Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Abstract

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.

FIG. 1.

MβCD treatment inhibits the kinetics of poliovirus growth. (A) MβCD treatment reduces cellular cholesterol levels. Cholesterol levels were measured from equal numbers of untreated HeLa cells or HeLa cells after one or two cycles of MβCD treatment. (B) Effects of MβCD treatment on cellular cholesterol levels are long lasting. Cholesterol was extracted from equivalent numbers of untreated cells, MβCD-treated cells, and MβCD-treated cells that were grown for 12 h in serum-free medium. (C) Virus infection (MOI = 5) of MβCD-treated (♦) and untreated control (○) HeLa cells. Infected cells were harvested at the indicated times, and viral titers were measured by plaque assays.

FIG. 2.

MβCD treatment reduces the frequency of poliovirus-infected cells. MβCD-treated or control cells were infected at high multiplicities (MOI = 5) with either poliovirus (A to D) or VSV (E and F) and analyzed in infectious center assays (A, C, and E) or by flow cytometry (B, D, and F). For flow cytometry, histograms shown are of poliovirus-infected untreated (no fill) and MβCD-treated (grey filled) cells. The percentage of cells with fluorescent intensities above those of uninfected, MβCD-treated, or untreated cells is indicated in the insert table. (A and B) Poliovirus-infected HeLa cells; (C and D) poliovirus-infected Hep2c cells; (E and F) VSV-infected HeLa cells.

FIG. 3.

Cholesterol replenishment reverses the effects of MβCD treatment. (A) Cholesterol levels were measured from equal numbers of control cells, MβCD-treated cells, or MβCD-treated cells which were subsequently incubated with MβCD-cholesterol (MβCD-Chol) complexes. (B) Control, MβCD-treated, and MβCD-cholesterol-treated cells were infected with poliovirus (MOI = 5) and harvested at 6 h p.i. Titers were measured, and percentage yields were determined with titers from infected untreated (control) cells normalized to 100%. (C) Flow cytometry analysis of infected untreated control (dotted line), MβCD-treated (solid line), and MβCD-cholesterol complex-treated (grey filled) cells. Cells were infected with polio-EGFP (MOI = 5), harvested at 5 h p.i., and analyzed by flow cytometry. The range of positive fluorescent intensities above that of uninfected cells is shown. The percentage of GFP-positive cells is shown in the insert table.

FIG. 4.

MβCD inhibition of virus infection is multiplicity dependent. Control and MβCD-treated cells were infected with polio-EGFP at varying multiplicities, and the percentages of GFP-positive cells were measured by flow cytometry. Because the fraction of infected cells in the control untreated population changed with MOI, the percentage of infected cells is the percentage of GFP-positive cells in MβCD-treated samples divided by the percentage of GFP-positive cells in the control untreated samples.

FIG. 5.

PVR and poliovirus localize to the detergent-soluble membrane fractions. (A) Detergent-soluble (S) and -insoluble (I) membranes were isolated from MβCD-treated (+) or untreated (−) cells and analyzed by Western blotting for the presence of CD55 and the ganglioside GM1. (B) Detergent-soluble (S) and -insoluble (I) membranes were isolated from MβCD-treated (+) or untreated (−) infected cells expressing the EGFP-tagged PVR at 0 and 30 min p.i. Similar membrane fractions were also isolated from infected MβCD-treated cells. The isolated fractions were analyzed by Western blotting to detect capsid proteins VP1 and VP4 using capsid-specific polyclonal antibodies or to detect PVR using anti-EGFP antibody.

FIG. 6.

MβCD inhibition occurs during early stages of poliovirus infection. (A) MβCD (5 mM) was added to HeLa cells at the indicated times before or during the course of viral infection (MOI = 5) for a period of 1 h. The percentage of GFP-positive cells (▪) was measured by flow cytometry at 5 h p.i. Viral titers (░⃞) were determined by plaque assays of cell-associated virus at 6 h p.i. Virus yield at 6 h p.i. from infected untreated control cells was considered 100%. (B) At the indicated multiplicities, ³⁵S-labeled poliovirus was bound to control (▪) or MβCD-treated (░⃞) HeLa cell monolayers for 1 h, and the cell-associated radioactivity was measured by scintillation counting. (C) Control or MβCD-treated CHO cells were infected with 135S virus particles (MOI = 5). The percentages of infected cells and resultant virus yields at 6 h were measured by infectious center assays (▪) and plaque assays on HeLa cells (░⃞). The titer from control untreated cells was considered 100%.

FIG. 7.

MβCD inhibits RNA delivery during virus entry. (A) RNA was extracted at various times p.i. from control cells or cells pretreated with MβCD and analyzed by RT-PCR using primers specific for viral RNA or β-actin mRNA sequences. (B) MβCD or GuHCl was added to infected cells at 30 min p.i. RNA was extracted at various times and analyzed by RT-PCR. (C) Control cells or cells pretreated with MβCD or MβCD-cholesterol complexes were infected with poliovirus (MOI = 5). At various times p.i., RNA was extracted and analyzed by RT-PCR to detect viral RNA or β-actin mRNA.