A Novel Fluorescent Labelling Approach to Detect Early Events in RSV-infected Living Cells

Abstract

FEBRUARY 2011 AB22 Abstracts S A T U R D A Y 70 Respiratory Syncytial Virus Docking on Lipid Rafts as revealed by Single Virus Tracking H. G. San Juan Vergara, V. P. Sampayo Escobar, B. Cha, L. Pacheco Lugo, N. Reyes Reyes, S. S. Mohapatra; University of South Florida, Tampa, FL, Universidad del Norte, Barranquilla, COLOMBIA, Universidad de Cartagena, Cartagena, COLOMBIA. RATIONALE:Respiratory syncytial virus (RSV) infection has been associated with the development of respiratory diseases. The precise mechanism of its entry to normal human bronchial epithelial (NHBE) cells has not been elucidated. We developed a single particle tracking approach to investigate the mechanism of RSV membrane fusion in NHBE cells. METHODS: Purified virions were dual-labeled with lipophilic fluorescent dyes R18 and DiOC18 and were allowed to attach to NHBE cells grown in a 35 mm glass bottom culture dish at 48C for 20 min. Then, cells were placed on a micro-incubator to maintain them at 37C during live imaging for 1 h with a Olympus FV1000 laser scanning confocal microscope. Cells were scanned with 488 nm laser and both emissions, 500 to 530 nm and 555 to 618 nm, were simultaneously detected. Alexa 647-labeled markers were used to pinpoint the cellular regions targeted during the virus fusion. Lipid rafts role during infection was evaluated by cholesterol sequestration. RESULTS: The fusion events were made visible by a sudden increase in the green fluorescence and were quantified. Non-fused virions were seen as red particles. The fluorescence switch from red to green occurred on the cell membrane. Labeled virions co-localized significantly with Cholera Toxin subunit B suggesting docking at lipid rafts. Moreover, cholesterol sequestration reduced RSV infection. CONCLUSIONS: Taken together, these results indicate that RSV fusion predominantly occurs at the lipid rafts on the cell membrane. Learning about the fusion location and mechanism is expected to lead to the search of therapeutic alternatives to prevent RSV infection.