1. Imaging Poliovirus Entry in Live Cells Brandenburg 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.

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 internalized before 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. Which endocytic mechanism is used by PV?

11. Endocytosis depends on tyrosine kinases

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