FIG. 2.

vhs is largely insoluble in infected cells. (A) PNS were prepared from HSV PAA^R5-infected cells at 16 h postinfection and incubated on ice for 0.5 h either alone (−) or in the presence of 0.5 M NaCl or 1% TX-100 or NaCl and TX-100 together as shown at the top of the figure. Following centrifugation at 18,500 × g, the resulting pellet (lanes P) and supernatant (lanes S) were subjected to SDS-PAGE and Western blotting and probed with antibodies against vhs, TfR, and β-COP, as indicated at the right side of the panel. (B) Cells were infected with HSV strain tsProt.A for 18 h at 31 or 39°C as indicated, and then PNS were collected and treated as described for panel A, followed by Western blotting with anti-vhs antibodies. (C) To confirm that capsid maturation had been blocked in the experiment described for panel B, samples of the PNS prepared as described for panel B were assayed for levels of PFU. (D) PNS from HSV PAA^R5-infected cells was incubated with 100 mM Na₂CO₃ (pH 11.5) on ice for 0.5 h and loaded at the bottom of a sucrose flotation gradient (see Materials and Methods). After centrifugation and fractionation, an equal volume of each fraction was subjected to SDS-PAGE and Western blotting, as indicated at the right side of the panel. Gradient fractions are numbered from the top (lane 1) to the bottom (lane 12).