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. 2009 Jul 29;83(19):10224–10233. doi: 10.1128/JVI.00589-09

FIG. 5.

FIG. 5.

Orf64 can effectively cleave both K48 and K63 Ub linkages. (A) pcDEF3 or pcDEF3-Orf64FL expression plasmids were cotransfected with expression plasmids for WT-Ub, K48-Ub, or K63-Ub into HEK-293 cells. Cells were harvested 48 h posttransfection and subjected to Western blot analysis with an anti-HA antibody. Orf64FL was able to deubiquitinate WT-Ub and K48- and K63-linked ubiquitinated cellular proteins. Actin blots show equal loading of protein lysates, and a Western blot with an anti-Flag antibody shows expression of Orf64FL. (B) pcDEF3 or pcDEF3-Orf64N expression plasmids were cotransfected with expression plasmids for WT-Ub, K48-Ub, or K63-Ub into HEK-293 cells. Cells were harvested 48 h posttransfection and subjected to Western blot analysis with an anti-HA antibody. Orf64N was able to deubiquitinate WT-Ub and K48- and K63-linked ubiquitinated cellular proteins. Western blots for actin show equal loading of protein lysates, and a Western blot with an anti-Flag antibody shows expression of Orf64N. (C) Cotransfection of pcDEF3, pcDEF3-Orf64N, and pcDEF3-Orf64N-C29G with WT-Ub expression plasmids in HEK-293 cells. Cells were harvested 48 h posttransfection and subjected to Western blot analysis with an anti-HA antibody. Orf64N can deubiquitinate cellular proteins, but the DUB activity of Orf64N is ablated by the C29G mutation, suggesting that this cysteine is essential for deubiquitination mediated by Orf64. Western blots for actin show equal loading of protein lysates, and a Western blot with an anti-Flag antibody shows expression of both Orf64N and Orf64N-C29G. (D) Cotransfection of pcDEF3, pcDEF3-Orf64FL, and pcDEF3- Orf64FL-C29G with WT-Ub expression plasmids in HEK-293 cells. Cells were harvested 48 h posttransfection and subjected to Western blot analysis with an anti-HA antibody. Orf64FL can deubiquitinate cellular proteins, but the DUB activity of Orf64FL is ablated by the C29G mutation, suggesting that this cysteine is essential for deubiquitination mediated by Orf64. Western blots for actin show equal loading of protein lysates, and a Western blot with an anti-Flag antibody shows expression of both Orf64FL and Orf64FL-C29G.