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.
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.
Affiliation: MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, Scotland, United Kingdom.
- Herpes Simplex/genetics*/immunology*/metabolism/virology
- Herpesvirus 1, Human/physiology*
- Host-Pathogen Interactions*
- Immunity, Innate/genetics*
- Protein Inhibitors of Activated STAT/chemistry/genetics*
- Amino Acid Motifs
- Amino Acid Sequence
- Cell Line
- DNA Replication
- DNA, Viral
- Disease Progression
- Gene Expression
- Genome, Viral
- Immediate-Early Proteins/metabolism
- Nuclear Proteins/metabolism
- Protein Binding
- Protein Interaction Domains and Motifs
- Protein Transport
- Recombinant Fusion Proteins
- SUMO-1 Protein/metabolism
- Transcription Factors/metabolism
- Tumor Suppressor Proteins/metabolism
- Ubiquitin-Protein Ligases/metabolism
- Virus Replication
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Figure 4: Overexpression of eYFP-PIAS4 disrupts SUMO pathway homeostasis. (A) Western blots show the levels of HMW-conjugated or free SUMO1 or -2/3 in transgenic HFt cells induced (+) to express eYFP or eYFP.PIAS4 wild-type (wt) or catalytically inactive mutant (C342A) proteins or not (−). Whole-cell lysates were collected at 4, 8, or 24 h postinduction. Membranes were probed for SUMO1 or -2/3, eYFP, or actin as a loading control. (B) Bar graphs show the average levels of HMW-conjugated or free SUMO1 or -2/3 in transgenic cells induced (+) or not (−) to express eYFP or eYFP.PIAS4 wild-type or catalytically inactive mutant proteins. The intensities of protein bands corresponding to SUMO1 or -2/3 quantitated from Western blots as in panel A were normalized to their respective loading control and expressed relative to the levels in uninduced cells (1.0). Means and SEM are shown (n = 2 [HMW] or n = 3 [free]). (C) Western blots show the SUMO modification of PML or Sp100 in transgenic cells induced (+) or not (−) to express eYFP or eYFP.PIAS4 wild-type or catalytically inactive mutant proteins for 24 h. Membranes were probed for SUMO2/3, eYFP, PML, Sp100, or actin as a loading control. (D) Confocal images show PML-NBs in transgenic cells induced to express eYFP or eYFP.PIAS4 wild-type or catalytically inactive mutant proteins. PML-NBs were identified by PML accumulation; PML was visualized by indirect immunofluorescence (red). (E) Box-and-whisker chart shows the number of PML-NBs per nuclei in transgenic cells induced (+DOX) for 24 h or not (−DOX) to express eYFP or eYFP.PIAS4 wild-type or catalytically inactive mutant proteins. Boxes, 25th to 75th percentile; black line, median number of PML-NBs per nucleus; whiskers, absolute range. The total number of cells analyzed per sample is indicated by each box from ≥10 fields of view per sample collected over two independent experiments. ***, P < 0.0001 (Mann-Whitney U test). (F) A Western blot shows the SUMO modification of PML in transgenic cells induced to express eYFP.P4 wt in the presence or absence of His-SUMO2. HFt cells stably transduced with eYFP.P4 expression vectors were transduced with a second lentiviral vector to constitutively express His-tagged SUMO2 or with an empty vector control. Expression of eYFP.P4 was induced for 0, 4, 8, or 24 h prior to the collection of whole-cell lysates. Membranes were probed for His, eYFP, PML, or actin as a loading control. Molecular mass markers are indicated, in kilodaltons.
EYFP.PIAS4 protein levels increased substantially with time, without considerable alteration to endogenous PIAS4 expression (Fig. 3C and D). As noted in other studies, however, high levels of ectopic PIAS4 had detrimental cellular effects (66, 68). Overexpression of eYFP.PIAS4 drastically affected SUMO signaling homeostasis, increasing HMW SUMO-conjugated protein levels at least 4-fold and decreasing free SUMO levels 5-fold following 24 h of induction (Fig. 4A and B). Consequently, dynamic SUMO modification was affected, which resulted in a loss of constitutive PML and Sp100 SUMOylation and disrupted PML-NBs (Fig. 4C to E), a phenotype similar to that in Ubc9-depleted cells (18). Consistently, the disruption of SUMO pathway homeostasis was attributed to PIAS4-mediated global depletion of free SUMO pools, as these effects did not occur when catalytically inactive PIAS4 was expressed or when His-tagged SUMO2 was expressed concomitantly with eYFP.PIAS4 to supplement the free SUMO2 pools (Fig. 4). To avoid gross disruption of SUMO pathway equilibrium, expression of the wild-type and mutant eYFP.PIAS4 proteins was typically induced for only 6 to 8 h, which did not substantially alter SUMO pathway homeostasis (Fig. 4A and B).