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. 2022 Sep 23:13:983502.
doi: 10.3389/fimmu.2022.983502. eCollection 2022.

Herpes simplex virus type 2 inhibits TNF-α-induced NF-κB activation through viral protein ICP22-mediated interaction with p65

Huimin Hu  1   2 , Ming Fu  1   3 , Chuntian Li  1   2 , Binman Zhang  1   2 , Yuncheng Li  1   2 , Qinxue Hu  1   4 , Mudan Zhang  1
Affiliations

Herpes simplex virus type 2 inhibits TNF-α-induced NF-κB activation through viral protein ICP22-mediated interaction with p65

Huimin Hu et al. Front Immunol. .

Abstract

Herpes simplex virus type 2 (HSV-2) is a prevalent human pathogen and the main cause of genital herpes. After initial infection, HSV-2 can establish lifelong latency within dorsal root ganglia by evading the innate immunity of the host. NF-κB has a crucial role in regulating cell proliferation, inflammation, apoptosis, and immune responses. It is known that inhibition of NF-κB activation by a virus could facilitate it to establish infection in the host. In the current study, we found that HSV-2 inhibited TNF-α-induced activation of NF-κB-responsive promoter in a dose-dependent manner, while UV-inactivated HSV-2 did not have such capability. We further identified the immediate early protein ICP22 of HSV-2 as a vital viral element in inhibiting the activation of NF-κB-responsive promoter. The role of ICP22 was confirmed in human cervical cell line HeLa and primary cervical fibroblasts in the context of HSV-2 infection, showing that ICP22 deficient HSV-2 largely lost the capability in suppressing NF-κB activation. HSV-2 ICP22 was further shown to suppress the activity of TNF receptor-associated factor 2 (TRAF2)-, IκB kinase α (IKK α)-, IKK β-, IKK γ-, or p65-induced activation of NF-κB-responsive promoter. Mechanistically, HSV-2 ICP22 inhibited the phosphorylation and nuclear translocation of p65 by directly interacting with p65, resulting in the blockade of NF-κB activation. Furthermore, ICP22 from several alpha-herpesviruses could also inhibit NF-κB activation, suggesting the significance of ICP22 in herpesvirus immune evasion. Findings in this study highlight the importance of ICP22 in inhibiting NF-κB activation, revealing a novel mechanism by which HSV-2 evades the host antiviral responses.

Keywords: HSV-2; ICP22; NF-κB; immune evasion; p65.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Productive HSV-2 infection suppresses TNF-α-induced NF-κB activation. (A) Productive HSV-2 infection suppresses TNF-α-induced NF-κB activation. HeLa cells were seeded in 24-well plates overnight and co-transfected with the reporter plasmids pNF-κB-Luc and phRL-TK. At 4 h post-transfection, cells were mock infected or infected with HSV-2 or UV-inactivated HSV-2 (UV HSV-2) at an MOI of 1, 0.6, 0.3, or 0.1. At 20 h post-infection, cells were stimulated with or without TNF-α (20 ng/ml) for 6 h. Reporter activities were determined by DLR assay. (B) Detection of viral protein expression in HSV-2-infected cells. HeLa cells were infected with HSV-2 or UV-inactivated HSV-2 at an MOI of 1, 0.6, 0.3, or 0.1 for 24 h. The expression of viral protein was detected by western blot using the anti-HSV-2 Ab. β-actin was used as a loading control. (C) HSV-2 infection inhibits CXCL10 mRNA production. HeLa cells seeded in 6-well plates were infected with HSV-2 or UV-inactivated HSV-2. At 24 h post-infection, cells were stimulated with TNF-α (20 ng/ml) for 6 h. Cells were harvested and total RNA was extracted. The expression of CXCL10 and GAPDH genes was evaluated by relative real-time quantitative PCR. For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate. For images, one representative experiment out of three is shown. *p < 0.05, ***p < 0.001. ns, not significantly. Rel, Relative.
Figure 2
Figure 2
HSV-2 ICP22 inhibits TNF-α-induced NF-κB activation. (A) HSV-2 ICP22 suppresses TNF-α-induced NF-κB activation. HEK 293T cells were seeded in 24-well plates overnight and co-transfected with the reporter plasmids pNF-κB-Luc and phRL-TK together with ICP22-expressing plasmid. At 24 h post-transfection, cells were stimulated with TNF-α (20 ng/ml) for 6 h. Reporter activities were determined by DLR assay. The expression of ICP22 was detected by western blot using the anti-Flag Ab. (B) HSV-2 ICP22 suppresses the production of CXCL10 mRNA. HEK 293T cells seeded in 6-well plates were transfected with vector or ICP22-expressing plasmid. At 24 h post-transfection, cells were stimulated with TNF-α (20 ng/ml) for 6 h. Cells were harvested and total RNA was extracted. The expression of CXCL10 and GAPDH genes was evaluated by relative real-time quantitative PCR. (C, D). ICP22 knockout impairs the inhibitory activity of HSV-2 on NF-κB activation. HeLa cells or primary human cervical fibroblasts were seeded in 24-well plates overnight and transfected with the reporter plasmids pNF-κB-Luc and phRL-TK, followed by infection with HSV-2 or us1 del HSV-2. After stimulation with TNF-α for 6 h, reporter activities were determined by DLR assay. (E, F). HSV-2 ICP22 knockout impairs the inhibitory activity of HSV-2 on CXCL10 mRNA production. HeLa cells or primary human cervical fibroblasts seeded in 6-well plates were infected with HSV-2 or us1 del HSV-2 at an MOI of 1. After stimulation with TNF-α for 6 h, cells were harvested and total RNA was extracted. The expression of CXCL10 and GAPDH genes was evaluated by relative real-time quantitative PCR. (G) ICP22s from several alpha-herpesviruses significantly inhibit NF-κB activation. HEK 293T cells were co-transfected with the reporter plasmids pNF-κB-Luc and phRL-TK together with ICP22-expressing plasmid of HSV-1, PRV or VZV, or expression plasmid of HSV-2 UL46. At 24 h post-transfection, cells were stimulated with TNF-α (20 ng/ml) for 6 h. Reporter activities were determined by DLR assay. The expressions of HSV-1 ICP22-Flag, HSV-2 ICP22-Flag, PRV ICP22-Flag, VZV ICP22-Flag and HSV-2 UL46-Flag were detected by western blot. Asterisk indicated the locations of proteins. For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate. For images, one representative experiment out of three is shown. *p < 0.05, ***p<0.001, ns, not significantly. Rel, Relative.
Figure 3
Figure 3
HSV-2 ICP22 inhibits NF-κB activation by acting on the downstream of p65. (A–E). HSV-2 ICP22 inhibits TRAF2, IKK α, IKK β, IKK γ and p65-induced NF-κB activation. HEK 293T cells were seeded in 24 well plates overnight and co-transfected with the reporter plasmids pNF-κB-Luc and phRL-TK, and plasmid expressing TRAF2, IKK α, IKK β, IKK γ or p65, together with empty vector or ICP22-expressing plasmid. At 30 h post-transfection, the reporter activities were determined by DLR assay. (F). HSV-2 ICP22 has no effect on the expression of TRAF2, IKK α, IKK β, IKK γ and p65 or degradation of IκB α. HEK 293T cells were seeded in 6 well plates overnight and transfected with plasmid expressing ICP22 or empty vector. At 24 h post-transfection, cells were stimulated with or without TNF-α (20 ng/ml) for 6 h. The expressions of TRAF2, IKK α, IKK β, IKK γ, IκB α, p65 and phospho-p65 were detected by western blot. For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate. For images, one representative experiment out of three is shown. *p<0.05, **p<0.01, ***p<0.001, ns, not significantly.
Figure 4
Figure 4
HSV-2 ICP22 inhibits the phosphorylation and nuclear translocation of p65. (A, B). HSV-2 ICP22 inhibits the phosphorylation of p65. HeLa cells seeded in the 6 well plates were transfected with empty vector or ICP22-expressing plasmid. At 4 h post-transfection, cells were mock infected or infected with HSV-2 or us1 del HSV-2 at an MOI of 1. At 20 h post-infection, cells were stimulated with or without TNF-α (20 ng/ml) for 6 h. The phosphorylated p65 in the cytoplasm and nucleus were detected by western blot. β-actin and PCNA were used as loading controls for cytoplasmic and nuclear proteins, respectively. (C, D). HSV-2 ICP22 significantly inhibits the nuclear translocation of p65. HeLa cells were transfected with ICP22-expressing plasmid or empty vector. At 24 h post-transfection, cells were stimulated with or without TNF-α (20 ng/ml) for 6 h. Cells were stained using the mouse anti-Flag and the rabbit anti-p65 Ab. Alexa Fluor 488-conjugated goat anti-mouse (green) and Alexa Fluor 647-conjugated goat anti-rabbit (red) were used as secondary antibodies. Cell nuclei (blue) were stained with DAPI. The images were obtained by fluorescence microscopy using a 60× objective. The percentage of p65-positive nuclei was quantified in a number of fields (D). The scale bar indicates 20 μm. For graphs, data shown are mean ± SD of three independent experiments with each condition performed in triplicate. For images, one representative experiment out of three is shown. ***p < 0.001.
Figure 5
Figure 5
HSV-2 ICP22 directly interacts with p65. (A) HSV-2 ICP22 interacts with endogenous p65. (B) Endogenous p65 interacts with HSV-2 ICP22. HeLa cells seeded in the 6 well plates were transfected with empty vector or ICP22-expressing plasmid. At 24 h post-transfection, cells were mock-treated or treated with TNF-α (20 ng/ml) for 6 h. Cell lysates were then subjected to co-immunoprecipitation assays using the anti-Flag (A) or anti-p65 Ab (B). The mouse (A) or rabbit (B) non-specific antibody was used as negative control. ICP22 and p65 were detected by western blot using the anti-Flag or anti-p65 Ab, respectively. (C) HSV-2 ICP22 directly interacts with p65. The kinetics of binding was performed on a Forte-Bio Octet Red System. 5 μg/mL rabbit anti-p65 Ab was coupled to Protein A biosensors. 25 μg/mL recombinant p65 was bound to Biosenors and immersed in different concentration of ICP22 (62.5, 125, 250, 500 or 1000 nM) for association and disassociation. The response in nm shift was recorded as a function of time. KD (M) = 2.18E-07. (D) Schematic representation of p65 truncations. (E) HSV-2 ICP22 interacts with the three truncation mutants Δ1, Δ2 and Δ3 of p65. Empty vector or ICP22-expressing plasmid and plasmid expressing full-length or truncated p65 were co-transfected into HEK 293T cells. At 24 h post-transfection, cells were mock-treated or treated with TNF-α (20 ng/ml) for 6 h. Cell lysates were then subjected to co-immunoprecipitation assays using the anti-Flag mAb. ICP22, truncated p65 were detected by western blot using the anti-Flag or anti-p65 Ab, respectively. Asterisk indicated the locations of proteins. One representative experiment out of three is shown.
Figure 6
Figure 6
A schematic model of the mechanism by which HSV-2 ICP22 blocks TNF-α-induced NF-κB activation. The host recognizes foreign PAMPs and activates the innate immune system to secret cytokines such as TNF-α. TNF-α subsequently binds TNF receptor (TNFR), resulting in the activation of the IKK complex. The inhibitory protein of NF-κB, IκB α, is then phosphorylated, degraded and detached from NF-κB. NF-κB dimers are then released and phosphorylated, and subsequently translocate to the nucleus to activate the expression of immunomodulatory genes. In the case of HSV-2 infection, the viral immediate early protein ICP22 directly interacts with p65 to block p65 phosphorylation and nuclear translocation, leading to an inhibition of NF-κB activation.
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