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. 2010 Sep;84(18):9140-8.
doi: 10.1128/JVI.00717-10. Epub 2010 Jun 30.

Alpha/beta interferon (IFN-alpha/beta)-independent induction of IFN-lambda1 (interleukin-29) in response to Hantaan virus infection

Malin Stoltz  1 , Jonas Klingström
Affiliations

Alpha/beta interferon (IFN-alpha/beta)-independent induction of IFN-lambda1 (interleukin-29) in response to Hantaan virus infection

Malin Stoltz et al. J Virol. 2010 Sep.

Abstract

Type III interferons ([IFNs] IFN-lambda and interleukin-28 and -29 [IL-28/29]) are recently recognized cytokines with innate antiviral effects similar to those of type I IFNs (IFN-alpha/beta). Like IFN-alpha/beta, IFN-lambda-expression can be induced by viruses, and it is believed that type I and III IFNs are regulated in the same manner. Hantaviruses are weak IFN-alpha/beta inducers and have surprisingly been shown to activate IFN-alpha/beta-independent IFN-stimulated gene (ISG) expression. Here, we show that in Hantaan virus (HTNV)-infected human epithelial A549 cells, induction of IFN-lambda1 preceded induction of MxA and IFN-beta by 12 and 24 h, respectively, and IFN-alpha was not induced at all. Furthermore, induction of IFN-lambda1 and MxA was observed in HTNV-infected African green monkey epithelial Vero E6 cells, a cell line that cannot produce type I IFNs, clearly showing that HTNV can induce IFN-lambda1 and ISGs in the complete absence of IFN-alpha/beta. In HTNV-infected human fibroblast MRC-5 cells, which lack the IFN-lambda receptor, induction of MxA coincided in time with IFN-beta-induction. UV-inactivated HTNV did not induce any IFNs or MxA in any cell line, showing that activation of IFN-lambda1 is dependent on replicating virus. Induction of both IFN-beta and IFN-lambda1 in A549 cells after poly(I:C)-stimulation was strongly inhibited in HTNV-infected cells, suggesting that HTNV can inhibit signaling pathways used to simultaneously activate types I and III IFNs. In conclusion, we show that HTNV can cause type I IFN-independent IFN-lambda1 induction and IFN-lambda1-specific ISG induction. Importantly, the results suggest the existence of specific signaling pathways that induce IFN-lambda1 without simultaneous type I IFN induction during virus infection.

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Figures

FIG. 1.
FIG. 1.
HTNV induces expression of IFN-λ1 prior to IFN-β and MxA in epithelial A549 cells. A549 cells were infected with HTNV (MOI of 3), and then cellular RNA was analyzed for levels of mRNA by Q-PCR, supernatants were analyzed for progeny virus titers by titration on Vero E6 cells, and total levels of bioactive IFNs in the supernatants were determined by an NDV-GFP assay. (A) mRNA expression of IFNs and MxA in HTNV-infected A549 cells at 6, 12, 24, 48, 72, and 96 h after infection was evaluated by Q-PCR. The data were normalized using β-actin and are presented as relative expression compared to the uninfected control. Error bars represent standard deviations of the means from two independent experiments. (B) Supernatants from infected cells were collected at 24, 48, 72, and 96 h after infection, and viral titers were determined. Error bars represent standard deviations of the means. (C) Supernatants drawn at 6, 12, 24, 48, 72, and 96 h after HTNV infection were UV inactivated and then analyzed for total antiviral capacity using an NDV-GFP assay. Data are presented as percent inhibition of NDV-enhanced GFP replication for supernatants from HTNV-infected compared to noninfected cells collected at the indicated times after HTNV infection. Human recombinant IFN-β and IFN-λ1 were used as controls. Error bars represent standard deviations of the means from one experiment.
FIG. 2.
FIG. 2.
IFN-λ1 is secreted from HTNV-infected cells. IFN-λ1 levels in supernatants from HTNV-infected (MOI of 1) MRC-5 fibroblasts at 6, 12, 24, 48, 72, and 96 h after infection were measured by ELISA. Error bars represent standard deviations of the means from two independent experiments.
FIG. 3.
FIG. 3.
Induction of MxA coincides in time with induction of IFN-β in HTNV-infected MRC-5 cells. MRC-5 cells were infected with HTNV (MOI of 1); then cellular RNA was analyzed for levels of mRNA by Q-PCR, and supernatants were analyzed for progeny virus titers by titration on Vero E6 cells. (A) mRNA expression of IFNs and MxA in HTNV-infected MRC-5 cells at 6, 12, 24, 48, 72, and 96 h after infection was evaluated by Q-PCR. The data were normalized using β-actin and are presented as relative expression compared to the uninfected control. Error bars represent standard deviations of the means from one representative experiment. (B) Supernatants from infected cells at 24, 48, 72, and 96 h after infection were collected, and viral titers were determined. Error bars represent standard deviations of the means from two independent experiments.
FIG. 4.
FIG. 4.
HTNV induces IFN-λ1 and MxA in the complete absence of type I IFNs. Vero E6 cells, that lack the capacity to produce type I IFNs due to a chromosomal deletion (7) were tested for their capacity to respond to and produce IFN-λ1 and then used to test if HTNV infection can induce IFN-λ1 and MxA in the complete absence of type I IFN production. (A) Nuclear translocation of phosphorylated STAT1 (STAT1-P) 60 min after treatment of Vero E6 cells with IFN-λ1. Glass slides with Vero E6 cells were treated with 100 ng/ml human recombinant IFN-λ1 (upper) or left untreated (lower) for 60 min and then fixed and stained for phosphorylated STAT1 (red) and nuclei (blue). (B) Vero E6 cells were stimulated with 100 ng/ml of human recombinant IFN-λ1 (or 50 ng/ml of IFN-β as a control), and MxA mRNA expression was evaluated by Q-PCR at 6, 12, 24, and 48 h after treatment. The data were normalized using β-actin and are presented as relative expression compared to the unstimulated control. Error bars represent standard deviations of the means from one representative experiment. (C) Vero E6 cells were stimulated with 10 or 100 μg/ml of poly(I:C) or with medium alone as a control. mRNA expression of IFN-λ1 and MxA in the cells at 6, 12, 24, and 48 h after treatment was then determined by Q-PCR. The data were normalized using β-actin and are presented as relative expression compared to the unstimulated control. Error bars represent standard deviations of the means from one representative experiment. (D) mRNA expression of IFNs and MxA in HTNV-infected (MOI of 4) Vero E6 cells was evaluated by Q-PCR at 24 and 48 h after infection. The data were normalized using β-actin and are presented as relative expression compared to the noninfected control. Error bars represent standard deviations of the means from one representative experiment.
FIG. 5.
FIG. 5.
IFN-λ1 inhibits HTNV replication in Vero E6 cells. The antiviral capacity of IFN-λ1 against HTNV in the complete absence of IFN-α/β was analyzed in Vero E6 cells. Cells were either stimulated with 10 or 100 ng/ml of recombinant IFN-λ1 (or with 10 or 100 ng/ml of IFN-β as control) before infection or infected with high and low doses of HTNV in order to analyze the effect of exogenously added IFN-λ1 and endogenously produced IFN-λ on HTNV-replication. (A) Production of progeny virus in supernatants from Vero E6 cells treated with recombinant human IFN-λ1. Vero E6 cells were stimulated with IFN-λ1 or IFN-β or incubated with only medium as a control for 24 h before infection (MOI of 4). Supernatants from infected cells were then collected at 30 hpi, and viral titers were determined. Error bars represent standard deviations of the means from one representative experiment. (B) Vero E6 cells were infected with high (MOI of 40) or low (MOI of 0.04) doses of HTNV. Supernatants were then collected at 2, 7, 9, and 11 days after infection, and viral titers were determined. Error bars represent standard deviations of the means from two independent experiments. (C) Vero E6 cells were infected with high (MOI of 40) or low (MOI of 0.04) doses of HTNV. HTNV S RNA levels in infected cells were evaluated by Q-PCR at 12 h and at 1, 2, 4, 7, 9, and 11 days after infection. The data were normalized using β-actin and are presented as relative expression compared to levels of HTNV S RNA at 6 h after infection for infection with a high dose or low dose, respectively. Error bars represent standard deviations of the means from one representative experiment. (D) mRNA expression of IFN-λ1 and MxA in Vero E6 cells, infected with a high (MOI of 40) or low (MOI of 0.04) dose of HTNV, at 1, 2, 4, and 7 days after infection was evaluated by Q-PCR. The data were normalized using β-actin and are presented as relative expression compared to the noninfected control. Error bars represent standard deviations of the means from one representative experiment.
FIG. 6.
FIG. 6.
HTNV inhibits poly(I:C)-mediated induction of IFN-β and IFN-λ1 in A549 cells. Infected (MOI of 5) and noninfected cells were stimulated with poly(I:C) (10 μg/ml) 48 h after HTNV infection or incubated with only medium as a control. Induction of IFN-β and IFN-λ1 mRNA expression at 1.5, 3, and 6 h after poly(I:C) stimulation was determined by Q-PCR. The data were normalized using β-actin, and relative expression in treated compared to nontreated cells was calculated for infected and noninfected cells, respectively. Data are presented as percent inhibition of relative expression for HTNV-infected compared to noninfected cells. Error bars represent standard deviations of the means from one representative experiment.
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