FIG. 8.

Virion assembly of ICP0 requires its RING finger and its C terminus. (A) Line drawing of the ICP0 mutants used in this study. (B) U2OS cells were infected with all the viruses shown in panel A together with the Δ22 mutant virus at a multiplicity of infection of 1 and harvested 20 h later. Immunoprecipitations were carried out using a VP22 antibody, and the resulting complexes were analyzed by Western blotting for VP22 and ICP0. (C) Equivalent amounts of extracellular virions from the viruses shown in panel A, together with the ICP0 deletion mutant (dl1403), were analyzed by Western blotting for VP5, VP16, VP22, and ICP0. (D) Extracellular virions from the USP7 binding site mutant D12 were centrifuged through a 5 to 15% Ficoll gradient, and fractions were taken across the gradient. Each sample was analyzed for infectivity (top) or subjected to Western blotting for the major capsid protein VP5 or ICP0.