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 11: Endogenous PIAS4 contributes to the cellular restriction of ICP0- mutant HSV-1. (A) Western blots show the levels of PIAS4 or PML in transgenic HFt cells that express short hairpin RNAs against PIAS4 (shPIAS4), PML (shPML), or a control sequence (shCtrl). Membranes were probed for PIAS4, PML, or actin as a loading control. (B) A bar graph shows the average relative levels of PIAS4 or PML mRNA in transgenic HFt cells that express shCtrl, shPIAS4, or shPML. PIAS4 or PML mRNA levels were determined using the TaqMan system of quantitative RT-PCR. Values normalized to 18S expression using the ΔΔCT method are expressed relative to cells that expressed shCtrl (1.0). Values represent means and SD from three independent rounds of RT using mRNA isolated from one representative experiment. (C) A bar graph shows the average relative plaque formation efficiency (PFE) of wild-type or ICP0- mutant (ΔICP0) HSV-1 in transgenic cells that express shCtrl, shPIAS4, or shPML, as indicated. The PFE for each strain in cells that express shPIAS4 or shPML was normalized to the respective PFE in cells that express shCtrl (1). Means and SD are shown (n ≥ 3). (D and E) Western blots show the levels of viral protein expression during wild-type or ICP0- mutant (ΔICP0) HSV-1 infection of transgenic HFt cells that express shCtrl or shPIAS4. Transgenic cells mock infected or infected with 10 PFU of wild-type (D) or ICP0- mutant (E) HSV-1 per cell in the presence (+) or absence (−) of MG132 were harvested at 3, 6, or 9 hpi. Membranes were probed for ICP4, ICP0, UL42, or VP5 to monitor the progression of infection or actin as a loading control. Molecular mass markers are shown, in kilodaltons. (F) A bar graph shows the average relative levels of PML or PIAS4 mRNA in transgenic cells that express shCtrl, shPIAS4, or shPML alone or in combination, as indicated. Analysis was performed as for panel B. Neo, neomycin resistance; Puro, puromycin resistance. (G) A bar graph shows the average relative PFE of wild-type or ICP0- mutant HSV-1 in transgenic HFt cells that express shCtrl, shPIAS4, or shPML alone or in combination, as indicated. PFE analysis was done as described for panel C. Means and SD are shown (n ≥ 3).
PIAS4 expression increased during HSV-1 infection, and it localized to nuclear domains that contained viral genomes. Furthermore, PIAS4 colocalized with known intrinsic antiviral factors during ICP0- mutant HSV-1 infection. To test the functional significance of PIAS4 during infection, replication of wild-type or ICP0- mutant HSV-1 was evaluated in cells depleted of PIAS4 or of PML as a positive control (50). To this end, HFt cells were transduced with lentiviral vectors that expressed short hairpin RNAs (shRNAs) against PIAS4, PML, or a nontargeted scrambled control. Cells that expressed PML-specific shRNAs had a marked depletion in PML protein and mRNA levels, while cells that expressed PIAS4-specific shRNAs had considerable, although not complete, depletion of PIAS4 protein and mRNA levels (Fig. 11A and B). As PIAS4 is integral for cell division (45), the degree of PIAS4 depletion varied with each round of transduction, and after limited passaging, PIAS4 protein levels recovered to those in cells transduced with control shRNAs (data not shown). All functional experiments were therefore conducted over multiple rounds of independent transduction with minimal passaging following the isolation of stably transduced cells.