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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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Related in: MedlinePlus

HMW SUMO-conjugated proteins do not accumulate when HSV-1 DNA does not replicate. (A) Western blots show the levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. HFt cells infected with 10 PFU of wild-type or ICP0- mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132 were harvested at 3, 6, or 9 h postinfection (hpi). Membranes were probed for SUMO1 or -2/3, the viral protein ICP0 or UL42 to monitor infection progression, or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (B) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots as shown in panel A; quantitated levels were normalized to their respective loading controls and are expressed relative to the levels in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown (n ≥ 4). (C) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 in the presence of either of the DNA replication inhibitors PAA or ACG, MG132, or no drug (ND). Infections equivalent to those for panel A were performed in the presence or absence of the indicated drugs. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots; quantitated values were normalized to their respective loading controls and are expressed relative to the levels in untreated mock-infected cells at 9 hpi. Means and SEM are shown (n ≥ 3). *, P < 0.05; **, P < 0.01 (Student's two-tailed t test).
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Figure 1: HMW SUMO-conjugated proteins do not accumulate when HSV-1 DNA does not replicate. (A) Western blots show the levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. HFt cells infected with 10 PFU of wild-type or ICP0- mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132 were harvested at 3, 6, or 9 h postinfection (hpi). Membranes were probed for SUMO1 or -2/3, the viral protein ICP0 or UL42 to monitor infection progression, or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (B) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots as shown in panel A; quantitated levels were normalized to their respective loading controls and are expressed relative to the levels in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown (n ≥ 4). (C) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 in the presence of either of the DNA replication inhibitors PAA or ACG, MG132, or no drug (ND). Infections equivalent to those for panel A were performed in the presence or absence of the indicated drugs. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots; quantitated values were normalized to their respective loading controls and are expressed relative to the levels in untreated mock-infected cells at 9 hpi. Means and SEM are shown (n ≥ 3). *, P < 0.05; **, P < 0.01 (Student's two-tailed t test).

Mentions: Whether or not the steady-state levels of unconjugated free SUMO were altered during HSV-1 infection along with the global changes in SUMOylation was evaluated. Consistent with previous reports (18), HMW SUMO1- or SUMO2/3-conjugated protein levels decreased during wild-type HSV-1 infection, while they increased during ICP0- mutant HSV-1 infection (Fig. 1A and B, HMW). Although the levels of HMW SUMO-conjugated proteins were clearly altered in an ICP0-dependent manner, the levels of unconjugated free SUMO1 or -2/3 remained relatively stable (Fig. 1A and B, free). In HFt cells infected with 10 PFU per cell of wild-type or ICP0- mutant HSV-1, the cellular pools of free SUMO1 or -2/3 were depleted by 20 to 30% at 9 hpi (Fig. 1A and B, free). In contrast, inhibition of the proteasome, in either the presence or absence of ICP0, was sufficient to deplete the pools of free SUMO1 or -2/3 by 70 to 80% (Fig. 1A and B, +MG132). Together, these data indicate that free SUMO levels are likely regulated during infection, irrespective of the broad depletion or accumulation of HMW SUMO-conjugated proteins during wild-type or ICP0- mutant HSV-1 infection, respectively. Moreover, these data suggest that free SUMO levels are unlikely to be a limiting factor for de novo SUMOylation, supporting the concept that dynamic SUMO modification and signaling occur throughout infection (28).


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)

HMW SUMO-conjugated proteins do not accumulate when HSV-1 DNA does not replicate. (A) Western blots show the levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. HFt cells infected with 10 PFU of wild-type or ICP0- mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132 were harvested at 3, 6, or 9 h postinfection (hpi). Membranes were probed for SUMO1 or -2/3, the viral protein ICP0 or UL42 to monitor infection progression, or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (B) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots as shown in panel A; quantitated levels were normalized to their respective loading controls and are expressed relative to the levels in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown (n ≥ 4). (C) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 in the presence of either of the DNA replication inhibitors PAA or ACG, MG132, or no drug (ND). Infections equivalent to those for panel A were performed in the presence or absence of the indicated drugs. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots; quantitated values were normalized to their respective loading controls and are expressed relative to the levels in untreated mock-infected cells at 9 hpi. Means and SEM are shown (n ≥ 3). *, P < 0.05; **, P < 0.01 (Student's two-tailed t test).
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Figure 1: HMW SUMO-conjugated proteins do not accumulate when HSV-1 DNA does not replicate. (A) Western blots show the levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. HFt cells infected with 10 PFU of wild-type or ICP0- mutant (ΔICP0) HSV-1 per cell in the presence (+) or absence (−) of the proteasome inhibitor MG132 were harvested at 3, 6, or 9 h postinfection (hpi). Membranes were probed for SUMO1 or -2/3, the viral protein ICP0 or UL42 to monitor infection progression, or actin as a loading control. Molecular mass markers are indicated, in kilodaltons. (B) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 during wild-type or ICP0- mutant HSV-1 infection. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots as shown in panel A; quantitated levels were normalized to their respective loading controls and are expressed relative to the levels in mock-infected cells at 9 hpi (1.0). Means and standard error of the means (SEM) are shown (n ≥ 4). (C) Bar graphs show the average relative levels of HMW-conjugated or free SUMO1 or -2/3 in the presence of either of the DNA replication inhibitors PAA or ACG, MG132, or no drug (ND). Infections equivalent to those for panel A were performed in the presence or absence of the indicated drugs. The intensities of SUMO1 or -2/3 protein bands were quantitated from Western blots; quantitated values were normalized to their respective loading controls and are expressed relative to the levels in untreated mock-infected cells at 9 hpi. Means and SEM are shown (n ≥ 3). *, P < 0.05; **, P < 0.01 (Student's two-tailed t test).
Mentions: Whether or not the steady-state levels of unconjugated free SUMO were altered during HSV-1 infection along with the global changes in SUMOylation was evaluated. Consistent with previous reports (18), HMW SUMO1- or SUMO2/3-conjugated protein levels decreased during wild-type HSV-1 infection, while they increased during ICP0- mutant HSV-1 infection (Fig. 1A and B, HMW). Although the levels of HMW SUMO-conjugated proteins were clearly altered in an ICP0-dependent manner, the levels of unconjugated free SUMO1 or -2/3 remained relatively stable (Fig. 1A and B, free). In HFt cells infected with 10 PFU per cell of wild-type or ICP0- mutant HSV-1, the cellular pools of free SUMO1 or -2/3 were depleted by 20 to 30% at 9 hpi (Fig. 1A and B, free). In contrast, inhibition of the proteasome, in either the presence or absence of ICP0, was sufficient to deplete the pools of free SUMO1 or -2/3 by 70 to 80% (Fig. 1A and B, +MG132). Together, these data indicate that free SUMO levels are likely regulated during infection, irrespective of the broad depletion or accumulation of HMW SUMO-conjugated proteins during wild-type or ICP0- mutant HSV-1 infection, respectively. Moreover, these data suggest that free SUMO levels are unlikely to be a limiting factor for de novo SUMOylation, supporting the concept that dynamic SUMO modification and signaling occur throughout infection (28).

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