Modulation of Dengue virus infection in human cells by alpha, beta, and gamma interferons

Abstract

A role for interferon (IFN) in modulating infection by dengue virus (DV) has been suggested by studies in DV-infected patients and IFN receptor-deficient mice. To address how IFN modulates DV type 2 infection, we have assayed IFN-alpha, -beta, and -gamma for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assay that quantitates positive and negative strands of viral RNA, a flow cytometric assay that measures viral antigen, and a plaque assay that analyzes virion production. Our data suggest that IFN-alpha and -beta protect cells against DV infection in vitro. Treatment of hepatoma cells with IFN-alpha or -beta decreases viral RNA levels greater than 1, 000-fold, the percentage of cells infected 90 to 95%, and the amount of infectious virus secreted 150- to 100,000-fold. These results have been reproduced with several cell types and viral strains, including low-passage isolates. In contrast, IFN-gamma has a more variable effect depending on the cell type and pathway of infection. Quantitative RT-PCR experiments indicate that IFN inhibits DV infection by preventing the accumulation of negative-strand viral RNA.

FIG. 1

Dose response of cytokine pretreatment on DV2 infection of HepG2 cells. HepG2 cells were preincubated with increasing concentrations of IFN-β or -γ or TNF-α for 24 h and then exposed to DV2 (strain 16681) at an MOI of 2; 72 h later, cells (6 × 10⁵) were detached and subjected to immunofluorescent flow cytometry after incubation with FITC-labeled anti-DV2 antibody. The results are expressed as the percentage of cells that express the E protein of DV2; 48% of cells that were infected without cytokine pretreatment (negative media control) were positive by flow cytometry. One representative experiment of two is shown.

FIG. 2

Time course of the effect of IFN pretreatment on DV2 infection of HFF cells. HFF cells were exposed to DV2 (strain 16681) and incubated for 72 h. Cells (2 × 10⁵) were processed for flow cytometry (A), and supernatants were harvested for plaque assays (B) using BHK-21 cells. In each case, cells were exposed to medium IFN-γ (10 ng/ml), IFN-β (10 ng/ml), or IFN-β+γ (10 ng/ml) either 24 or 4 h before (pre) or after (post) incubation with virus. The flow cytometric data are presented as the percentage of cells that express the E protein of DV2, and the plaque assay data are expressed as the number of PFU per milliliter. One representative experiment of three is shown.

FIG. 3

Time course of the effect of IFN pretreatment on DV2 infection by low-passage viral isolates. HepG2 cells were exposed to DV2 (Thai strains C0477 and K0049; Nicaraguan strain N9622) at an MOI of 2 and incubated for 72 h. In each case, cells were exposed to medium or combinations of IFN-β and -γ (10 ng/ml each) as described for Fig. 2. One representative experiment of two is shown.

FIG. 4

Effect of IFN pretreatment on antibody-dependent DV2 infection of THP-1 and U937 cells. THP-1 and U937 cells were exposed to DV2 (strain 16681) at an MOI of 10 in the absence (No Ab) or presence (4G2) of an enhancing MAb. After incubation for 96 h, cells were processed for flow cytometry (A), and supernatants were harvested for plaque assays (B). In each case, cells were pretreated 24 h prior to exposure to virus with medium, IFN-α (100 IU/ml), IFN-β (10 ng/ml), IFN-γ (10 ng/ml), or IFN-β+γ (each at 10 ng/ml). The data are expressed as in Fig. 2. One representative experiment of three is shown.

FIG. 5

Time course of the effect of IFN pretreatment on antibody-dependent DV2 infection of K562 cells. K562 cells were exposed to DV2 (strain 16681) at an MOI of 0.005 in the presence of an enhancing MAb (3H5). After incubation for 96 h, cells (10⁶) were processed for flow cytometry. In each case, cells were exposed to medium, IFN-α (100 IU/ml), IFN-β (10 ng/ml), IFN-γ (10 ng/ml), or a combination of IFNs as described for Fig. 2. One representative experiment of two is shown.

FIG. 6

IFN effect on the levels of positive and negative strands of DV2 RNA in HepG2 cells. HepG2 cells were exposed to DV2 (strain 16681) at an MOI of 2 and incubated for 24 h. In each case, cells were exposed to medium, IFN-γ (10 ng/ml), IFN-β (10 ng/ml), or IFN-β+γ (each at 10 ng/ml) 24 h prior to incubation with virus. Cells (6 × 10⁵) were harvested, total RNA was isolated, and quantitative asymmetric RT-PCR was performed with fixed amounts of cellular RNA in the presence of 10-fold decreasing concentrations of positive- or negative-strand competitor. The RT-PCR product was subjected to agarose gel electrophoresis. M refers to the molecular weight marker, C denotes an RT-PCR with only competitor RNA, and the number above each lane represents the log number of copies of competitor used. The amount of viral RNA was determined from the competitor concentration that produces competitor and DV bands of equal intensity (denoted by asterisk). RNA per cell is calculated as defined in Materials and Methods. For the positive strand, the equivalence point and RNA copies per cell were as follows: media, 10⁷ and 833; IFN-γ, 5 × 10⁶ and 417; IFN-β, 10⁴ and 0.8; IFN-β+γ, 10⁴ and 0.8. For the negative strand, the corresponding values were as follows: media, 10⁶ and 110; IFN-γ, 10⁵ and 11; IFN-β, 10³ and 0.1; IFN-β+γ, undetectable.

FIG. 7

Time course of IFN effect on the levels of positive and negative strands of DV2 RNA in HepG2 cells. HepG2 cells were exposed to DV2 (Thai strain K0049) at an MOI of 2 and incubated for 72 h. In each case, cells were exposed to medium or IFN-β+γ (each at 10 ng/ml) either 24 or 4 h before (pre) or after (post) incubation with virus. Cells (6 × 10⁵) were harvested, total RNA was isolated, and asymmetric RT-PCR was performed with fixed amounts of cellular RNA in the presence of 10-fold decreasing concentrations of positive- or negative-strand competitor. The amount of viral RNA per cell (A) was determined after agarose gel electrophoresis (B) as described for Fig. 6.

FIG. 8

Time course of the levels of positive and negative strands of DV2 RNA after IFN treatment of HepG2 cells. HepG2 cells were exposed to DV2 (strain 16681) at an MOI of 2 and incubated as indicated prior to harvest. In each case, cells were pretreated with medium or IFN-β+γ (each at 10 ng/ml) for 24 h prior to exposure to virus. At each time point after infection, cells were harvested and counted by hemocytometry, and total RNA was isolated. Subsequently, asymmetric RT-PCR was performed with fixed amounts of cellular RNA in the presence of 10-fold decreasing concentrations of positive- or negative-strand competitor. The product was subjected to agarose gel electrophoresis, and the amount of viral RNA per cell was determined as described for Fig. 6.