HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression

Abstract

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

Keywords: herpes simplex virus; immune evasion; innate immunity; type I IFN.

Figure 1. HSV‐1 harbors a mechanism to inhibit type I IFN expression, which is dependent on viral gene expression but independent of ICP0

1. THP1 cells were infected with the shown strains of HSV‐1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 h for measurement of type I IFN bioactivity.

2. THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS and F HSV‐1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

3. THP1 cells were treated with infectious or UV‐inactivated HSV‐1 (strain KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

4. MDMs were infected with ICP0‐deficient or revertant HSV‐1 (strain KOS, MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

5. THP1‐derived cells deficient for cGAS or STING were infected with the shown strains of HSV‐1 (MOI 3) or stimulated with poly(I:C) (2 μg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

Data information: Data are presented as means of triplicates ± SD; symbols for P‐values: **0.001 < P < 0.01; ***P < 0.001; ns, not significant.

Figure 2. HSV‐1 does not replicate productively in macrophages, but ICP27 expression correlates inversely with type I IFN production

1. MDMs were infected for 18 h with HSV‐1 (KOS, MOI 1). Cytosolic viral proteins were detected by mass spectrometry using iTRAQ labeling. Identified viral proteins are shown.

2. MDMs were infected with the KOS or F strains of HSV‐1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β‐actin were determined by Western blotting.

3. HFFs and MDMs were infected with HSV‐1 (KOS, MOI 0.1, and 1.0). Culture medium was replaced 3 hpi and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates ± SD.

4. THP1 cells were infected with the indicated strains of HSV‐1 (MOI 3) for 12 h. Cell lysates were isolated, and levels of ICP27 and β‐actin were determined by Western blotting.

Source data are available online for this figure.

Figure 3. ICP27 inhibits the cGAS–STING pathway in macrophages

1. THP1 cells were infected with HSV‐1 KOS or an ICP27‐deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 h for measurement of type I IFN bioactivity.

2. MDMs were infected with HSV‐1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

3. THP1‐derived cells deficient for cGAS or STING were infected with HSV‐1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

4. HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2 × 10⁴ cells per well). Reporter gene activity was measured in lysates isolated 24 h post‐transfection.

5. THP1 cells were infected with HSV‐1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity.

Data information: Data are presented as means of triplicates ± SD; symbols for P‐values: ***P < 0.001; ns, not significant.

Figure 4. ICP27 inhibits the cGAS–STING pathway downstream of TBK1 phosphorylation but upstream of IRF3 phosphorylation

1. A

   THP1 cells were infected with HSV‐1 KOS or ΔICP27 (MOI 3). Cytoplasmic and nuclear extracts were isolated at the indicated time points post‐infection, and levels of ICP27, RCC1, and GAPDH were determined by Western blotting.

2. B

   THP1 cells were infected with HSV‐1 KOS or ΔICP27 (MOI 10). The cells were fixed at the indicated time points post‐infection, stained with DAPI and an antibody against ICP27, and visualized by confocal microscopy. Scale bar, 10 μm.

3. C, D

   THP1 cells were infected with HSV‐1 KOS, ΔICP27, or HSV‐1 ΔNLS. The localization of the major NLS is illustrated in (C). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

4. E, F

   THP1 cells were infected with HSV‐1 KOS or ΔICP27 (MOI 3). Cytoplasmic (E) and nuclear extracts (F) were isolated at the indicated time points post‐infection. Western blotting was used to determine levels of phospho‐ and total TBK1 were determined in the cytoplasmic fractions and levels of phospho‐IRF3 and RCC1 in the nuclear fractions.

5. G–I

   HEK293T cells were (G) transfected with STING, TBK1, IRF3‐5D, and ICP27, (H) transfected with STING and stimulated for 16 h with cGAMP (4 μg/ml) or (I) transfected with TRIF, MAVS, STING, and ICP27, together with the reporter gene constructs indicated. Reporter gene activity was measured 24 h post‐transfection.

Data information: Data are presented as means of triplicates ± SD; symbols for P‐values: *0.01 < P < 0.05; **0.001 < P < 0.01; ns, not significant.Source data are available online for this figure.

Figure 5. ICP27 interacts with the active STING–TBK1 signalsome

1. A

   Lysates from THP1 cells infected with HSV‐1 KOS (MOI 10) were subjected to immunoprecipitation using an anti‐STING antibody, and the presence of TBK1, STING, and ICP27 in the precipitates was detected by Western blotting.

2. B, C

   THP1 cells were infected with HSV‐1 KOS (MOI 10) for 6 and 8 h. The cells were fixed and stained with DAPI and anti‐ICP27 together with anti‐pTBK1 (B), or anti‐STING (C). The stainings were visualized by confocal microscopy. Arrowheads, co‐localizations between ICP27 and pTBK1/STING. Scale bar, 10 μm.

3. D

   TBK1 was immunoprecipitated from whole‐cell lysates from HEK293T cells transfected with ICP27 and each of the adaptor proteins TRIF, MAVS, and STING. The precipitates were immunoblotted for TBK1, ICP27, and STING.

4. E

   THP1 control and STING KO cells were infected with HSV‐1 KOS (MOI 10) for 6 and 8 h, and total lysates were generated. TBK1 was immunoprecipitated and subjected to Western blotting using antibodies against ICP27, phospho‐TBK1 (pTBK1), and TBK1.

5. F

   THP1 control and TBK1 KO cells were infected with HSV‐1 KOS (MOI 10) for 8 h, and total lysates were generated. STING was immunoprecipitated and subjected to Western blotting using antibodies against ICP27 and STING.

6. G

   THP1 cells were infected with HSV‐1 KOS (MOI 10) for 8 h in the presence or absence of the TBK1 inhibitor BX795 (BX, 200 nM). Total lysates were generated and STING was immunoprecipitated and subjected to Western blotting using antibodies against ICP27 and STING.

7. H

   THP1 cells were infected with HSV‐1 KOS (MOI 10) for 6 and 8 h, and total lysates were generated. Phospho‐TBK1 was immunoprecipitated and subjected to Western blotting using antibodies against ICP27 and total TBK1.

8. I

   TBK1 was immunoprecipitated from whole‐cell lysates from HEK293T cells transfected with ICP27 and STING (either WT or S366A). The precipitates were immunoblotted for TBK1, ICP27, and STING.

Source data are available online for this figure.

Figure 6. ICP27 from the simplex virus genera of alpha‐herpesviruses inhibits IFN production in a manner dependent on the RGG box

1. A, B

   Alignment of the ICP27 homologs of the human herpesviruses (A) and a series of alpha‐herpesviruses (B). The diagram to the right illustrates the degree of similarity with HSV‐1 ICP27 and the distribution within the molecules.

2. C

   THP1 cells were infected with HSV‐1 (KOS), HSV‐2 (HG52), HSV‐1 ΔICP27, or HSV‐1 ΔICP27 rescued with HSV‐2 ICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

3. D

   RAW264.7 cells were infected with HSV‐1 (KOS), PRV (vBecker3) and the corresponding mutants lacking ICP27 (UL54 in the case of PRV). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity.

4. E

   Alignment of the RGG box of in ICP27 proteins of the simplex virus genera of alpha‐herpesviruses.

5. F

   THP1 cells were infected with HSV‐1 (KOS), ΔRGG, or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. ICP27 expression in the infected cells was determined by Western blotting.

6. G

   THP1 cells were infected with HSV‐1 KOS or HSV‐1 ΔRGG (MOI 10) for 8 h, and total lysates were generated. STING was immunoprecipitated and subjected to Western blotting using antibodies against ICP27 and STING.

Data information: Data in (C, D, and F) are presented as means of triplicates ± SD; symbols for P‐values: *0.01 < P < 0.05; **0.001 < P < 0.01; ns, not significant.Source data are available online for this figure.