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Imaging Poliovirus Entry in Live Cells

Imaging Poliovirus Entry in Live Cells. 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. Brandenburg et al PLoS Biology 2007. Poliovirus (PV). Simple model system

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Imaging Poliovirus Entry in Live Cells

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  1. Imaging Poliovirus Entry in Live Cells 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. Brandenburg et al PLoSBiology 2007

  2. Poliovirus (PV) • Simple model system • Infection pathway? • Difficult to characterize virus entry pathways.

  3. 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)

  4. RNA release is efficient

  5. RNA is released near cell membrane TIRF: Total internal reflection fluorescence microscopy. Isolates nearest 100-200 nm.

  6. Virus is internalizedbefore RNA release pH-sensitive dye: No fluorescence at pH>9 Living cells maintain constant pH => only PV outside cells are pH-sensitive

  7. Neutral red-dependent infectious center (NRIC) assay R78206: blocks RNA release. Brefeldin A: Blocks RNA replication during later stages.

  8. RNA release is ATP dependent NaAz and deoxyglucose depletes cells of ATP

  9. RNA release depends on actin Nocodazole: Inhibits microtubule-dependent transport

  10. Whichendocyticmechanism is used by PV?

  11. Endocytosisdependsontyrosinekinases

  12. 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

  13. Model of PV entry

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