Herpes simplex virus type 1 (HSV-1)-induced apoptosis in human dendritic cells as a result of downregulation of cellular FLICE-inhibitory protein and reduced expression of HSV-1 antiapoptotic latency-associated transcript sequences

Abstract

Herpes simplex virus type 1 (HSV-1) is one of the most frequent and successful human pathogens. It targets immature dendritic cells (iDCs) to interfere with the antiviral immune response. The mechanisms underlying apoptosis of HSV-1-infected iDCs are not fully understood. Previously, we have shown that HSV-1-induced apoptosis of iDCs is associated with downregulation of the cellular FLICE-inhibitory protein (c-FLIP), a potent inhibitor of caspase-8-mediated apoptosis. In this study, we prove that HSV-1 induces degradation of c-FLIP in a proteasome-independent manner. In addition, by using c-FLIP-specific small interfering RNA (siRNA) we show for the first time that downregulation of c-FLIP expression is sufficient to drive uninfected iDCs into apoptosis, underlining the importance of this molecule for iDC survival. Surprisingly, we also observed virus-induced c-FLIP downregulation in epithelial cells and many other cell types that do not undergo apoptosis after HSV-1 infection. Microarray analyses revealed that HSV-1-encoded latency-associated transcript (LAT) sequences, which can substitute for c-FLIP as an inhibitor of caspase-8-mediated apoptosis, are much less abundant in iDCs as compared to epithelial cells. Finally, iDCs infected with an HSV-1 LAT knockout mutant showed increased apoptosis when compared to iDCs infected with the corresponding wild-type HSV-1. Taken together, our results demonstrate that apoptosis of HSV-1-infected iDCs requires both c-FLIP downregulation and diminished expression of viral LAT.

FIG. 1.

Kinetics of c-FLIP degradation and apoptosis in iDCs infected with HSV-1. iDCs were infected with live (HSV-1) or heat-inactivated (hi-HSV-1) virus (MOI = 1.5; strain F). At different time points, as indicated, cells were harvested and either used for immunoblot analysis (A) or flow cytometry (B and C). (A) Immunoblots were stained for c-FLIP_L and β-actin (loading control). The relative intensities of c-FLIP_L bands were determined by densitometry and normalized to β-actin. The value obtained with HSV-1-infected iDCs at 2 h p.i. was set to 100%. One out of three independent experiments is shown. (B) Relative apoptosis was assessed by measuring the number of Annexin V-positive iDCs after exclusion of propidium iodide (PI)-positive cells. The number of apoptotic cells obtained with HSV-1-infected iDCs at 20 h p.i. was set to 100%. Mean values ± standard deviations (SD) derived from three independent experiments are shown. At time points marked with an asterisk, apoptosis in HSV-1-infected iDC is significantly higher than in iDCs treated with heat-inactivated HSV-1 (P ≤ 0.05). (C) Dot plots from one representative experiment out of three used for the calculation in panel B are shown. Numbers in each quadrant indicate the percentage of cells. On the x and y axes, the fluorescence intensity (log scale, 4 decades) is given.

FIG. 2.

Mechanism of c-FLIP downregulation in HSV-1-infected iDCs. (A) Increased c-FLIP_L transcript number in the cytoplasm and nucleus of HSV-1-infected iDCs. iDCs were left uninfected, treated with lipopolysaccharide (LPS) for 24 h, or infected for 18 h with either heat-inactivated HSV-1 (hi-HSV-1), UV-inactivated HSV-1 (uv-HSV-1), or live virus (HSV-1). The relative abundance of c-FLIP_L mRNA in the cytosolic and nucleic fraction was analyzed by quantitative RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, and compared with LPS-treated cells that were set to 100. The results shown represent the means of three independent experiments ± SD, except for uv-HSV-1, which is representative for two experiments. (B) Proteasome-independent c-FLIP downregulation in HSV-1-infected iDCs. Cells were left uninfected or infected with HSV-1 (MOI = 3; strain KOS). MG132 was added at 1 μM (5.25 h p.i.) or at 10 μM (7.5 h p.i.). iDCs were lysed at 10 h p.i. and subjected to immunoblot analysis. The c-FLIP band intensity was analyzed by densitometry and normalized to β-actin. The value obtained for uninfected cells in the absence of MG132 was set to 100%. One representative experiment out of three is shown. (C) Degradation of c-FLIP by a viral or a virus-induced cellular protease. Lysates were prepared from A549 cells transfected with a mutated form of c-FLIP_L (left side, upper panel) that does not aggregate when overexpressed in cells (26) or c-FLIP_S-GFP (left side, lower panel). One aliquot was kept at −20°C, and another aliquot was incubated for 2 h at 37°C. The two remaining aliquots were mixed with lysates of HSV-1-infected but untransfected A549 cells and incubated at 37°C for 2 h in the presence or absence of proteasome inhibitor MG132 (50 μM). Immunoblots were stained for c-FLIP and β-actin (loading control). The c-FLIP band intensity was analyzed by densitometry and normalized to β-actin. The value obtained for untreated lysates kept at −20°C was set to 100%.

FIG. 3.

Apoptosis of iDCs after c-FLIP downregulation by siRNA. CD14-positive selected monocytes from healthy buffy coats were left untreated (co) or transfected with control siRNA (siC) and siRNA targeting c-FLIP (siFLIP), respectively. Subsequently, cells were differentiated into iDCs for 3 to 6 days. (A) Knockdown of c-FLIP was analyzed by immunoblotting. (B) Apoptosis of iDCs and iDCs treated in addition with agonistic anti-CD95 monoclonal antibody (clone CH-11) for 12 h was measured. Cells were stained with Annexin V-FITC and propidium-iodide (PI) and subsequently analyzed by flow cytometry. On the x and y axes, the fluorescence intensity (log scale, 4 decades) is given. The percentage of cells in each quadrant is given in the upper left corner. Results shown are representative of three independent experiments.

FIG. 4.

Amount of c-FLIP protein (A) and extent of apoptosis (B) in different HSV-1-infected cell types. Cells were infected with HSV-1 strain F at MOIs of 1 (iDCs), 1.5 (fibroblasts, keratinocytes), 3 (HUVECs), or 5 (L428 and L1236). As a control, cells were either left uninfected or were incubated with heat-inactivated (hi-HSV-1) or UV-inactivated (uv-HSV-1) virus. After 18 h (fibroblasts, 16.5 h p.i.), cells were harvested. (A) One aliquot was used for immunoblot analysis of c-FLIP_L and β-actin (loading control). The c-FLIP band intensity was analyzed by densitometry and normalized to β-actin. Values obtained for uninfected cells were set to 100%. (B) A second aliquot of cells was used for flow cytometric analysis of apoptosis and necrosis by staining with Annexin V-FITC and propidium iodide (PI). On the x and y axes, the fluorescence intensity (log scale, 4 decades) is given. Numbers in each quadrant indicate the percentage of cells. Data are representative of four (iDCs, HUVECs), three (primary keratinocytes, L428), or two (primary fibroblasts, L1236) experiments.

FIG. 5.

HSV-1-induced apoptosis in iDCs as compared to epithelial cells (HaCaT and HeLa cells). Cells were infected with heat-inactivated (hi-HSV-1) or live virus (HSV-1) for 18 h (MOI = 1.5; strain KOS). (A) Infection was verified by staining for viral gD and flow cytometric analysis (hi-HSV-1, filled curve; live HSV-1, open curve). (B) Apoptosis was visualized by staining with Annexin V-FITC and propidium iodide (PI) and flow cytometric analysis. On the x and y axes, the fluorescence intensity (log scale, 4 decades) is given. Numbers in each quadrant indicate the percentage of cells. Data from one representative experiment are shown. Data from all samples used for microarray analyses are summarized in Table 1. (C) Cells were left uninfected (dot plots, upper panel) or infected with an apoptosis-inducing ICP27 deletion mutant of HSV-1 (ΔICP27; dot plots, lower panel) for 18 h (MOI = 1.5). Apoptosis was visualized as described for panel B. One representative experiment out of three is shown.

FIG. 6.

Diagram of the LAT region of the HSV-1 genome showing the location of LAT probes used for microarray analyses and quantitative RT-PCR. Selected restriction sites, the region associated with antiapoptotic function of LAT (10), and the deletion in 17N/H, an HSV-1 LAT deletion mutant (7) are indicated. Positions of probes used in the microarray (69) are marked with filled arrows, and positions of probes used in quantitative RT-PCR are marked with open arrows. Below the map of the genome, important LAT RNA species are depicted based on data published by Devi-Rao et al. (15). For better orientation, base numbers of the total HSV-1 DNA sequence (accession no. X14112) are given for selected positions.

FIG. 7.

Differential expression of viral genes in iDCs compared to HaCaT or HeLa cells. Total RNA was extracted at 10 h after infection with HSV-1 strain KOS at an MOI of 1.5 and subjected to HSV-1 microarray analysis. Expression of each viral gene or coterminal viral transcript family was normalized to the total net HSV-1-specific fluorescence signal to obtain the relative abundance. Subsequently, the median of relative abundance values from different experiments was calculated. The median relative abundance of each HSV-1 transcript in iDCs was divided by the median relative abundance of the corresponding transcript in HaCaT or HeLa cells, respectively (data not shown). Log₁₀ values of the obtained ratios are shown. A log₁₀ value of 0.3 corresponds to a 2-fold difference in relative abundance, and a log₁₀ value of 0.6 corresponds to a 4-fold difference. Probes are depicted in the order of the respective genes on the HSV-1 genome. The results shown are derived from microarray analysis performed two or three times with RNA from two or three independent infections.

FIG. 8.

Time course of expression of antiapoptotic HSV-1 genes in iDCs in comparison to HaCaT or HeLa cells. The median relative abundance detected with the HSV-1 microarray for each gene or coterminal transcript family at different time points is shown as a percentage of total HSV-1 transcripts (data not shown). The results shown as mean ± SD are derived from microarray analyses performed two or three times with RNA from two or three independent infections (#, P ≤ 0.05 for iDCs compared to HaCaT cells; *, P ≤ 0.05 for iDCs compared to HeLa cells).

FIG. 9.

Increased apoptosis of iDCs infected with LAT deletion mutant virus despite unaltered infection efficiency and upregulated expression of antiapoptotic genes other than LAT. iDCs from five different blood donors (D1 to D5) were infected with either HSV-1 wild-type virus (wt; strain 17) or LAT deletion mutant virus (ΔLAT; 17N/H) at an MOI of 1.5 or treated with a mixture of heat-inactivated wt and mutant virus (hi-HSV-1). (A) At 18 h p.i., apoptosis was quantified by Annexin V staining and subsequent flow cytometric analysis. Propidium iodide (PI)-positive (necrotic) cells were excluded. (B) The infection efficiency was measured by staining viral gD and subsequent flow cytometric analysis. Background as determined by staining of uninfected cells with the anti-gD antibody was subtracted out. (C) Mean values ± SD of relative copy numbers determined by quantitative RT-PCR probing different LAT regions (LAT-A, LAT-B, and LAT-C; see Fig. 5 for location in the LAT region) are shown. (D) Relative copy numbers of six antiapoptotic HSV-1 genes (US3, US5, US6, UL54, US1, and US11) and, for comparison, one glycoprotein-encoding virus gene (UL10) were determined by quantitative RT-PCR. Mean values ± SD of wt HSV-1 and the LAT deletion mutant are depicted.