We use a variety of proteomics methods coupled to mass spectroscopy to identify cellular and viral protein interactions in human cells, which points us to the functional roles and mechanisms of action of these proteins. For example, we were among the first to apply tandem affinity purification (TAP)-tagging methods to human cells, in which two purification tags are used to isolate the protein of interest and its binding partners from cells. We used this approach to reveal stable interactions of EBNA1 with USP7 and CK2, as well as to identify the previously unstudied MCM-BP protein as a component of the MCM replicative helicase complex. We also use affinity column approaches to reveal specific interactions which can be either transient or stable in nature. This involves coupling increasing amounts of purified protein to a fix amount of resin and using this resin to isolate interacting proteins from cell extracts. We used this approach to identify interactions of EBNA1 with NAP1 and TAF-I and the interaction of USP7 with GMP synthetase. Most recently, we identify protein interactions by expressing triple FLAG-tagged proteins in human cells, using the tag to recover the protein and its binding partners. An adenovirus delivery system enables low level expression of the tagged protein in most cell lines and has allowed us to profile protein interactions in a variety of cells and conditions. By comparing data sets for multiple proteins, specific interactions can be distinguished from nonspecific ones. This approach was used to determine EBNA1 interactions in nasopharyngeal and gastric carcinoma cells in the context of latent and lytic EBV infection. It was also used to discover an interaction of CMV UL35 with the CUL4/DCAF1 E3 ubiquitin ligase complex.

Malik-Soni, N and Frappier, L 2012 Proteomic Profiling of EBNA1-Host Interaction in Latent and Lytic Epstein-Barr Virus Infections. J. Virol. 86, 6999-7002. PMID: 22496234

Salsman, J., Jagannathan, M., Paladino, P., Chan, P.-K., Dellaire, G., Raught, B. and Frappier, L. 2012 Proteomic Profiling of the Human Cytomegalovirus UL35 Gene Products Reveals a Role for UL35 in the DNA Repair Response. J. Virol. 86, 806-820. PMID: 22072767 (cover for vol 86 issue 4)

Sakwe, A.M., Nguyen, T., Athanasopoulos, V., Shire, K. and Frappier, L. 2007. Identification and characterization of a novel component of the human MCM complex. Mol Cell Biol. 27, 3044-3055. PMID:17296731

Hegde, N.R., Chevalier, M.S., Wisner, T.W., Denton, M.C., Shire, K. Frappier, L. and Johnson, D.C. 2006. The role of Bip in ER-associated degradation of major histocompatibility complex class I heavy chain induced by cytomegalovirus proteins. J. Biol. Chem. 281, 20910-20919. PMID: 16731524

Saridakis, V., Sheng, Y., Sarkari, F., Holowaty, M.N., Shire, K., Nguyen, T., Zhang, R.G., Liao, J., Lee, W., Edwards, A.M., Arrowsmith, C.H. and Frappier, L. 2005 Structure of the p53-binding domain of HAUSP/USP7 bound to Epstein-Barr Nuclear Antigen 1: Implications for EBV-mediate immortalization. Mol. Cell 18, 25-36. PMID:15808506

Holowaty, M.N., Zeghouf, M., Wu, H., Tellam, J., Athanasopoulos, V., Greenblatt, J. and Frappier, L. 2003. Protein profiling with Epstein-Barr nuclear antigen 1 reveals an interaction with the herpesvirus associated ubiquitin specific protease, HAUSP/USP7. J.Biol. Chem. 278, 29987-29994. PMID: 12783858

Shire, K., Ceccarelli, D.F., Avolio-Hunter, T.M. and Frappier, L. 1999 EBP2, a human protein that interacts with sequences of the Epstein-Barr nuclear antigen 1 important for plasmid maintenance. J. Virol. 73, 2587-2595. PMID: 10074103