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.

Poliovirus (PV)

• Simple model system• Infection pathway?• Difficult to characterize virus entry pathways.

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)

RNA release is efficient

RNA is released near cell membrane

TIRF: Total internal reflection fluorescence microscopy. Isolates nearest 100-200 nm.

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

Neutral red-dependent infectious center (NRIC) assay

R78206: blocks RNA release.

Brefeldin A: Blocks RNA replication during later stages.

RNA release is ATP dependent

NaAz and deoxyglucose depletes cells of ATP

RNA release depends on actin

Nocodazole: Inhibits microtubule-dependent transport

Which endocytic mechanism is used by PV?

Endocytosis depends on tyrosine kinases

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

Model of PV entry