TABLE 1.

Details of the GST fusion protein constructs used in this studyf

PlasmidaDerivationbActivationcFK2dE3 ligasee
pGEX262p110262(+++)+++
pGEX262del106-149p110FXE
pGEX241This study(+++)ND+
pGEX211This study(+++)ND+
pGEX241K144Ep110K144E+
pGEX241W146Ap110DR36c+++++++
pGEX241Q148Ep110Q148E++++
pGEX241N151Dp110N151D++
pGEX241ins150p110F1
pGEX241ins162p110R2
pGEX241ins188p110E8+
pGEX241ins197p110E13+
pGEX241del9-76p110D1++ND+
pGEX241del129/130p110DR40
pGEX241del162-188p110D22
  • a The names of the various GST fusion proteins expressed from pGEX vector plasmids indicate the background ICP0 segment (262 is residues 241 plus 21 residues encoded in intron 2; 241 is residues 1 to 241; 211 is residues 1 to 211) plus the various deletion, insertion, or substitution mutations.

  • b The names of the plasmids (as referenced in Materials and Methods) that were used as sources of the mutations present in the pGEX series plasmids.

  • c Activation refers to the ability of otherwise full-length versions of ICP0 carrying these lesions to activate gene expression from either the ICP6 promoter (mutants FXE, K144E, W146A, Q148E, and N151D [12]), the tk promoter (mutant DR40 [32]), or the gD promoter (all other mutants [2, 3]). −, less than 10% of wild-type ICP0 activity; +, 10 to 40%; ++, 40 to 70%; +++, >70%. The entries shown in parentheses indicate that these proteins contain segments of wild-type ICP0.

  • d The effects of these mutations on the ability of ICP0 to induce colocalizing conjugated ubiquitin in transfected and (where known) infected cells, as described in reference 4. The scoring system is a qualitative comparison of the intensity of induced signal compared to a wild-type, full-length ICP0 control. −, undetectable activity. ND, not done.

  • e Ability of the protein expressed by the relevant pGEX plasmid to stimulate the production of high-molecular-weight ubiquitin chains in vitro.

  • f The effects of the mutations on activation of gene expression and production of colocalizing conjugated ubiquitin induced by full-length ICP0 in vivo are compared with the results of in vitro ubiquitin E3 ligase assays using GST fusion proteins containing the same mutations.