Depletion of Host and Viral Sphingomyelin Impairs Influenza Virus Infection

Abstract

Influenza A virus (IAV) is a major human respiratory pathogen causing annual epidemics as well as periodic pandemics. A complete understanding of the virus pathogenesis and host factors involved in the viral lifecycle is crucial for developing novel therapeutic approaches. Sphingomyelin (SM) is the most abundant membrane sphingolipid. It preferentially associates with cholesterol to form distinct domains named lipid rafts. Sphingomyelinases, including acid sphingomyelinase (ASMase), catalyzes the hydrolysis of membrane SM and consequently transform lipid rafts into ceramide-enriched membrane platforms. In this study, we investigated the effect of SM hydrolysis on IAV propagation. Depleting plasma membrane SM by exogenous bacterial SMase (bSMase) impaired virus infection and reduced virus entry, whereas exogenous SM enhanced infection. Moreover, the depletion of virus envelope SM also reduced virus infectivity and impaired its attachment and internalization. Nonetheless, inhibition of ASMase by desipramine did not affect IAV infection. Similarly, virus replication was not impaired in Niemann-Pick disease type A (NPA) cells, which lack functional ASMase. IAV infection in A549 cells was associated with suppression of ASMase activity starting at 6 h post-infection. Our data reveals that intact cellular and viral envelope SM is required for efficient IAV infection. Therefore, SM metabolism can be a potential target for therapeutic intervention against influenza virus infection.

Keywords: acid sphingomyelinase; bacterial sphingomyelinase; influenza virus; lipid rafts; plasma membrane; sphingomyelin; viral envelope.

FIGURE 1

Plasma membrane SM depletion suppresses IAV replication in vitro. (A,B) The effect of exogenous bSMase on ceramide accumulation was studied using confocal microscopy. A549 cells were treated with 0.1 U/ml bSMase or with the vehicle (HBSS⁺) for 2 h at 37°C. Cells were then washed, fixed, and stained with mouse anti-ceramide antibody followed by Alexa Fluor 488 conjugated anti-mouse antibody (green). The cell nuclei were stained with DAPI (blue). The images were taken using confocal microscopy with a 40x oil objective lens (A). Fluorescent intensities of confocal images were measured, as shown in (B) using ZEN software. (C) Cytotoxicity of exogenous bSMase on A549 cells was assessed using the MTT assay. A549 cells were treated with increasing concentrations of bSMase for 2 h, washed with PBS^+, and incubated with VIM for 24 h. MTT assay was then performed at 24 h post-treatment to determine cell viability in treated and untreated cells. (D–G) The effect of SM depletion on IAV infection was determined by the plaque assay (D–F) or RT-PCR (G). A549 cells were pretreated with increasing bSMase concentrations or vehicle for 2 h followed by infection with either PR8 at 1 MOI (D,G) and 0.01 MOI (E) or Cal07 at 1 MOI (F). Viral titers were then determined using the plaque assay at the indicated time points (D) or at 24 hpi (E,F) and plotted relative to the vehicle-treated cells. Variation in PR8 total viral RNA in the supernatant of bSMase-treated and untreated cells was assessed at 24 hpi by RT-PCR using One-Step RT-PCR AgPath-ID (G) and plotted relative to the vehicle-treated cells. The results shown are representatives of two independent experiments. Means ± SD are shown. Statistical significance between untreated and bSMase-treated cells was assessed using the t-test (^∗corresponds to a P-value < 0.05, and ^∗∗∗corresponds to a P-value < 0.001).

FIGURE 2

Exogenous SM enhances IAV replication. (A) Cytotoxicity of SM on A549 cells was assessed using MTT assay. A549 cells were treated with increasing SM concentrations up to 50 μM, and MTT assay was then performed at 24 h post-treatment to determine cell viability in treated and untreated cells. (B) The effect of SM on IAV infection was studied. A549 cells were pretreated with 50 μM of exogenous SM or solvent for 24 h and inoculated with PR8 at 0.01 MOI. Virus titers were then assessed at 24 hpi using the plaque assay and plotted relative to vehicle-treated cells. Means ± SD are shown. Results shown are representative of two independent experiments. Statistical significance was assessed using the t-test (^∗corresponds to a P-value < 0.01).

FIGURE 3

Cellular SM depletion restricts IAV entry into A549 cells. A549 cells were pretreated with 1 U/ml bSMase (bSMase + IAV) for 2 h at 4°C or left untreated (IAV) prior to inoculation with PR8 at MOI of 15. Inoculated cells were kept at 4°C for 1 h to allow virus adsorption and then washed with cold PBS⁺ (A–D). The attached virus was labeled with mouse anti-influenza A (H1N1) antibody followed by Alexa Fluor 488 conjugated anti-mouse antibody (green) (A,B). Alternatively, after viral adsorption at 4°C for 1 h, the cells were placed at 37°C for an additional 30 min to allow virus entry, and internalized virions were assessed using mouse anti-NP antibody followed by Alexa Fluor 488 conjugated anti-mouse antibody (green) (C,D). Mock cells were left untreated/uninfected. The cell nuclei were stained with DAPI (blue). The images were taken using confocal microscopy with a 63x oil objective lens (A,C). The variation of fluorescence intensities was measured using ZEN software (B,D). Means ± SD are shown. Results are representative of two independent experiments. Statistical significance between infected (IAV) and treated infected (bSMase + IAV) cells was assessed using the t-test (*corresponds to a P-value < 0.05).

FIGURE 4

ASMase is dispensable for IAV infection (A,B) Cytotoxicity of desipramine (Des) in A549 cells was assessed using the MTT assay. A549 cells were treated with increasing desipramine concentrations for 24 h (A) or with desipramine at 12.5 μM or 25 μM for the indicated durations (B). (C) The effect of desipramine on ASMase activity in A549 cells was determined. A549 cells were treated with the vehicle or 25 μM desipramine, and ASMase activity was then assessed at 1 and 24 h post-treatment using Amplex^® Red Sphingomyelinase Assay Kit. (D) The impact of ASMase inhibition on IAV infection was assessed using the plaque assay and plotted relative to the vehicle-treated cells. A549 cells were either treated with desipramine for 1 h or left untreated prior to inoculation with PR8 (1 MOI) for 1 h in the presence or absence of the inhibitor. Cells were then incubated with or without the drug for 24 hpi, and viral titers were determined using the plaque assay. (E) Primary human fibroblasts deficient in ASMase activity (GM00112 and GM13205) and normal human primary fibroblasts (GM00038) were infected with PR8 at 1 MOI for the indicated durations. Normal human primary fibroblasts were used as a control. The virus yield was determined using plaque assay and plotted relative to the control. The results are representative of two independent experiments. Statistical significance was measured using t-test (*correspond to a P-value < 0.05 and ***correspond to a P-value < 0.001).

FIGURE 5

IAV infection restricts ASMase activity at later time-points post-infection. A549 cells were infected with PR8 at 1 MOI or left uninfected. Whole-cell lysates were used to assess ASMase activity at different durations post-infection using Amplex Red Sphingomyelinase Assay Kit. Results shown are fold changes of fluorescence in infected cells compared to their time-matching controls (uninfected cells). The average results of two independent experiments are shown. Statistical significance was measured between infected cells and their time-matching controls using t-test (*correspond to a P-value < 0.05 and ***correspond to a P-value < 0.001).

FIGURE 6

Virus SM depletion reduces IAV infectivity. The IAV PR8 stock was treated with 0.1 U/ml bSMase or with the vehicle (HBSS⁺) at 37°C for 1 h. (A) Treated or untreated PR8 was inoculated onto A549 cells after dilution in VIM to obtain an MOI of 1. Control cells were inoculated with untreated virus particles. Virus titers were assessed at 24 hpi using the plaque assay, and the virus yield was compared to control cells. (B) Alternatively, the infectivity of bSMase-treated or untreated IAV PR8 was assessed by plaque reduction assay using MDCK cells. Confluent monolayers of MDCK cells were inoculated with 90 PFU of bSMase-treated or untreated virus, and plaques were counted at 72 hpi. The % of infectivity was calculated relative to the control cells. Means ± SD are shown. (C) Hemagglutination assay was performed to assess the HA levels in the bSMase-treated virus. The results are representative of two independent experiments. Statistical significance between bSMase-treated and vehicle-treated IAV was assessed using the t-test (*corresponds to a P-value < 0.05, **corresponds to a P-value < 0.01).

FIGURE 7

Virus SM depletion impairs attachment and entry. The IAV PR8 stock was treated with 0.1 U/ml bSMase (bSMase-treated IAV) or with the vehicle (HBSS⁺) (IAV) at 37°C for 1 h. (A–D) Treated or untreated PR8 was inoculated onto A549 cells after dilution in VIM to obtain an MOI of 15. Control cells were inoculated with untreated virus particles. Cells were infected for 1 h at 4°C to allow virus adsorption and then washed with cold PBS⁺. After adsorption, attached virions were labeled using mouse anti-influenza A (H1N1) antibody followed by Alexa Fluor 488 conjugated anti-mouse antibody (green) (A,B). Alternatively, following viral adsorption, the cells were placed at 37°C for an additional 30 min to allow virus entry and internalized virions were assessed using mouse anti-NP antibody followed by Alexa Fluor 488 conjugated anti-mouse antibody (green) (C,D). Mock cells were left untreated/uninfected and used to confirm the specificity of the antibodies. DAPI (blue) was used as a nuclear stain. The images were taken using confocal microscopy with a 63x oil objective lens (A,C). The variation of fluorescence intensities was measured using ZEN software (B,D). Means ± SD are shown. The results are the average of two independent experiments. Statistical significance between bSMase-treated and Mock-treated IAV was assessed using the t-test (*corresponds to a P-value < 0.05, **corresponds to a P-value < 0.01).