Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells

Abstract

Lipid rafts play critical roles in many aspects of the influenza A virus life cycle. Cholesterol is a critical structural component of lipid rafts, and depletion of cholesterol leads to disorganization of lipid raft microdomains. In this study, we have investigated the effect of cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) treatment on influenza virus budding. When virus-infected Madin-Darby canine kidney cells were treated with MbetaCD at the late phase of infection for a short duration, budding of virus particles, as determined by protein analysis and electron microscopy, increased with increasing concentrations and lengths of treatment. However, infectious virus yield varied, depending on the concentration and duration of MbetaCD treatment. Low concentrations of MbetaCD increased infectious virus yield throughout the treatment period, but higher concentrations caused an initial increase of infectious virus titer followed by a decrease with a longer duration. Relative infectivity of the released virus particles, on the other hand, decreased with increasing concentrations and durations of MbetaCD treatment. Loss of infectivity of virus particles is due to multiple effects of MbetaCD-mediated cholesterol depletion causing disruption of lipid rafts, changes in structural integrity of the viral membrane, leakage of viral proteins, a nick or hole on the viral envelope, and disruption of the virus structure. Exogenous cholesterol increased lipid raft integrity, inhibited particle release, and partially restored the infectivity of the released virus particles. These data show that disruption of lipid rafts by cholesterol depletion caused an enhancement of virus particle release from infected cells and a decrease in the infectivity of virus particles.

FIG. 1.

Cholesterol release from MDCK cells resulting from MβCD treatment. Error bars, standard deviations (SD) (n = 4).

FIG. 2.

Effect of MβCD treatment on virus budding. (A) PFU titers of viruses released from virus-infected (MOI of 3) MDCK cells after MβCD treatment (at 12 h p.i.). (B) ³⁵S-labeled virus particles released from MβCD-treated (at 12 h p.i.) cells were purified, and viral proteins were separated in SDS-PAGE. M1/HA ratios are shown at the bottom of the panel. (C and D) Intensities of the HA (C) and NP (D) protein bands shown in panel B are plotted in arbitrary units against durations (in minutes) of MβCD treatment. Error bars in panels A, C, and D, SD (n = 5).

FIG. 3.

Effect of MβCD treatment on infectivity of virus particles. Error bars, SD (n = 4).

FIG. 4.

Increased release of virus particles from MβCD-treated cells by negative stain EM. Virus particles released from mock- or MβCD-treated (for 45 min at 12 h p.i.) virus-infected (MOI of 3) MDCK cells were purified and examined by negative-stain EM. A portion of a representative micrograph is shown on the left (magnification, ×19,000). The rectangle on the left is magnified (×72,000) on the right. ‖ or =, beads; single arrowheads, normal virus particles; double arrowheads, disrupted virus particles.

FIG. 5.

Thin-section EM of budding virus particles in (A) mock-treated and (B) MβCD-treated (30 mM, 45 min) MDCK cells. Cell profiles were randomly selected and photographed at a magnification of ×19,000. single arrowheads, virus particles; double arrowheads, villi.

FIG. 6.

Negative-stain electron microscopy of MβCD- or HβCD-treated virus particles. Virus particles were mock treated or treated with MβCD or HβCD for 30 min and were examined (photographed at ×36,000 magnification) by negative-stain EM. Single arrowheads, normal virus particles; double arrowheads, nicked or disrupted virus particles.