Cholesterol Removal by Methyl- (cid:2) -Cyclodextrin Inhibits Poliovirus Entry

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 myristy- lated 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- (cid:1) -cyclodextrin (M (cid:1) CD), 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 M (cid:1) CD 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 M (cid:1) CD. We further demonstrate that treatment with M (cid:1) CD 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 virus were generated from coupled vaccinia virus-poliovirus cDNA transfections using DOTAP trans- fection methods instead of electroporation (35). Vaccinia virus-T7-infected cells were transfected with 10 (cid:4) g of pPVM1-EGFP cDNA using 45 (cid:4) g of DOTAP liposomal transfection reagent (Roche, Indianapolis, Ind.) and transferred to plates (10 6 cells/ml). Cells were harvested at 12 h posttransfection and lysed in reticulocyte standard buffer (10 mM Tris-HCl [pH 7.4], 10 mM NaCl, 1.5 mM MgCl 2 )–1% NP-40 at 4°C. Virus was concentrated from the postnuclear super- natant by centrifugation at 50,000 rpm fo r 1 h at 4°C in a Beckman 70.1 Ti rotor and partially puriﬁed by 15-to-30% sucrose density gradients. Fractions consti- tuting the 160S virus peaks were identiﬁed using a parallel gradient of [ 35 S]me-thionine-labeled poliovirus. These fractions were pooled and dialyzed against phosphate-buffered saline (PBS), and the working stocks of polio-EGFP (pas-sage 2 [P2]) were generated from this seed stock in a single low-multiplicity passage. The low-multiplicity-of-infection (MOI) cells were harvested when the majority of cells showed green ﬂuorescence, and virus was released from infected cell pellets by three cycles of freeze-thaw and used without further puriﬁcation. Typical polio-EGFP stocks had titers of 2 (cid:3) 10 8 PFU/ml. Stocks beyond the P2 passage often contained viruses which had lost the EGFP sequence from the viral genome. Therefore, the P2 passage was used for all experiments conducted here. Vesicular stomatitis virus (VSV) and VSV-expressing GFP were obtained from Whitt and were propagated by low- multiplicity passages on BHK cells. blots were developed using Nitro Blue Tetrazolium and 5-bromo-4-chloro-3- indolylphosphate (Fisherbiotech, Fair Lawn, N.J.). GM1 was detected as follows: horseradish peroxidase-labeled (cid:2) -subunit of cholera toxin (10 (cid:4) g/ml; Sigma) was added fo r 1 h atroom temperature in TBST-milk. The blot was developed using Renaissance Western Blot Chemiluminescence Reagent Plus (NEN).