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Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response.

Conn KL, Wasson P, McFarlane S, Tong L, Brown JR, Grant KG, Domingues P, Boutell C - J. Virol. (2016)

Bottom Line: Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined.Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate.The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling.

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Affiliation: MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, Scotland, United Kingdom.

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ICP0 disrupts PIAS4 SIM-dependent recruitment to nuclear domains associated with HSV-1 genome entry. (A to I) Confocal images show the nuclear localization of eYFP or eYFP.PIAS4 wild-type or mutant proteins (as indicated) with respect to infecting ICP0- mutant (ΔICP0) HSV-1 genomes, visualized by the asymmetric nuclear edge localization of ICP4 or PML (6, 69). Transgenic HFt cells were infected with 1 PFU of ICP0- mutant HSV-1 per cell for 16 h prior to DOX induction of eYFP or eYFP.PIAS4 wild-type or mutant protein expression for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. (J) Validation of PIAS4 pAb (cyan) detection of eYFP.P4 wt at nuclear domains that contain infecting HSV-1 genomes. (K and M) Confocal images show the typical (K) or atypical (M) localization of endogenous PIAS4 (red) with respect to eYFP.ICP4 at a nuclear edge associated with ICP0- mutant HSV-1 genome nuclear entry. (L) Emission spectra depict pixel intensity and colocalization between eYFP.ICP4 and PIAS4 in selected foci that correspond to the numbered lines in panel K. (N) Confocal images show the localization of eYFP.P4 wt with respect to PML or ICP4 in cells at the periphery of a developing wild-type HSV-1 plaque. Transgenic HFt cells were infected with 0.002 PFU of wild-type HSV-1 per cell for 16 h prior to induction of eYFP.P4 wt for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. The arrow highlights the asymmetric redistribution of eYFP.P4 wt or PML to the nuclear edge prior to the detectable expression of ICP4. (O) Confocal images show the localization of eYFP.P4 wt with respect to ICP0 or PML prior to ICP0-mediated degradation of PML. Transgenic HFt cells were infected as for panel N. ICP0 (red) and PML (cyan) were visualized by indirect immunofluorescence. Nuclei were visualized by DAPI (blue).
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Figure 8: ICP0 disrupts PIAS4 SIM-dependent recruitment to nuclear domains associated with HSV-1 genome entry. (A to I) Confocal images show the nuclear localization of eYFP or eYFP.PIAS4 wild-type or mutant proteins (as indicated) with respect to infecting ICP0- mutant (ΔICP0) HSV-1 genomes, visualized by the asymmetric nuclear edge localization of ICP4 or PML (6, 69). Transgenic HFt cells were infected with 1 PFU of ICP0- mutant HSV-1 per cell for 16 h prior to DOX induction of eYFP or eYFP.PIAS4 wild-type or mutant protein expression for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. (J) Validation of PIAS4 pAb (cyan) detection of eYFP.P4 wt at nuclear domains that contain infecting HSV-1 genomes. (K and M) Confocal images show the typical (K) or atypical (M) localization of endogenous PIAS4 (red) with respect to eYFP.ICP4 at a nuclear edge associated with ICP0- mutant HSV-1 genome nuclear entry. (L) Emission spectra depict pixel intensity and colocalization between eYFP.ICP4 and PIAS4 in selected foci that correspond to the numbered lines in panel K. (N) Confocal images show the localization of eYFP.P4 wt with respect to PML or ICP4 in cells at the periphery of a developing wild-type HSV-1 plaque. Transgenic HFt cells were infected with 0.002 PFU of wild-type HSV-1 per cell for 16 h prior to induction of eYFP.P4 wt for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. The arrow highlights the asymmetric redistribution of eYFP.P4 wt or PML to the nuclear edge prior to the detectable expression of ICP4. (O) Confocal images show the localization of eYFP.P4 wt with respect to ICP0 or PML prior to ICP0-mediated degradation of PML. Transgenic HFt cells were infected as for panel N. ICP0 (red) and PML (cyan) were visualized by indirect immunofluorescence. Nuclei were visualized by DAPI (blue).

Mentions: During ICP0- mutant HSV-1 infection, eYFP.PIAS4 also localized to nuclear domains that contained infecting viral genomes (Fig. 8B) (6, 8, 69). This localization was SIM dependent (Fig. 8C to I), consistent with the SIM-dependent recruitment of evaluated constituent PML-NB antiviral proteins (17). However, high levels of eYFP.PIAS4 expression disrupted the SIM-dependent recruitment of constituent PML-NB antiviral proteins without disrupting its own recruitment (Fig. 9). The global depletion of free SUMO pools as a consequence of high levels of eYFP.PIAS4 expression likely precluded the de novo SUMOylation events that mediate recruitment of constitutive PML-NB antiviral proteins (Fig. 4A and B and 9) (17, 18). In contrast, de novo SUMOylation, higher levels of free SUMO, or constituent PML-NB antiviral proteins were not required for the recruitment of eYFP-PIAS4 to domains that contained infecting HSV-1 genomes (Fig. 9A). Moreover, under conditions of depleted free SUMO pools, eYFP.PIAS4 was not sufficient to recruit constituent PML-NB antiviral proteins to nuclear domains that contained infecting HSV-1 genomes (Fig. 9).


Novel Role for Protein Inhibitor of Activated STAT 4 (PIAS4) in the Restriction of Herpes Simplex Virus 1 by the Cellular Intrinsic Antiviral Immune Response.

Conn KL, Wasson P, McFarlane S, Tong L, Brown JR, Grant KG, Domingues P, Boutell C - J. Virol. (2016)

ICP0 disrupts PIAS4 SIM-dependent recruitment to nuclear domains associated with HSV-1 genome entry. (A to I) Confocal images show the nuclear localization of eYFP or eYFP.PIAS4 wild-type or mutant proteins (as indicated) with respect to infecting ICP0- mutant (ΔICP0) HSV-1 genomes, visualized by the asymmetric nuclear edge localization of ICP4 or PML (6, 69). Transgenic HFt cells were infected with 1 PFU of ICP0- mutant HSV-1 per cell for 16 h prior to DOX induction of eYFP or eYFP.PIAS4 wild-type or mutant protein expression for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. (J) Validation of PIAS4 pAb (cyan) detection of eYFP.P4 wt at nuclear domains that contain infecting HSV-1 genomes. (K and M) Confocal images show the typical (K) or atypical (M) localization of endogenous PIAS4 (red) with respect to eYFP.ICP4 at a nuclear edge associated with ICP0- mutant HSV-1 genome nuclear entry. (L) Emission spectra depict pixel intensity and colocalization between eYFP.ICP4 and PIAS4 in selected foci that correspond to the numbered lines in panel K. (N) Confocal images show the localization of eYFP.P4 wt with respect to PML or ICP4 in cells at the periphery of a developing wild-type HSV-1 plaque. Transgenic HFt cells were infected with 0.002 PFU of wild-type HSV-1 per cell for 16 h prior to induction of eYFP.P4 wt for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. The arrow highlights the asymmetric redistribution of eYFP.P4 wt or PML to the nuclear edge prior to the detectable expression of ICP4. (O) Confocal images show the localization of eYFP.P4 wt with respect to ICP0 or PML prior to ICP0-mediated degradation of PML. Transgenic HFt cells were infected as for panel N. ICP0 (red) and PML (cyan) were visualized by indirect immunofluorescence. Nuclei were visualized by DAPI (blue).
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Figure 8: ICP0 disrupts PIAS4 SIM-dependent recruitment to nuclear domains associated with HSV-1 genome entry. (A to I) Confocal images show the nuclear localization of eYFP or eYFP.PIAS4 wild-type or mutant proteins (as indicated) with respect to infecting ICP0- mutant (ΔICP0) HSV-1 genomes, visualized by the asymmetric nuclear edge localization of ICP4 or PML (6, 69). Transgenic HFt cells were infected with 1 PFU of ICP0- mutant HSV-1 per cell for 16 h prior to DOX induction of eYFP or eYFP.PIAS4 wild-type or mutant protein expression for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. (J) Validation of PIAS4 pAb (cyan) detection of eYFP.P4 wt at nuclear domains that contain infecting HSV-1 genomes. (K and M) Confocal images show the typical (K) or atypical (M) localization of endogenous PIAS4 (red) with respect to eYFP.ICP4 at a nuclear edge associated with ICP0- mutant HSV-1 genome nuclear entry. (L) Emission spectra depict pixel intensity and colocalization between eYFP.ICP4 and PIAS4 in selected foci that correspond to the numbered lines in panel K. (N) Confocal images show the localization of eYFP.P4 wt with respect to PML or ICP4 in cells at the periphery of a developing wild-type HSV-1 plaque. Transgenic HFt cells were infected with 0.002 PFU of wild-type HSV-1 per cell for 16 h prior to induction of eYFP.P4 wt for 6 to 8 h. ICP4 (red) and PML (cyan) were visualized by indirect immunofluorescence. The arrow highlights the asymmetric redistribution of eYFP.P4 wt or PML to the nuclear edge prior to the detectable expression of ICP4. (O) Confocal images show the localization of eYFP.P4 wt with respect to ICP0 or PML prior to ICP0-mediated degradation of PML. Transgenic HFt cells were infected as for panel N. ICP0 (red) and PML (cyan) were visualized by indirect immunofluorescence. Nuclei were visualized by DAPI (blue).
Mentions: During ICP0- mutant HSV-1 infection, eYFP.PIAS4 also localized to nuclear domains that contained infecting viral genomes (Fig. 8B) (6, 8, 69). This localization was SIM dependent (Fig. 8C to I), consistent with the SIM-dependent recruitment of evaluated constituent PML-NB antiviral proteins (17). However, high levels of eYFP.PIAS4 expression disrupted the SIM-dependent recruitment of constituent PML-NB antiviral proteins without disrupting its own recruitment (Fig. 9). The global depletion of free SUMO pools as a consequence of high levels of eYFP.PIAS4 expression likely precluded the de novo SUMOylation events that mediate recruitment of constitutive PML-NB antiviral proteins (Fig. 4A and B and 9) (17, 18). In contrast, de novo SUMOylation, higher levels of free SUMO, or constituent PML-NB antiviral proteins were not required for the recruitment of eYFP-PIAS4 to domains that contained infecting HSV-1 genomes (Fig. 9A). Moreover, under conditions of depleted free SUMO pools, eYFP.PIAS4 was not sufficient to recruit constituent PML-NB antiviral proteins to nuclear domains that contained infecting HSV-1 genomes (Fig. 9).

Bottom Line: Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined.Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate.The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling.

View Article: PubMed Central - PubMed

Affiliation: MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, Scotland, United Kingdom.

Show MeSH
Related in: MedlinePlus