Critical role of the lipid rafts in caprine herpesvirus type 1 infection in vitro

Abstract

The fusion machinery for herpesvirus entry in the host cells involves the interactions of viral glycoproteins with cellular receptors, although additional viral and cellular domains are required. Extensive areas of the plasma membrane surface consist of lipid rafts organized into cholesterol-rich microdomains involved in signal transduction, protein sorting, membrane transport and in many processes of viruses infection. Because of the extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to the lipid rafts, we investigated the effect of cholesterol depletion by methyl-β-cyclodextrin (MβCD) on caprine herpesvirus 1 (CpHV.1) in three important phases of virus infection such as binding, entry and post-entry. MβCD treatment did not prejudice virus binding to cells, while a dose-dependent reduction of the virus yield was observed at the virus entry stage, and 30 mM MβCD reduced infectivity evidently. Treatment of MDBK after virus entry revealed a moderate inhibitory effect suggesting that cholesterol is mainly required during virus entry rather than during the post-entry stage. Alteration of the envelope lipid composition affected virus entry and a noticeable reduction in virus infectivity was detected in the presence of 15 mM MβCD. Considering that the recognition of a host cell receptor is a crucial step in the start-up phase of infection, these data are essential for the study of CpHV.1 pathogenesis. To date virus receptors for CpHV.1 have not yet been identified and further investigations are required to state that MβCD treatment affects the expression of the viral receptors.

Keywords: Cholesterol; Goat; Herpesvirus; Infectivity; Plasma membrane.

Fig. 1

Cellular cholesterol determined with a cholesterol assay kit. (a) MDBK cells were treated with different concentration of MβCD and cholesterol was determined in comparison to untreated cells. MβCD treatment resulted in a dose-dependent reduction of the cholesterol content. (b) MDBK cells pre-treated with 30 mM MβCD were tested after cholesterol replenishment in increasing amounts. Exogenous cholesterol partially recovered cellular cholesterol. Experiments were repeated three times and the error bars indicate the standard deviations of the two independent experiments.

Fig. 2

Viral cholesterol determined with a cholesterol assay kit. (a) Viral suspensions were treated with 6–20 mM MβCD and the cholesterol content in MβCD-treated and untreated CpHV.1 suspensions was determined. A dose-dependent decrease was observed and 20 mM MβCD reduced viral cholesterol. (b) Cholesterol depleted virions were replenished with exogenous cholesterol in increasing amounts. Cholesterol content was partially recovered. Experiments were repeated three times and the error bars indicate the standard deviations of the two independent experiments.

Fig. 3

Cell membrane cholesterol depletion at the virus-binding phase. MDBK cells were mock treated or treated with 30 mM MβCD, infected with CpHV.1 in ice-cold medium and then titrated. Cholesterol depletion did not affect CpHV.1 binding to target cells. Experiments were repeated three times and the error bars indicate the standard deviations of the two independent experiments.

Fig. 4

Depletion (a) and replenishment (b) of cell membrane cholesterol at the virus entry stage. (a) MDBK cells were pre-treated with 6–30 mM MβCD then incubated with CpHV.1. Virus yield was determined with virus titration assay. Cholesterol depletion at the virus entry stage reduced the efficiency of CpHV.1 infection in a dose-dependent manner. b) MDBK depleted cells were supplemented with 400–800 μg/ml cholesterol then incubated with CpHV.1 and titrated. In the cholesterol depleted cells replenished with exogenous cholesterol, the inhibitory effect was partially restored. Experiments were repeated three times and the error bars indicate the standard deviations of the two independent experiments.

Fig. 5

Cholesterol depletion from cell membrane at the post-entry stage. MDBK cells were infected with CpHV.1 and then treated with 30 mM MβCD (test 1). In the control 1 and in the control 2 MDBK cells were mock treated and treated with 30 mM MβCD before virus infection. Cholesterol depletion at the post-entry stage had only a mild inhibitory effect on CpHV.1 production. Experiments were repeated three times and the error bars indicate the standard deviations of the two independent experiments.

Fig. 6

Cholesterol depletion (a) and replenishment (b) from viral envelope. a) CpHV.1 suspensions containing 10^6.25 TCID₅₀/50 μl were treated with 6–20 mM MβCD or mock treated. MβCD treatment resulted in a dose dependent inhibitory effect. b) CpHV.1 suspensions were mock pre-treatment or pre-treatment of with 15 mM MβCD, then mock replenished or replenished with 400–600 μg/ml exogenous cholesterol. Cholesterol replenishment partially re-established virus infectivity. Experiments were repeated three times and the error bars indicate the standard deviations of the two independent experiments.