Imaging Poliovirus Entry in Live Cells
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Imaging Poliovirus Entry in Live CellsBrandenburg et al PLoS Biology 2007
Purpose: To chacterize early stages of poliovirus infection in living cells. How does virus enter the cell, and where is RNA released?
Method: Fluorescent labelling.
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Poliovirus (PV)
• Simple model system• Infection pathway?• Difficult to characterize virus entry pathways.
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Experiment
• Dual-labeled PV• RNA: Syto82 (55-65%), green, RNA unfolding reduces
signal• Kapsel: Cy5 (100%), red
• 300-400 virus particles per cell = 1 pfu/cell• Live-cell fluorescence microscopy• Human cells (HeLa S3)
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RNA release is efficient
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RNA is released near cell membrane
TIRF: Total internal reflection fluorescence microscopy. Isolates nearest 100-200 nm.
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Virus is internalized before RNA release
pH-sensitive dye: No fluorescence at pH>9
Living cells maintain constant pH => only PV outside cells are pH-sensitive
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Neutral red-dependent infectious center (NRIC) assay
R78206: blocks RNA release.
Brefeldin A: Blocks RNA replication during later stages.
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RNA release is ATP dependent
NaAz and deoxyglucose depletes cells of ATP
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RNA release depends on actin
Nocodazole: Inhibits microtubule-dependent transport
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Which endocytic mechanism is used by PV?
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Endocytosis depends on tyrosine kinases
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Summary
• RNA release is rapid and efficient• RNA release takes place after internalization,
and near the cell membrane• RNA release depends on temperature, energy
(ATP), actin and tyrosine kinases
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Model of PV entry