Advances in Rapid Isolations of Protein Complexes:Revealing the Dynamic Viral-Host Interactome

Abstract

Isolation of protein complexes, if performed appropriately, can provide an invaluable shortcut to uncovering protein interactions and to gaining clues towards their biological functions. The literature is, justifiably so, replete with approaches designed for the study of protein interactions. Advances are, nevertheless, highly desirable. An ideal isolation would maintain the protein complex as close as possible to its original state in the cell. To date, achieving this ideal isolation remains a challenge. The identification of transient or weak interacting partners and the stoichiometry within a complex present difficulties. We have recently reported an approach for the rapid and efficient isolation of protein complexes. We demonstrated that a combination of cryogenic-based cell lysis and fast immunoaffinity purifications helps to maintain interactions, minimizing nonspecific associations and maximizing the recovery of transiently interacting partners. This presentation will underline some of the technical aspects that were found to be important in studying macromolecular assemblies. Highlights will be shown from our studies of the dynamic viral-host protein interactions during the course of infections with Sindbis and human ytomegalovirus (CMV) Our results revealed cellular pathways utilized by these viruses to manipulate host systems. For example, our studies of the Sindbis virus, an Alphavirus genus member that in humans causes arthritis, indicated that Sindbis may utilize G3BP, at least in part, to interfere with the host cellular responses to stress. Most recently, we generated a library of 155 HCMV viruses, each containing a different C-terminal epitope tagged ORF, and initiated a comprehensive study of the HCMV interactions. One important finding from our interactome data was that the HCMV UL38 protein interacts with the tuberous sclerosis protein complex (SC1/2) to block its negative regulation of mTORC1, a growth regulatory pathway, and facilitate viral replication.