Role of Sphingomyelin in Alphaherpesvirus Entry

Abstract

Bovine herpesvirus 1 (BoHV-1) is an alphaherpesvirus that causes disease in cattle populations worldwide. Sphingomyelin (SM) is the most abundant sphingolipid in the mammalian cell membrane, where it preferentially associates with cholesterol to form lipid raft domains. SM is a substrate for the lysosome-resident enzyme acid sphingomyelinase, which plays a role in cell membrane repair following injury. Treatment of cells with noncytotoxic concentrations of Staphylococcus aureus-derived sphingomyelinase successfully reduced cell surface-exposed sphingomyelin but did not significantly inhibit BoHV-1 entry and infection, as measured by the beta-galactosidase reporter assay. Interestingly, entry of the porcine alphaherpesvirus pseudorabies virus (PRV) was inhibited by sphingomyelin-depletion of cells. Treatment of BoHV-1 particles with sphingomyelinase inhibited viral entry activity, suggesting that viral SM plays a role in BoHV-1 entry, while cellular SM does not. Treatment of cells with noncytotoxic concentrations of the functional inhibitors of host acid sphingomyelinase, imipramine and amitriptyline, which induce degradation of the cellular enzyme, did not significantly inhibit BoHV-1 entry. In contrast, inhibition of cellular acid sphingomyelinase inhibited PRV entry. Entry of the human alphaherpesvirus herpes simplex virus 1 (HSV-1) was independent of both host SM and acid sphingomyelinase, in a manner similar to BoHV-1. Together, the results suggest that among the alphaherpesviruses, there is variability in entry requirements for cellular sphingomyelin and acid sphingomyelinase activity.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is an ubiquitous pathogen affecting cattle populations worldwide. Infection can result in complicated, polymicrobial infections due to the immunosuppressive properties of the virus. Available vaccines limit disease severity and spread but do not prevent infection. The financial and animal welfare ramifications of BoHV-1 are significant. In order to develop more effective prevention and treatment regimens, a more complete understanding of the initial steps in viral infection is necessary. We recently identified a low pH endocytosis pathway for BoHV-1. Here, we examine the role of cellular factors responsible for membrane integrity and repair in alphaherpesviral entry. This study allows comparisons of the BoHV-1 entry pathway with those of other alphaherpesviruses (pseudorabies virus [PRV] and herpes simplex virus 1 [HSV-1]). Lastly, this is the first report of sphingomyelin and lysosomal sphingomyelinase playing a role in the entry of a herpesvirus. The results may lead to the development of more effective prevention and treatment regimens.

Keywords: bovine herpesvirus 1; endocytosis; herpes simplex virus; herpesviruses; membranes; pseudorabies virus; sphingomyelin; viral entry.

FIG 1

Effect of sphingomyelin depletion on BoHV-1 and PRV entry into cells. MDBK or PK15 cells were treated with SMase at the indicated concentrations and incubated at 37°C for 45 min. Cells were washed with PBS. BoHV-1 or PRV lacZ-positive strains were added, and at 45 min p.i. noninternalized virus was inactivated with medium buffered to pH 4.7. Infection proceeded for a total of 6.5 h in the presence of normal culture medium. The beta-galactosidase expression is calculated as a percentage of the activity in mock-treated cells. Values shown are the means of three independent experiments with standard errors. The P values were determined using Student’s t test. (*, P < 0.05). Cytotoxicity is shown as percent LDH activity. LDH activity was <2% for all tested concentrations of SMase on both MDBK and PK15 cells.

FIG 2

SMase treatment of MDBK cells reduces sphingomyelin levels. Confluent MDBK monolayers were treated with DMEM (A) or 10 U/ml S. aureus SMase (B) for 45 min at 37°C. Cells were fixed with 6% paraformaldehyde and incubated with 0.5 μM lysenin for 2 h. Rabbit polyclonal antibody to lysenin was added and then detected with Alexa Fluor 594-conjugated goat anti-rabbit antibody (red). Nuclei were counterstained with DAPI. Cells were visualized by fluorescence microscopy. Magnification, ×40. ImageJ software was used to measure the mean fluorescence intensity from five equal areas per sample, each containing ∼150 to 250 cells. Results are representative of two independent experiments. The P value was determined using Student’s t test. (*, P < 0.0005).

FIG 3

BoHV-1 entry activity is dependent on viral envelope sphingomyelin. Concentrated BoHV-1 virions were incubated at the indicated concentrations for 45 min at 37°C. Virus was diluted to a final concentration of 0.1 U/ml or less. Cells were infected with treated virus at an MOI of 2 for 6.5 h. The beta-galactosidase expression was calculated as a percentage of activity of untreated virus. Values are the means of three independent experiments with standard errors. The P value was determined using Student’s t test. (*, P < 0.0005).

FIG 4

Effect of cellular acid sphingomyelinase inactivation by FIASMAs on BoHV-1 infection. Cells were treated with imipramine (A) or amitriptyline (B) at the indicated concentrations for 1 h at 37°C. MDBK or PK15 cells were infected with BoHV-1 or PRV, respectively, in the continued presence of drug. At 6.5 h p.i., the beta-galactosidase expression was calculated as a percentage of activity in untreated, infected cells. Cytotoxicity is shown as percent LDH activity. Values are the means of two or three independent experiments with standard errors. The P value represents PRV on MDBK cells and was determined using Student’s t test. (*, P < 0.009).

FIG 5

Effects of cellular sphingomyelin depletion and FIASMAs on HSV-1 entry. (A) SMase was added to Vero cells at 37°C for 45 min. Treated and mock-treated cells were washed with PBS. HSV-1 KOS tk12 (lacZ-positive) was added, and at 45 min p.i., noninternalized virus was inactivated with medium buffered to pH 4.7. The infection proceeded for total of 6.5 h in the presence of normal culture medium. (B) Vero cells were treated with imipramine or amitriptyline for 1 h at 37°C. Cells were infected with HSV-1 in the continued presence of drugs. At 6.5 h p.i., the beta-galactosidase expression was calculated as a percentage of activity in untreated, infected cells. Values are the means of three independent experiments with standard errors. Cytotoxicity is shown as percent LDH activity.