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-␤-cyclodextrin (M␤CD) treatment on influenza virus budding. When virus-infected Madin-Darby canine kidney cells were treated with M␤CD 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 M␤CD treatment. Low concentrations of M␤CD 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 M␤CD treatment. Loss of infectivity of virus particles is due to multiple effects of M␤CD-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.

Chemical–Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation

“…Besides, cholesterol is known to play a important role in maintaining membrane integrity, trafficking, signal transduction and fluidity [31,32]. Cholesterol depletion results in the disorganization of lipid raft microdomains and also in the dissociation of proteins [47] that are bound to the lipid raft. Cholesterol accumulation is known to be associated in many tumors,…”

Role of Membrane Rafts in Viral Infection

“…It seems that gangliosides, major components of rafts, are not essential for the influenza virus life cycle. This suggestion for virus assembly and budding is supported by evidence that mutant viruses possessing alteration of raftbinding domains in HA and NA have the ability to produce infectious progeny virus [59,155] and evidence that membrane raft disruption enhances virus budding from MDCK cells [160]. How do membrane rafts help the influenza virus life cycle?…”

“…Therefore, M2 results in poor incorporation into the progeny viral membrane [157]. Moreover, membrane raft disruption causes a decrease in progeny virus infectivity concomitantly with an enhancement of virus particle release from infected cells [160]. Taken together, the results suggest that the role of membrane rafts in the influenza virus life cycle contributes to efficient incorporation of raft-associated viral proteins into the progeny viral membrane, which enhances progeny virus infectivity, rather than specific sorting and assembly of viral structural components and pinching-off of viral membrane from the plasma membrane.…”

“…The role of membrane rafts in the assembly and budding of enveloped viruses has been investigated for influenza virus [59,63,[150][151][152][153][154][155][156][157][158][159][160], HIV-1 [63,[161][162][163][164][165][166][167][168][169][170][171][172][173], HTLV-1 [174], measles virus (Paramyxoviridae) [63,175,176], Sendai virus [Paramyxoviridae] [177,178], Newcastle disease virus (NDV; Paramyxoviridae) [179,180], RSV [147,148,[181][182][183][184], HSV [185,186], murine cytomegalovirus (MCMV; Herpesviridae) [187], Ebola virus [70,188], Marburg virus [70], and vesicular stomatitis virus (VSV; Rhabdoviridae) …”

Regulation of NADPH oxidase (Nox2) by lipid rafts in breast carcinoma cells

“…However, there are no such studies in breast cancer cells. Cholesterol depletion causes the disorganization of lipid raft microdomains and also the dissociation of raft-bound proteins (13). Cholesterol sequestering agents, like filipin, nystatin or methyl-ß-cyclodextrin (MßCD), remove cholesterol and cause disruption of lipid rafts (14).…”