Induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 contributes to inhibition of the interferon signaling pathway

Abstract

We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-alpha4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-beta, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection.

FIG. 1.

Effects of infection with HSV-1 strain VR3, a UL41-deficient mutant (d41), or a UL13-deficient mutant (d13) on IFN-α-induced tyrosine phosphorylation of STAT1 (A) and induction of 2-5AS mRNA (B) in FL cells. FL cells were infected with HSV-1 VR3 or the d41 or d13 mutant at MOI 5 for 3 h. (A) The infected cells were treated with 1,000 IU of IFN-α/ml for 10 or 20 min. The cell lysates were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5% polyacrylamide slab gel and analyzed by Western blotting using anti-STAT1 and anti-tyrosine phosphorylated STAT1 (STAT1-pY) antibodies. C, uninfected control. (B) The infected cells were treated with 1,000 IU of IFN-α/ml for 6 or 12 h, after which total cellular RNA was extracted. 2-5AS mRNA levels were determined by RT-PCR. GAPDH mRNA levels were determined as a control.

FIG. 2.

Induction of SOCS3 protein by HSV-1 infection. (A and B) FL cells were infected with HSV-1 VR3 or the d41 or d13 mutant at MOI 5 for various periods. (A) The infected cell lysates were then analyzed by Western blotting using anti-SOCS3 antibody. Actin was employed as a control for protein loading. (B) Each band was quantified, and the results of the experiments performed in triplicate (means ± standard deviations) are shown. (C) FL cells were infected with HSV-1 VR3 or UV-inactivated virus for various periods. (D) FL cells were infected with VR3 at various MOIs for 2 h, and the levels of SOCS3 protein were then determined. The expression of SOCS3 protein was detected by Western blotting (arrow). *, nonspecific binding; C, uninfected control.

FIG. 3.

Induction of SOCS3 mRNA by HSV-1 infection. (A) FL cells were infected with HSV-1 VR3 at MOI 5 for various periods. (B) FL cells were infected with VR3 at various MOIs for 30 min. The levels of SOCS3 mRNA were determined by semiquantitative RT-PCR. (C) FL cells were infected with HSV-1 VR3 or the d41 or d13 mutant at MOI 5 for various periods. The SOCS1, SOCS3, and CIS mRNA levels in the infected cells were analyzed by semiquantitative RT-PCR. GAPDH was determined as a control. C, uninfected control.

FIG. 4.

Overexpression of SOCS3 suppresses IFN-α-induced Tyr-phosohorylation of STAT1. FL cells were transiently transfected with the SOCS3 expression plasmid (pSOCS3). (A) The expression of SOCS3 was determined by Western blotting. (B) After 24 h of transfection, the cells were treated with IFN-α (1,000 IU/ml) for 10 or 20 min. The cell lysates were then analyzed for the Tyr phosphorylation status of STAT1 by Western blotting. Actin and STAT1 were used as controls. *, nonspecific binding.

FIG. 5.

Semiquantitative RT-PCR analysis of IRF-3, IRF-7, IFN-β, and IFN-α4 mRNA (A) and Western blotting analysis of IRF-3 and IRF-7 (B) in FL cells during infection with HSV-1 VR3 or the d41 or d13 tegument protein-deficient mutant. FL cells were infected with virus at MOI 5 for various periods. The phosphorylation status of IRF-3 was determined by Western blotting using an anti-IRF-3 antibody. Unphosphorylated and phosphorylated forms of IRF-3 are indicated by U and P, respectively. In contrast, total protein levels of IRF-7 are shown. C, uninfected control. Actin mRNA and protein (RT-PCR and Western blotting) and GAPDH mRNA (RT-PCR) were determined as controls.