FIG. 3.

ATP depletion does not affect the rate or extent of capsid scaffold cleavage. Vero cells were infected with tsProt.A and incubated as shown in Fig. 1. At 6.5 h postinfection cells were mock treated (ND, nondepleted control cells) or treated with an ATP depletion mixture (D, cells depleted of ATP), incubated for a further 0.5 h at 39°C, harvested, and processed as follows. (A) Cell extracts were prepared immediately or after successive times of incubation at 31°C (as indicated above the figure in minutes), subjected to SDS-PAGE, and Western blotted for ICP35. (B) Cell extracts were prepared immediately or after 150 min of incubation, subjected to SDS-PAGE, Western blotted, and probed with antisera specific for the amino-terminal portion of Pra. The positions of Pra and VP24 are indicated at the left of the figure. (C) Cell extracts were chilled, solubilized in Triton X-100, and subjected to several high-salt extractions in order to release viral capsids. Capsids were then pelleted through a sucrose cushion, and the pellet was subjected to SDS-PAGE, Western blotted, and probed for ICP35. The positions of Pra and ICP35 are indicated by the upper and lower pair of arrowheads, respectively. The uppermost of each pair of arrowheads indicates the position of the unprocessed form of the scaffold polypeptide. The autoradiograph is deliberately overexposed to show the low levels of uncleaved ICP35c,d and Pra in the capsid pellet. The reactive bands at the top of the gel appear to be capsid specific and may represent aggregated or multimeric Pra.