In vivo analysis of protein sumoylation induced by a viral protein: Detection of HCMV pp71-induced Daxx sumoylation

Small ubiquitin-like modifiers (SUMOs) are covalently conjugated to target proteins to regulate numerous biological processes, including subcellular localization, protein-protein interactions, and transactivational activities. While the majority of identified SUMO targets are cellular proteins, SUMO modified viral proteins have also been identified. In addition, there are a growing number of examples where viruses alter the sumoylation status of host cell proteins. Work from our laboratory has previously demonstrated that the human cytomegalovirus (HCMV) virion tegument protein pp71 binds to Daxx, a cellular transcriptional co-repressor, and promotes its sumoylation. Here we describe the in vivo techniques used to detect pp71-induced sumoylation of Daxx in a cotransfection system as well as the endogenous SUMO modified form of Daxx in HCMV-infected cells. The approaches we describe can be easily adapted to infections with other viruses and for the detection of sumoylation of other proteins. (C) 2011 Elsevier Inc. All rights reserved.