Cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus

Abstract

Cholesterol is a major constituent of detergent-resistant membrane microdomains (DRMs). We localized transmissible gastroenteritis virus (TGEV) spike (S) protein in DRMs in the viral envelope. Though S protein was not solubilized by cold non-ionic detergents, this behavior was unchanged when cholesterol was depleted from viral membrane by methyl-β-cyclodextrin (MβCD) and the protein did not comigrate with cellular DRM marker proteins in flotation analyses. Therefore, the S protein is not anchored in the viral membrane DRMs as they are known to occur in the plasma membrane. Cholesterol depletion from viral membrane may not affect the adsorption process as neither the sialic acid binding activity nor the binding to aminopeptidase N was reduced post-MβCD treatment. Reduced infectivity of cholesterol-depleted TGEV was observed only when the adsorption process occurred at 37°C but not when the virus was applied at 4°C. Cholesterol is important for a post-adsorption step, allowing membrane rearrangements that facilitate virus entry.

Fig. 1

Solubility of viral proteins. The S protein of TGEV treated with or without 10 mM MβCD and VSV G protein were extracted with 1% Triton X-100 at 4 °C and were detected by Western blot by corresponding antibody. DRMs is the abbreviation of detergent-resistant microdomains (Panel A); The infectivity of TGEV (2 × 10⁶ pfu/ml) treated with 10 mM MβCD was compared with that of mock-treated TGEV (Panel B). Recovery of TGEV infectivity after exogenous cholesterol addition to cells treated with 10 mM MβCD is shown (Panel C). Symbol “**” means highly significant difference in statistics, “***” means extremely significant difference in statistics.

Fig. 2

Flotation density of viral proteins by sucrose density gradient centrifugation. The flotation density of either TGEV S protein or VSV G protein (with or without 10 mM MβCD) was analyzed using a 5–40% sucrose gradient after treatment of 1% Triton X-100 at 4 °C The drug concentrations, viral protein and gradient sequence are indicated.

Fig. 3

Effect of MβCD-treatment of virions on infectivity and HA actvity of TGEV. TGEV was harvested from neuraminidase-treated ST cells and the resulting viruses (2 × 10⁵ pfu/ml) were treated with MβCD (0, 4 and 10 mM) at 37 °C for 30 min. The treated viruses were used to determine the infectivity and the HA activity.

Fig. 4

The effect of viral cholesterol depletion on binding between TGEV S and pAPN. Cholesterol of neuraminidase treated TGEV was depleted with MβCD (0, 2, 4, 8 and 10 mM) at 37 °C for 30 min and then the viruses were used to bind neuraminidase treated ST cells. The binding activity between them was analyzed by indirect ELISA. The OD value was measured at 405 nm wavelength (Panel A); The infectivity of TGEV (2 × 10⁴ pfu/ml) treated with various concentrations of MβCD was reflected by virus plaque-reduction assays (Panel B). The OD₄₀₅ is from three independent experiments and each experiment was performed in triplicate.

Fig. 5

Binding of TGEV virions. Desialylated ST cells were incubated with untreated or MβCD-treated TGEV (2 × 10⁶ pfu/ml) harvested from NA-treated ST cells for 1 h at 4 °C or 37 °C prior to further incubation at 37 °C for subsequent infection assay. The infectivity of the treated viruses was compared with that of mock-treated viruses at 4 °C (A) or 37 °C (B), respectively. Each sample was done in triplicate, and bars indicate standard deviation. Symbol “**” means highly significant difference in statistics.