1. Volume 23, Issue 2, Pages 254-265.e7 (February 2018)
Herpes Simplex Virus 1 VP22 Inhibits AIM2-Dependent Inflammasome Activation to Enable Efficient Viral Replication 
Yuhei Maruzuru, Takeshi Ichinohe, Ryota Sato, Kensuke Miyake, Tokuju Okano, Toshihiko Suzuki, Takumi Koshiba, Naoto Koyanagi, Shumpei Tsuda, Mizuki Watanabe, Jun Arii, Akihisa Kato, Yasushi Kawaguchi 
Cell Host & Microbe 
Volume 23, Issue 2, Pages e7 (February 2018)
DOI: /j.chom
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2. Cell Host & Microbe 2018 23, 254-265. e7DOI: (10. 1016/j. chom. 2017
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3. Figure 1
Identification of HSV-1 VP22 as an Inhibitor of AIM2-Dependent Inflammasome Activation
(A) The inhibitory effect of each HSV-1 protein on AIM2-dependent inflammasome-mediated IL-1β secretion was calculated by dividing the mean value (Figure S1A) for the effect of each HSV-1 protein on IL-1β secretion in the presence of NLRP3 by the mean value (Figure S1A) in the presence of AIM2.
(B and C) The inhibitory effect of VP22 on IL-1β secretion was analyzed in the AIM2 or NLRP3 inflammasome reconstitution system (B). Expression levels of the indicated proteins were analyzed by immunoblotting (C).
(D) 293FT cells transfected with expression plasmids encoding ASC alone or both ASC and AIM2, along with expression plasmids encoding VP22 or an empty plasmid for 24 hr, were examined by confocal microscopy. Bars, 5 μm.
(E) Percentage of cells with ASC speck structures in the experiments in (D).
Each value is the mean ± standard error of the results of three independent experiments (B and E). Statistical analysis was performed by the two-tailed Student’s t test. n.s., not statistically significant; ∗∗p < The data are representative of at least three independent experiments (C and D). See also Figures S1 and S2.
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4. Figure 2 VP22 Blocked DNA-Mediated Inflammasome Activation
(A) J774/VP22 and J774/Ct cells were analyzed by immunoblotting.
(B) J774/Ct and J774/VP22 cells were stimulated by treatment with LPS (1 μg/mL) and transfection with poly(dA-dT) (10 μg/mL) for 18 hr, and cell-free supernatants (sup) and cells (cell) were analyzed by immunoblotting.
(C) J774/Ct and J774/VP22 cells were stimulated by treatment with LPS (1 μg/mL) and transfection with poly(dA-dT) (10 μg/mL) for 18 hr. The release of IL-1β, IL-6, and TNF-α into the supernatants was analyzed by ELISA. Each value is the mean ± standard error of the results of four independent experiments.
Statistical analysis was performed by the two-tailed Student’s t test. n.s., not statistically significant; ∗p < 0.05 (C). The data are representative of at least three independent experiments (A and B). See also Figures S3 and S4.
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5. Figure 3
The VP22 Null Mutation Augmented AIM2-Dependent Inflammasome Activation in HSV-1-Infected Cells
(A and B) BMMs mock infected or infected with HSV-1 (WT), HSV-1ΔVP22 (ΔVP22), or HSV-1ΔVP22-repair (ΔVP22-repair) at an MOI of 3 for 12 hr were analyzed by immunoblotting.
(C) IL-1β secretion in experiments (A and B) was measured by ELISA. The dotted line indicates the detection limit (7.8 pg/mL) of the ELISA assays.
(D and E) WT (AIM2+/+) and AIM2−/− BMMs mock infected or infected with HSV-1 (WT), HSV-1ΔVP22 (ΔVP22), or HSV-1ΔVP22-repair (ΔVP22-repair) at an MOI of 3 for 12 hr were analyzed by immunoblotting.
(F and G) WT (AIM2+/+) and AIM2−/− BMMs were mock infected or infected with HSV-1 (WT), HSV-1ΔVP22 (ΔVP22), or HSV-1ΔVP22-repair (ΔVP22-repair) at an MOI of 3 for 12 hr in the absence (F) or presence (G) of 0.75 μg LPS/mL. IL-1β secretion was measured by ELISA. The dotted line indicates the detection limit (7.8 pg/mL) of the ELISA assays.
The data are representative of at least three independent experiments (A, B, D, and E). Each value is the mean ± standard error of the results of four independent experiments (C, F, and G). Statistical analysis was performed by one-way ANOVA and Tukey’s test (C) or the two-tailed Student’s t test (F and G). n.s., not statistically significant; ∗p < 0.05, ∗∗∗p < See also Figures S5 and S6.
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6. Figure 4
Virion-Associated VP22 Inhibited Inflammasome Activation in BMMs
(A) Schematic diagram of the procedure to produce HSV-1ΔVP22+/− (ΔVP22+/−) and HSV-1ΔVP22−/− (ΔVP22−/−).
(B) Vero/EGFP or Vero/VP22 cells were infected with HSV-1ΔVP22 at an MOI of 0.05 for 48 hr. Extracellular virions purified from the supernatants (Virion) and cell lysates (Cell) were analyzed by immunoblotting.
(C) Vero/VP22 cells were infected with HSV-1ΔVP22 (ΔVP22), and Vero/EGFP cells were infected with HSV-1 (WT), HSV-1ΔVP22 (ΔVP22), or HSV-1ΔVP22-repair (ΔVP22-repair) at an MOI of 0.05 for 48 hr. Extracellular virions purified from the supernatants were analyzed by immunoblotting.
(D and E) BMMs were mock -infected or infected with HSV-1ΔVP22+/− (ΔVP22+/−) or HSV-1ΔVP22−/− (ΔVP22−/−) at an MOI of 3 for 12 hr. Infected cells were analyzed by immunoblotting (D) and IL-1β secretion was measured by ELISA (E). The dotted line indicates the IL-1β detection limit (7.8 pg/mL) of the ELISA assays (E). Each value is the mean ± standard error of the results of five independent experiments.
Statistical significance was determined by the two-tailed Student’s t test, ∗p < The data are representative of two (C) or three (B and D) independent experiments.
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7. Figure 5
VP22 Was Required for Efficient Viral Replication in AIM2+/+ Mice but Not in AIM2−/− Mice
5- to 7-week-old female WT (AIM2+/+) and AIM2−/− mice were inoculated intracranially with 105 PFU HSV-1ΔVP22 or HSV-1ΔVP22-repair. At 4 days post infection, the brains of infected mice were harvested and virus titers were assayed. Each data point is the virus titer in the brain of one infected mouse: 15 WT (AIM2+/+) mice were infected with HSV-1ΔVP22, 14 AIM2−/− mice were infected with HSV-1ΔVP22, 11 WT (AIM2+/+) mice were infected with HSV-1ΔVP22-repair, and 8 AIM2−/− mice were infected with HSV-1ΔVP22-repair. The dashed line indicates the limit of detection (18 PFU/brain). The horizontal bar indicates the mean ± standard error for each group. Statistical significance was determined by the two-tailed Student’s t test with the Bonferroni adjustment for the four comparison analyses. n.s., not statistically significant; ∗∗p < (0.05/6). See also Figure S7.
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8. Figure 6 VP22 Interacted with AIM2
(A) 293FT cells co-transfected with expression plasmids encoding AIM2 and VP22 for 24 hr were lysed, immunoprecipitated (IP), and analyzed by immunoblotting.
(B) J774.A1 cells infected with WT HSV-1(F) or HSV-1/VP22(F) at an MOI 3 for 8 hr were lysed, immunoprecipitated, and analyzed by immunoblotting.
(C) Schematic diagrams showing the domain structures of AIM2 and the truncated mutants used in Figures 6D, 6F, 7D–7F, and S7C.
(D) The indicated GST fusion proteins immobilized on glutathione-Sepharose beads were reacted with lysates of 293FT cells transfected with an expression plasmid encoding Flag-VP22. The beads were analyzed by immunoblotting (top gel) and were also electrophoretically separated in a denaturing gel and stained with Coomassie brilliant blue (CBB; bottom gel).
(E) Schematic diagrams showing the truncated mutants of VP22 used in Figures 6F and 6G.
(F) GST-AIM2-HIN immobilized on glutathione-Sepharose beads were reacted with lysates of 293FT cells transfected with an expression plasmid encoding each of the indicated truncated mutants of Flag-VP22. The beads were then processed as described in (D).
(G) 293FT cells were transfected with expression plasmids encoding AIM2, ASC, pro-caspase-1, and pro-IL-1β together with Flag-EGFP or each of the indicated truncated mutants of Flag-VP22 for 24 hr. IL-1β secretion was analyzed by ELISA. Each value is the mean ± standard error of triplicate experiments.
The data are representative of at least three independent experiments (A, B, D, F, and G). See also Figure S7.
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9. Figure 7
VP22 Interacted with the AIM2 HIN Domain but Not the Pyrin Domain to Block Oligomerization
(A) 293FT cells co-transfected with an expression plasmid encoding AIM2-Flag and an expression plasmid encoding either VP22 or EGFP or with an expression plasmid encoding AIM2-Flag and an empty plasmid for 24 hr were examined by confocal microscopy. Bars, 5 μm.
(B) Percentage of cells with AIM2 filamentous aggregates in the experiments in (A).
(C) 293FT cells co-transfected with an expression plasmid encoding AIM2 and an expression plasmid encoding either VP22 or EGFP for 48 hr were subjected to sucrose density gradient sedimentations.
(D) 293FT cells co-transfected with an expression plasmid encoding AIM2-HIN-Flag or AIM2-pyrin-Flag and with an expression plasmid encoding VP22 or an empty plasmid for 24 hr were examined by confocal microscopy.
(E and F) Percentage of cells with AIM2-HIN-Flag (E) or AIM2-pyrin-Flag (F) aggregates in the cytoplasm in the experiments in (D).
Each value is the mean ± standard error of the results of three independent experiments (B, E, and F). Statistical analysis was performed by one-way ANOVA and Tukey’s test (B) or the two-tailed Student’s t test (E and F). n.s., not statistically significant; ∗∗p < 0.01, ∗∗∗∗p < The data are representative of at least three independent experiments (A, C, and D). See also Figure S7.
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