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. 2011 Jun;17(3):212-9.
doi: 10.1007/s13365-011-0031-8. Epub 2011 Apr 16.

IL-29/IL-28A Suppress HSV-1 Infection of Human NT2-N Neurons

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Free PMC article

IL-29/IL-28A Suppress HSV-1 Infection of Human NT2-N Neurons

Lin Zhou et al. J Neurovirol. .
Free PMC article

Abstract

The newly identified cytokines, IL-28/IL-29 (also termed type III IFNs), are able to inhibit a number of viruses. Here, we examined the antiviral effects of IL-29/IL-28A against herpes simplex virus type 1 (HSV-1) in human NT2-N neurons and CHP212 neuronal cells. Both IL-29 and IL-28A could efficiently inhibit HSV-1 replication in neuronal cells, as evidenced by the reduced expression of HSV-1 DNA and proteins. This inhibitory effect of IL-29 and IL-28A against HSV-1 could be partially blocked by antibody to IL-10Rβ, one of the key receptors for IL-29 and IL-28A. To explore the underlying antiviral mechanisms employed by IL-29/IL-28A, we showed that IL-29/IL-28A could selectively induce the expression of several Toll-like receptors (TLRs) as well as activate TLR-mediated antiviral pathway, including IFN regulatory factor 7, IFN-α, and the key IFN-α stimulated antiviral genes.

Figures

Fig. 1
Fig. 1
HSV-1 replication in human neuronal cells. Morphological view of NT2-N neuron (a) or CHP212 cells (b) infected with or without HSV-1 and observed at 72 h post-infection. Arrows indicate the characteristic cytopathic effect caused by HSV-1 infection in the neurons. NT2-N cells (c) and CHP212 cells (d) cultured in 24-well plates (5×105 cells/well) were then infected with HSV-1 17 syn+ (MOI=0.01) for 90 min. At 6, 12, 24, 48, and 72 h post-infection, genomic DNA was extracted as described in “Materials and methods” and subjected to quantitative real-time PCR to detect the HSV-1 gD level. Since the HSV-1 replication efficiency varies in different neuronal cells, data were expressed as HSV-1 gD DNA copies in 1E+4 GAPDH copies (NT2-N) (a) or 1 GAPDH copy (CHP212) (b). Representative data of three independent experiments are shown
Fig. 2
Fig. 2
Dose- and time-dependent anti-HSV-1 effect of IL-29 and IL-28A. a, b NT2-N cells and CHP212 cells were cultured in 24-well plates (5×105 cells/well) and pretreated with IL-29 or IL-28A at indicated concentrations for 24 h followed with HSV-1 17 syn+ (MOI=0.01) infection for 90 min. At 72 h post-infection, genomic DNA was extracted and subjected to quantitative real-time PCR for HSV-1 gD DNA. c, d Cells were pretreated with IL-29 (100 ng/ml) or IL-28A (100 ng/ml) for 24 h followed with infection with HSV-1 17 syn+ (MOI=0.01) for 90 min. At indicated time points post-infection, genomic DNA was extracted and subjected to quantitative real-time PCR for HSV-1 gD DNA. The replication of HSV-1 in treated cultures was expressed as percentage of HSV-1 gD DNA levels relative to control (without treatment, which is defined as 100%). Data are expressed as mean ± SD of three different experiments (*P<0.05; **P<0.01)
Fig. 3
Fig. 3
IL-29 and IL-28A inhibit HSV-1 protein expression in human neuronal cells. a, b Effect of IFNs on HSV-1 replication. NT2-N cells (a) and CHP212 cells (b) were cultured in 24-well plates at 5×105 cells per well and pretreated with IFN-α (100 U/ml), IFN-β (100 U/ml), IL-29 (100 ng/ml), or IL-28A (100 ng/ml) for 24 h. Cultures were then infected with HSV-1 17 syn+ (M.O.I=0.01) and further cultured for 72 h. Genomic DNA was extracted from infected cells and subjected to quantitative real-time PCR for HSV-1 gD DNA. The data are expressed as HSV-1 gD levels relative to control (without treatment, which is defined as 100%). The results are mean ± SD of triplicate cultures, representative of three experiments (*P<0.05; **P<0.01). c, d Immunofluorescence assay showing IL-29 and IL-28A inhibit HSV-1 protein expression. NT2-N cells (c) and CHP212 cells (d) pretreated with or without IL-29 (100 ng/ml) or IL-28A (100 ng/ml) for 24 h were infected with HSV-1 (MOI=0.01) for 90 min and washed. Uninfected cells are considered as control. At 72 h post-infection, cells were fixed and the expression of HSV-1 proteins was determined by immunofluorescence assay using a polyclonal anti-HSV-1 gD antibody. A representative experiment was shown (magnification ×200)
Fig. 4
Fig. 4
Neutralization effect of antibody against IL-10Rβ on IL-29/IL-28A-mediated anti-HSV-1 activity. a Expression of IL-10Rβ mRNA in neurons. RNA isolated from NT2-N, CHP212, Huh7, and BB19 cells was subjected to the real-time RT-PCR with the specific primers for IL-10Rβ and GAPDH. Huh7 cells were used as a positive control and BB19 cells were used as a negative control for IL-10Rβ expression. The RT-PCR-amplified products were analyzed by 2% agarose electrophoresis. b Expression of IL-10Rβprotein in NT2-N neurons by immunochemistry staining. NT2-N cells were incubated with polyclonal anti-IL-10Rβ antibody and subsequently observed under an immunofluorescence microscope. Scale bar, 50 μm. c NT2-N cells were incubated with or without IL-29 (100 ng/ml) or IL-28A (100 ng/ml) and/or antibody to IL-10Rβ (5 μg/ml) for 24 h prior to HSV-1 infection (MOI=0.01) for 90 min. Goat IgG antibody was used as control. The HSV-1 replication was measured by quantitative real-time PCR to detect HSV-1 gD level at 72 h post-infection. Values are the mean ± SD of three different cultures (*P<0.05)
Fig. 5
Fig. 5
Effect of IL-29 and IL-28A on TLR-mediated antiviral pathway. NT2-N cells were treated with IL-29 or IL-28A for 12 h, and total cellular RNA was extracted from cell cultures for quantitative real-time PCR assay using specific primers for TLRs (a), IRFs (b), type I IFN (c), and ISGs (d). Data are expressed as the type III IFN-mediated increase in induction (n-fold) relative to untreated control, which is regarded as 1. All the specimens are normalized based on GAPDH mRNA levels. Values are the mean ± SD of three different cultures (*P<0.05; **P<0.01)

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