Distinct pathways for tumor necrosis factor alpha and ceramides in human cytomegalovirus infection

Abstract

Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-alpha) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-alpha. To investigate the mechanisms involved in the inhibition of HCMV by TNF-alpha, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-alpha versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-alpha. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-alpha. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-alpha is independent of ceramides. In addition, our results suggest that TNF-alpha and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.

FIG. 1

Effect of TNF-α on the cell cycle of infected U373 MG cells. U373 MG cells were incubated with 100 U of TNF-α per ml (b and d) or without TNF-α (a and c) for 24 h prior to HCMV infection (c and d) or mock infection (a and b). The medium was replaced 24 h later, and 6 days p.i. the nuclei were stained with propidium iodide and analyzed by flow cytometry.

FIG. 2

Effect of C6-ceramide on the cell cycle of U373 MG cells. U373 MG cells were incubated for 24 h with 0 to 10 μM C6-ceramide prior to mock (A) or HCMV (B) infection. The inoculum was removed 24 h later, and the concentrations of ceramide were maintained throughout the experiment. At 6 days p.i., the nuclei were stained with propidium iodide and analyzed by flow cytometry.

FIG. 3

Increase in gB expression and HCMV production by C6-ceramide. U373 MG cells were incubated with 0 to 10 μM C6-ceramide prior to HCMV infection. Medium was replaced after 24 h infection, maintaining the concentrations of ceramide throughout the experiment. (A) On day 6 p.i., the expression of gB was estimated by intracytoplasmic immunofluorescent staining followed by flow cytometry analysis. (B) Cells were harvested and sonicated at 6 days p.i. The sonicate was used to infect an MRC5 fibroblast monolayer. Infection was estimated by plaque assay.

FIG. 4

Effect of TNF-α on ceramide and sphingomyelin levels. U373 MG cells were labelled with 1 μCi of [³H]palmitate (51 Ci/mmol) per dish for 48 h and then incubated for the indicated times with 2,000 U of TNF-α per ml (⧫) or 0.1 U of sphingomyelinase per ml (▪). The quantities of labelled sphingomyelin (A) and ceramide (B) were assessed, and the results are expressed as a percentage of total ³H-labelled lipids. Points on the TNF curve represent the mean ± standard deviation (n = 3).

FIG. 5

Synergy between TNF-α and sphingomyelinase on HCMV inhibition. (A) gB expression. U373 MG cells were preincubated for 24 h with 100 U of TNF-α per ml and/or treated with 0.1 U of sphingomyelinase (SMase) per ml 1 h before HCMV infection. Expression of gB was assessed by intracellular immunofluorescent staining followed by flow cytometry analysis. (B) Viral production. U373 MG cells were pretreated or not treated for 24 h with the indicated concentrations of TNF-α and/or treated with the indicated concentrations of sphingomyelinase (SM) 1 h prior to HCMV infection at a MOI of 5. On day 6 p.i., the cells were sonicated and the sonicate was used at a dilution of 1/100 to infect a monolayer of FSF1 cells. Intracellular IE expression was estimated 4 days later by flow cytometry analysis. The results are expressed as mean fluorescence.

FIG. 6

TNF-α alone increases JNK1 activity. U373 MG cells were either untreated (control) or treated with 500 U of TNF-α (TNF) per ml, 10 μM C6-ceramide (C6-cer), or 0.1 U of sphingomyelinase (SMase) per ml for 15 min prior to cell lysis. JNK1 was immunoprecipitated from cell lysates and incubated with recombinant c-Jun and [γ-³²P]ATP in a 20-min assay at 30°C. Phosphorylation of c-Jun was determined by autoradiography following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5% polyacrylamide). The bands were quantified by densitometry and expressed as fold increase compared to control. This data is from one of three experiments with similar results.

FIG. 7

Summary of the pathways used by TNF-α and ceramides in the control of in vitro HCMV replication. TNF-α inhibits HCMV replication through a ceramide-independent and NO-independent pathway (see the text). Activation of JNK1 may be involved in this inhibition. Long-chain ceramides inhibit HCMV infection through another pathway, which is also independent of NO. Permeant C6-ceramide uses yet another pathway to activate HCMV replication.