Cholesterol dependence of varicella-zoster virion entry into target cells

Abstract

The entry of inhaled virions into airway cells is presumably the initiating step of varicella-zoster infection. In order to characterize viral entry, we studied the relative roles played by lipid rafts and clathrin-mediated transport. Virus and target cells were pretreated with agents designed to perturb selected aspects of endocytosis and membrane composition, and the effects of these perturbations on infectious focus formation were monitored. Infectivity was exquisitely sensitive to methyl-beta-cyclodextrin (M beta CD) and nystatin, which disrupt lipid rafts by removing cholesterol. These agents inhibited infection by enveloped, but not cell-associated, varicella-zoster virus (VZV) in a dose-dependent manner and exerted these effects on both target cell and viral membranes. Inhibition by M beta CD, which could be reversed by cholesterol replenishment, rapidly declined as a function of time after exposure of target cells to VZV, suggesting that an early step in viral infection requires cholesterol. No effect of cholesterol depletion, however, was seen on viral binding; moreover, there was no reduction in the surface expression or internalization of mannose 6-phosphate receptors, which are required for VZV entry. Viral entry was energy dependent and showed concentration-dependent inhibition by chlorpromazine, which, among other actions, blocks clathrin-mediated endocytosis. These data suggest that both membrane lipid composition and clathrin-mediated transport are critical for VZV entry. Lipid rafts are likely to contribute directly to viral envelope integrity and, in the host membrane, may influence endocytosis, evoke downstream signaling, and/or facilitate membrane fusion.

FIG. 1.

The infectivity of a sonicated VZV preparation is attributable to free virions and can be inhibited by pretreatment of target cells with MβCD. (a) Infectious-focus formation by cell-free virus is inhibited by known antagonists of VZ virion entry. Cell-free virus (prepared as described in Materials and Methods) was inoculated onto HELF in the presence or absence of mannose 6-phosphate (10 mM) or heparin (100 μg/ml). Five days later, infectious foci were stained with an anti-VZV antibody and enumerated. Data shown are mean counts ± the standard error of triplicate wells, normalized to the control. (b) Transmission electron micrograph of a cell-free virus preparation, demonstrating apparently intact enveloped virions along with cellular debris and viral fragments. (c and d) Susceptibility to cell-free VZV infection is reduced in a concentration-dependent manner by pretreatment of target cells with MβCD. (c) Quantitation of infectious foci (mean ± standard error of triplicate wells, normalized to the control). Data are representative of three experiments. (d) Appearance of stained foci of VZV infection. Representative wells at a single input MOI are shown, with progressively greater concentrations of MβCD from the bottom (no drug) to the top (10 mM MβCD).

FIG. 2.

Mechanism of action of MβCD. Cholesterol depletion from target cells or virions inhibits viral entry. (a) Inhibition of viral infectivity by pretreatment with MβCD (2.5 mM) can be completely reversed by cholesterol replenishment (30 min at 0.1 mM compared with 0 mM cholesterol). (b) Inhibition of viral infectivity by overnight pretreatment of target cells with nystatin (25 μg/ml) and progesterone (10 μg/ml), compared with heparin (100 μg/ml, present in the viral inoculum). (c) Delayed addition of MβCD (2.5 mM) after inoculation of virus onto cells shows progressive loss of susceptibility to inhibition of infectivity, suggesting that cholesterol is required for an early step. (d) Viral infectivity is impaired by direct exposure of the viral inoculum to cholesterol-depleting concentrations of MβCD, suggesting a distinct role for envelope cholesterol in virion infectivity.

FIG. 3.

Virus binding is not affected by cholesterol depletion from target cells. The binding of cell-free VZV to target cells was analyzed by a FACS-based immunoassay (see Materials and Methods). Heparin completely inhibited virus binding (purple trace, lower panel), but MβCD pretreatment had no effect, even at concentrations that strongly inhibit viral infectivity.

FIG. 4.

Cholesterol depletion neither reduces the surface expression of the MPR^ci nor prevents its internalization. (A to F) Fluorescence microscopy following the incubation of live cells with antibodies to MPR^ci, with fixation before (A to C) or after (D to F) a 15-min (15′) chase. Green, anti-MPR^ci; blue, nuclei counterstained with bisbenzimide. (a to f) Corresponding FACS experiment with anti-MPR^ci and labeled secondary antibodies and analysis in the presence (gray fill) or absence (red line) of the membrane-impermeant quenching agent trypan blue as described in Materials and Methods. Similar results were obtained with Zenon-Alexa Fluor-labeled anti-MPR^ci antibody. A, D, a, and d, untreated target cells; B, E, b, and e, pretreatment with sodium azide (10 mM) and deoxyglucose (50 mM); C, F, c, and f, target cell pretreatment with MβCD (2.5 mM).

FIG. 5.

A possible role for endocytosis in VZV entry. (a) Metabolic poisoning with sodium azide (10 mM) and deoxyglucose (50 mM) prevents establishment of infection with VZV. (b) Target cell treatment with chlorpromazine produces a concentration-dependent reduction in VZV infectivity. (c) The chlorpromazine-dependent step occurs early in the infection process, compatible with a role in viral entry. Addition of chlorpromazine (final concentration, 25 μM) was delayed to the indicated times with respect to viral inoculation of target cells.

FIG. 6.

TEM images are compatible with endocytosis of VZ virions. (A) Intact enveloped VZ virions (arrows) are found extracellularly in proximity to the plasma membrane of a target cell. (B) An enveloped virion is found in a coated pit (arrow) formed by the plasma membrane of a target cell. (C) An enveloped virion is found in a vesicle in the cortical cytoplasm of a target cell. The appearance of the vesicle is compatible with that of an early endosome. (D) An unenveloped nucleocapsid is found in the region of the Golgi apparatus of a target cell. Bars = 200 nm. N, nucleus; G, Golgi apparatus; a, cortical actin filaments.

FIG. 7.

Cellular kinase activity is required for viral infectivity beyond an entry step. (a) A delay in the addition of genistein (100 μM) of up to 5 h after inoculation of virus onto target cells produced no diminution of its inhibitory effect on infectious focus formation. Treatment was continued until 20 h postinfection. (b) Target cells preincubated with increasing concentrations of the PI3K inhibitor wortmannin showed no consistent inhibition of VZV infectivity.

FIG. 8.

A suggested mechanism of VZ virion entry. The incoming virion initially attaches to cellular HSPG (step 1), facilitating specific interaction with a cellular receptor, e.g., MPR^ci (step 2). This leads to receptor-mediated (clathrin-dependent) endocytosis (step 3) and delivery to an endosomal compartment (step 4). Within this structure, the virion is exposed to cofactors for membrane fusion, possibly including IDE and/or an altered pH (step 5). Following triggered membrane fusion, the VZ nucleocapsid is delivered to the cytoplasm. Cholesterol may play a role at each stage.