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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.

View Article: PubMed Central - PubMed

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

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HFt cells stably transduced with DOX-inducible eYFP.PIAS4 wild-type or mutant protein expression constructs. (A) The illustration depicts the conserved PIAS4 domains (gray boxes) mutated within eYFP.PIAS4 (eYFP.P4) (Table 1). (B) Confocal images show the nuclear expression of eYFP, eYFP.P4 wild-type (wt) or mutant proteins (as indicated) in transgenic HFt cells after 8 h of induction (+DOX) or not (−DOX). Nuclei were visualized by DAPI (blue). (C) Western blot shows the levels of eYFP.PIAS4 wild-type (eYFP.P4 wt) or endogenous PIAS4 (PIAS4) in transgenic HFt cells after different lengths of DOX induction, as indicated. Membranes were probed for PIAS4 or actin as a loading control. The asterisk indicates a PIAS4 polyclonal antibody cross-reacting band. (D) A bar graph shows the average level of eYFP.P4 wt relative to endogenous PIAS4 in transgenic cells after various lengths of DOX induction. The intensities of PIAS4 protein bands from Western blots as in panel C were quantitated and normalized to their respective loading controls. Normalized levels of eYFP.P4 wt are expressed relative to the normalized levels of endogenous PIAS4 at each time. Means and standard deviations (SD) are shown (n = 3). (E) Whole-cell lysates collected from transgenic HFt cells 8 h post-DOX induction were analyzed by Western blotting for the levels of eYFP and eYFP.PIAS4 wild-type or mutant protein (as indicated) expression. Membranes were probed for eYFP or actin as a loading control. (F) Western blot shows the levels of eYFP.PIAS4 wild-type or catalytically inactive mutant (C342A) proteins at different times after translational inhibition. Expression of eYFP.P4 wt or eYFP.P4.C342A was DOX induced for 4 h prior to the replacement of DOX with the translation inhibitor cycloheximide (CHX) in the presence (+) or absence (−) of MG132. Whole-cell lysates were harvested at 0, 2, 4, 6, or 8 h following CHX addition. Membranes were probed with eYFP or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (G) A bar graph shows the average relative levels of eYFP.PIAS4 wild-type or catalytically inactive mutant proteins following translation inhibition. The intensities of protein bands corresponding to eYFP.P4 quantitated from Western blots as in panel F were normalized to their respective loading controls and expressed relative to the normalized levels at 0 h of CHX treatment (1.0). Means and SEM are shown (n = 3). (H) A bar graph shows the average relative levels of PIAS4 mRNA in transgenic cells that express eYFP.P4 wild-type or catalytically inactive mutant proteins. Total RNA was isolated after 4, 8, or 24 h of DOX induction. The levels of PIAS4 mRNA were determined using the TaqMan system of quantitative RT-PCR. Values were normalized to GAPDH expression using the threshold cycle (ΔΔCT) method and are expressed relative to the normalized level of PIAS4 mRNA at 0 h of DOX induction (1). Values represent the averages from two independent experiments. (I) A bar graph shows the average relative level of endogenous PIAS4 following translation inhibition. HFt cells were treated with CHX in the presence (+) or absence (−) of MG132, and whole-cell lysates were collected at 2, 4, or 8 h after CHX addition. Intensities of the protein bands corresponding to PIAS4 were quantitated and normalized to their respective loading controls and are expressed relative to the normalized level of PIAS4 at 0 h of CHX treatment (1.0). Means and SEM are shown (n ≥ 3).
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Figure 3: HFt cells stably transduced with DOX-inducible eYFP.PIAS4 wild-type or mutant protein expression constructs. (A) The illustration depicts the conserved PIAS4 domains (gray boxes) mutated within eYFP.PIAS4 (eYFP.P4) (Table 1). (B) Confocal images show the nuclear expression of eYFP, eYFP.P4 wild-type (wt) or mutant proteins (as indicated) in transgenic HFt cells after 8 h of induction (+DOX) or not (−DOX). Nuclei were visualized by DAPI (blue). (C) Western blot shows the levels of eYFP.PIAS4 wild-type (eYFP.P4 wt) or endogenous PIAS4 (PIAS4) in transgenic HFt cells after different lengths of DOX induction, as indicated. Membranes were probed for PIAS4 or actin as a loading control. The asterisk indicates a PIAS4 polyclonal antibody cross-reacting band. (D) A bar graph shows the average level of eYFP.P4 wt relative to endogenous PIAS4 in transgenic cells after various lengths of DOX induction. The intensities of PIAS4 protein bands from Western blots as in panel C were quantitated and normalized to their respective loading controls. Normalized levels of eYFP.P4 wt are expressed relative to the normalized levels of endogenous PIAS4 at each time. Means and standard deviations (SD) are shown (n = 3). (E) Whole-cell lysates collected from transgenic HFt cells 8 h post-DOX induction were analyzed by Western blotting for the levels of eYFP and eYFP.PIAS4 wild-type or mutant protein (as indicated) expression. Membranes were probed for eYFP or actin as a loading control. (F) Western blot shows the levels of eYFP.PIAS4 wild-type or catalytically inactive mutant (C342A) proteins at different times after translational inhibition. Expression of eYFP.P4 wt or eYFP.P4.C342A was DOX induced for 4 h prior to the replacement of DOX with the translation inhibitor cycloheximide (CHX) in the presence (+) or absence (−) of MG132. Whole-cell lysates were harvested at 0, 2, 4, 6, or 8 h following CHX addition. Membranes were probed with eYFP or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (G) A bar graph shows the average relative levels of eYFP.PIAS4 wild-type or catalytically inactive mutant proteins following translation inhibition. The intensities of protein bands corresponding to eYFP.P4 quantitated from Western blots as in panel F were normalized to their respective loading controls and expressed relative to the normalized levels at 0 h of CHX treatment (1.0). Means and SEM are shown (n = 3). (H) A bar graph shows the average relative levels of PIAS4 mRNA in transgenic cells that express eYFP.P4 wild-type or catalytically inactive mutant proteins. Total RNA was isolated after 4, 8, or 24 h of DOX induction. The levels of PIAS4 mRNA were determined using the TaqMan system of quantitative RT-PCR. Values were normalized to GAPDH expression using the threshold cycle (ΔΔCT) method and are expressed relative to the normalized level of PIAS4 mRNA at 0 h of DOX induction (1). Values represent the averages from two independent experiments. (I) A bar graph shows the average relative level of endogenous PIAS4 following translation inhibition. HFt cells were treated with CHX in the presence (+) or absence (−) of MG132, and whole-cell lysates were collected at 2, 4, or 8 h after CHX addition. Intensities of the protein bands corresponding to PIAS4 were quantitated and normalized to their respective loading controls and are expressed relative to the normalized level of PIAS4 at 0 h of CHX treatment (1.0). Means and SEM are shown (n ≥ 3).

Mentions: To investigate the roles of PIAS4 during HSV-1 infection, the domains that mediate recruitment into replication compartments were evaluated (Fig. 3A). While the SAP domain regulates MAR association (65, 66), the LxxLL sequence, PINIT domain, SIM, or carboxyl (C)-terminal acidic domain (AD) may mediate important protein-protein interactions that regulate PIAS4 localization during infection (Fig. 3A). Additionally, the catalytic activity of PIAS4 may regulate localization through the SUMOylation of substrates to influence relevant protein-protein interactions.


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)

HFt cells stably transduced with DOX-inducible eYFP.PIAS4 wild-type or mutant protein expression constructs. (A) The illustration depicts the conserved PIAS4 domains (gray boxes) mutated within eYFP.PIAS4 (eYFP.P4) (Table 1). (B) Confocal images show the nuclear expression of eYFP, eYFP.P4 wild-type (wt) or mutant proteins (as indicated) in transgenic HFt cells after 8 h of induction (+DOX) or not (−DOX). Nuclei were visualized by DAPI (blue). (C) Western blot shows the levels of eYFP.PIAS4 wild-type (eYFP.P4 wt) or endogenous PIAS4 (PIAS4) in transgenic HFt cells after different lengths of DOX induction, as indicated. Membranes were probed for PIAS4 or actin as a loading control. The asterisk indicates a PIAS4 polyclonal antibody cross-reacting band. (D) A bar graph shows the average level of eYFP.P4 wt relative to endogenous PIAS4 in transgenic cells after various lengths of DOX induction. The intensities of PIAS4 protein bands from Western blots as in panel C were quantitated and normalized to their respective loading controls. Normalized levels of eYFP.P4 wt are expressed relative to the normalized levels of endogenous PIAS4 at each time. Means and standard deviations (SD) are shown (n = 3). (E) Whole-cell lysates collected from transgenic HFt cells 8 h post-DOX induction were analyzed by Western blotting for the levels of eYFP and eYFP.PIAS4 wild-type or mutant protein (as indicated) expression. Membranes were probed for eYFP or actin as a loading control. (F) Western blot shows the levels of eYFP.PIAS4 wild-type or catalytically inactive mutant (C342A) proteins at different times after translational inhibition. Expression of eYFP.P4 wt or eYFP.P4.C342A was DOX induced for 4 h prior to the replacement of DOX with the translation inhibitor cycloheximide (CHX) in the presence (+) or absence (−) of MG132. Whole-cell lysates were harvested at 0, 2, 4, 6, or 8 h following CHX addition. Membranes were probed with eYFP or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (G) A bar graph shows the average relative levels of eYFP.PIAS4 wild-type or catalytically inactive mutant proteins following translation inhibition. The intensities of protein bands corresponding to eYFP.P4 quantitated from Western blots as in panel F were normalized to their respective loading controls and expressed relative to the normalized levels at 0 h of CHX treatment (1.0). Means and SEM are shown (n = 3). (H) A bar graph shows the average relative levels of PIAS4 mRNA in transgenic cells that express eYFP.P4 wild-type or catalytically inactive mutant proteins. Total RNA was isolated after 4, 8, or 24 h of DOX induction. The levels of PIAS4 mRNA were determined using the TaqMan system of quantitative RT-PCR. Values were normalized to GAPDH expression using the threshold cycle (ΔΔCT) method and are expressed relative to the normalized level of PIAS4 mRNA at 0 h of DOX induction (1). Values represent the averages from two independent experiments. (I) A bar graph shows the average relative level of endogenous PIAS4 following translation inhibition. HFt cells were treated with CHX in the presence (+) or absence (−) of MG132, and whole-cell lysates were collected at 2, 4, or 8 h after CHX addition. Intensities of the protein bands corresponding to PIAS4 were quantitated and normalized to their respective loading controls and are expressed relative to the normalized level of PIAS4 at 0 h of CHX treatment (1.0). Means and SEM are shown (n ≥ 3).
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Figure 3: HFt cells stably transduced with DOX-inducible eYFP.PIAS4 wild-type or mutant protein expression constructs. (A) The illustration depicts the conserved PIAS4 domains (gray boxes) mutated within eYFP.PIAS4 (eYFP.P4) (Table 1). (B) Confocal images show the nuclear expression of eYFP, eYFP.P4 wild-type (wt) or mutant proteins (as indicated) in transgenic HFt cells after 8 h of induction (+DOX) or not (−DOX). Nuclei were visualized by DAPI (blue). (C) Western blot shows the levels of eYFP.PIAS4 wild-type (eYFP.P4 wt) or endogenous PIAS4 (PIAS4) in transgenic HFt cells after different lengths of DOX induction, as indicated. Membranes were probed for PIAS4 or actin as a loading control. The asterisk indicates a PIAS4 polyclonal antibody cross-reacting band. (D) A bar graph shows the average level of eYFP.P4 wt relative to endogenous PIAS4 in transgenic cells after various lengths of DOX induction. The intensities of PIAS4 protein bands from Western blots as in panel C were quantitated and normalized to their respective loading controls. Normalized levels of eYFP.P4 wt are expressed relative to the normalized levels of endogenous PIAS4 at each time. Means and standard deviations (SD) are shown (n = 3). (E) Whole-cell lysates collected from transgenic HFt cells 8 h post-DOX induction were analyzed by Western blotting for the levels of eYFP and eYFP.PIAS4 wild-type or mutant protein (as indicated) expression. Membranes were probed for eYFP or actin as a loading control. (F) Western blot shows the levels of eYFP.PIAS4 wild-type or catalytically inactive mutant (C342A) proteins at different times after translational inhibition. Expression of eYFP.P4 wt or eYFP.P4.C342A was DOX induced for 4 h prior to the replacement of DOX with the translation inhibitor cycloheximide (CHX) in the presence (+) or absence (−) of MG132. Whole-cell lysates were harvested at 0, 2, 4, 6, or 8 h following CHX addition. Membranes were probed with eYFP or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (G) A bar graph shows the average relative levels of eYFP.PIAS4 wild-type or catalytically inactive mutant proteins following translation inhibition. The intensities of protein bands corresponding to eYFP.P4 quantitated from Western blots as in panel F were normalized to their respective loading controls and expressed relative to the normalized levels at 0 h of CHX treatment (1.0). Means and SEM are shown (n = 3). (H) A bar graph shows the average relative levels of PIAS4 mRNA in transgenic cells that express eYFP.P4 wild-type or catalytically inactive mutant proteins. Total RNA was isolated after 4, 8, or 24 h of DOX induction. The levels of PIAS4 mRNA were determined using the TaqMan system of quantitative RT-PCR. Values were normalized to GAPDH expression using the threshold cycle (ΔΔCT) method and are expressed relative to the normalized level of PIAS4 mRNA at 0 h of DOX induction (1). Values represent the averages from two independent experiments. (I) A bar graph shows the average relative level of endogenous PIAS4 following translation inhibition. HFt cells were treated with CHX in the presence (+) or absence (−) of MG132, and whole-cell lysates were collected at 2, 4, or 8 h after CHX addition. Intensities of the protein bands corresponding to PIAS4 were quantitated and normalized to their respective loading controls and are expressed relative to the normalized level of PIAS4 at 0 h of CHX treatment (1.0). Means and SEM are shown (n ≥ 3).
Mentions: To investigate the roles of PIAS4 during HSV-1 infection, the domains that mediate recruitment into replication compartments were evaluated (Fig. 3A). While the SAP domain regulates MAR association (65, 66), the LxxLL sequence, PINIT domain, SIM, or carboxyl (C)-terminal acidic domain (AD) may mediate important protein-protein interactions that regulate PIAS4 localization during infection (Fig. 3A). Additionally, the catalytic activity of PIAS4 may regulate localization through the SUMOylation of substrates to influence relevant protein-protein interactions.

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