, H5N1) Cells transiently expressing PB1 (or an E445A/E446A inactive mutant), AvianPr-PB2-E627K, PA, NP, and a negative-sense viral-like mini-replicon Firefly luciferase reporter construct. Bars represent mean values from triplicates (±SD). (Bottom) Quantification of SUMO1 nuclear foci for the conditions indicated above as determined by the mCherry-based mini-replicon reporter assay in MRC5s. For (B)–(F), cells were transfected for 36 hr prior to processing or fixation and immunostaining. Representative images are shown. Scale bars represent 5 μm. Statistical significance in panels (B), (C), (E), and (F) was determined using the Student’s t test (∗∗∗p < 0.0001; ns, non-significant). See also Figure S2.
Mentions: We used small-molecule inhibitors and UV-inactivation methods to map IAV-triggered SUMOylation to a process requiring viral genome replication and protein synthesis, but not genome nuclear export or later stages of the virus replication cycle, such as virion budding (Figure 2A). Given the tight association of active influenza virus replication complexes with nuclear processes (a distinguishing feature from cytoplasmic-replicating RNA viruses), we speculated that the stress of nuclear IAV polymerase activity may contribute to host SUMO remodeling. To test this hypothesis, we used a transfection-based mini-replicon reporter system, whereby viral ribonucleoprotein complexes (vRNPs) consisting of viral NP, PB1, PB2, and PA are assembled together in the nucleus following plasmid expression along with a negative-sense viral-like RNA genomic segment encoding mCherry. As the viral-like RNA cannot be transcribed into mRNA by cellular polymerases, mCherry protein is only produced in cells expressing all five viral components. Furthermore, the mCherry construct is unspliced such that this assay recapitulates IAV RNA transcription and replication, but not splicing.

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