Suppression of proinflammatory cytokine expression by herpes simplex virus type 1

Abstract

Viral immune evasion strategies are important for establishment and maintenance of infections. Many viruses are in possession of mechanisms to counteract the antiviral response raised by the infected host. Here we show that a herpes simplex virus type 1 (HSV-1) mutant lacking functional viral protein 16 (VP16)-a tegument protein promoting viral gene expression-induced significantly higher levels of proinflammatory cytokines than wild-type HSV-1. This was observed in several cell lines and primary murine macrophages, as well as in peritoneal cells harvested from mice infected in vivo. The enhanced ability to stimulate cytokine expression in the absence of VP16 was not mediated directly by VP16 but was dependent on the viral immediate-early genes for infected cell protein 4 (ICP4) and ICP27, which are expressed in a VP16-dependent manner during primary HSV infection. The virus appeared to target cellular factors other than interferon-induced double-stranded RNA-activated protein kinase R (PKR), since the virus mutants remained stronger inducers of cytokines in cells stably expressing a dominant-negative mutant form of PKR. Finally, mRNA stability assay revealed a significantly longer half-life for interleukin-6 mRNA after infection with the VP16 mutant than after infection with the wild-type virus. Thus, HSV is able to suppress expression of proinflammatory cytokines by decreasing the stability of mRNAs, thereby potentially impeding the antiviral host response to infection.

FIG. 1.

VP16 mutation potentiates HSV-1-induced cytokine expression. The cells (RAW264.7, A, B, C, and H; J774A.1, D and E; PCs, F; NIH 3T3, G) were seeded in triplicate cultures, left overnight to settle, and treated with a mock preparation or infected with wt HSV-1 or in1814 (A to F, MOI of 1; G, MOI of 2; H, MOI of 0.02 to 2) as indicated. At 8 (C) and 24 (A, B, and D to H) h later, the supernatants were harvested and RANTES, IL-6, and IL-12 p40 were measured by ELISA while IFN-α/β and TNF-α were measured by bioassays. The results are shown as means ± SEM. Essentially similar results were seen in more than five independent experiments for the results presented in panels A to E, G, and H and in two independent experiments for the result presented in panel F.

FIG. 2.

VP16 mutation potentiates expression of RANTES during HSV-1 infection in vivo. Eight-week-old female BALB/c mice were infected with wt or VP16-deficient HSV-1 (10⁶ PFU) or treated with a mock preparation. At 24 h later, PCs were harvested and either cultured for 24 h in RPMI 1640 medium supplemented with 5% FCS or lysed for subsequent RNA purification. (A) RANTES was measured in the cell culture supernatants by ELISA. The results are shown as means of five mice per group ± SEM. Essentially similar results were seen in two independent experiments. *, P < 0.05. (B) Total RNA was reverse transcribed and analyzed for the presence of RANTES and β-actin by PCR. Essentially similar results were seen in two independent experiments.

FIG. 3.

Down-regulation of cytokine expression by HSV-1 is dependent on viral IE and/or E genes. RAW264.7 cells were seeded in triplicate cultures and left overnight to settle. The cells received a mock preparation or wt or mutant HSV-1 (MOI of 1) as indicated. In panel B, some wells were treated with PAA (500 μg/ml) prior to infection. The cells were incubated for 24 h, and supernatants were harvested for measurement of IL-6 by ELISA. Essentially similar results were seen in three independent experiments. Results are shown as means ± SEM. *, P < 0.05.

FIG. 4.

Suppression of cytokine expression by HSV-1 is independent of PKR. RAW264.7-derived cell lines stably transfected with control plasmid (pBK CMV) or a dominant-negative PKR construct (PKR M7) were serum starved for 5 h before treatment with a mock preparation or infection with wt HSV-1 or the mutant in1814 (VP16), vi-13 (ICP4), or d27-1 (ICP27) (MOI of 1). Supernatants were harvested 24 h later, and IL-6 was measured by ELISA. The results are shown as means of triplicate cultures ± SEM. Essentially similar results were observed in three independent experiments.

FIG. 5.

HSV-1 down-modulates proinflammatory gene expression at the level of mRNA stability. (A) RAW264.7 cells were infected with HSV-1 or in1814, and RNA was harvested at the indicated time point p.i. IL-6 and β-actin mRNAs were amplified by RT-PCR and visualized by ethidium bromide staining of the agarose gel. Essentially similar results were seen in two independent experiments. (B) NIH 3T3 cells were transiently transfected with a RANTES promoter gene construct and treated as indicated. After 8 and 24 h, cells were lysed and luciferase activity was measured. The results are shown as means ± SEM. RLU, relative luciferase units. Essentially similar results were seen in two independent experiments. (C) J774A.1 cells were treated for 2 h with a mock virus preparation or infected with wt or mutant HSV-1 (MOI of 1) as indicated. Nuclear proteins were harvested, and NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay. Essentially similar results were seen in two independent experiments. (D) RAW264.7 cells were infected with HSV-1 or in1814 for 5 h, at which point ActD was added. RNA was extracted from cells at various intervals after the addition of ActD, and IL-6 and β-actin mRNAs were amplified by RT-PCR and visualized by ethidium bromide staining of the agarose gel. Essentially similar results were seen in two independent experiments.

FIG. 6.

Model of HSV-1-mediated suppression of cytokine expression. Accumulation of HSV IE genes correlates with induction of cytokines and chemokine expression. The data presented in this work demonstrate that two IE genes, ICP4 and ICP27, actively counteract the expression of many proinflammatory cytokines with antiviral potential by decreasing mRNA stability, a phenomenon that could potentially impede the host defense against HSV.