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Internalization of intact poliovirus by HeLa cells as shown by subcellular fractionation in isoosmotic Nycodenz gradients

¹ Department of Microbiology and Hygiene
and² Department of Cell Biology and Histology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium

HeLa cells were infected with radiolabelled poliovirus at different temperatures, and the intracellular distribution of input radioactivity was studied. To this end, homogenates were fractionated by rate zonal centrifugation in linear isoosmotic (2 to 30%) Nycodenz gradients. Further purification of subcellular fractions was achieved by recentrifugation to equilibrium in 10 to 30% Nycodenz. Temperatures were kept below 30 °C to prevent virus capsid modification. Under these conditions, the cell-associated virions remained fully infectious. Below 18 °C, most of the viral label was recovered from a bottom region (BR) of the rate zonal gradients. Marker enzyme analysis and antibody accessibility showed that the BR consisted of virions bound to the plasma membrane. Between 18 °C and 26 °C, viral label also accumulated in a top region (TR) of the rate zonal gradients. According to the criterion of antibody accessibility, the virions associated with the TR were present within intracellular structures, probably lipid membranes. Electron microscopy confirmed the presence of vesicles and tubules in this region of the gradient. No correlation was found between the TR and endosomal, lysosomal or plasma membrane markers. The TR equilibrated at low density (1·10 g/ml) in Nycodenz (free virus, 1·31 g/ml). The results confirm that intact poliovirions can enter the cell and do so via lipid-bound vesicles.

Received 17 June 1991; accepted 18 October 1991.

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