Imaging Poliovirus Entry in Live Cells

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.
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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 pHgt9 Living
cells maintain constant pH gt 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