Trapping mammalian protein complexes in viral particles

Abstract

Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes.

Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector–Host Protein–Protein Interactions

“…The development of techniques such as BioID and Virotrap (partially) alleviated these shortcomings by means of covalent modification and avoiding cell lysis, respectively [91,97]. More specifically, BioID on the one hand takes advantage of marking PPIs by means of the covalent attachment of biotin, a stable modification that is retained during cell lysis conditions, opposed to the necessity for the maintenance of interactions upon cell lysis in case of AP-MS analysis.…”

“…False positive interactions with GAG can be eliminated by comparing to a control setup. An appropriate VLP control setup, i.e., a representation of the complete VLP proteome in absence of the effector, is indispensable for Virotrap MS-based analysis as VLPs exhibit their own characteristic proteome content [97]. In the context of appropriate BioID controls, it is noteworthy that by using the power of genome editing, Vandemoortele et al designed a method to obtain isogenic cell lines expressing T2A-BirA* bait fusion or its corresponding BirA* control protein, both under the control of the endogenous bait promoter [105].…”

“…By allowing stringent IP conditions or the direct identification of biotinylated sites [96], BioID benefits from the covalent biotin modification to circumvent false positive or negative PPIs due to cell lysis and nonspecific interactions [87]. Conversely, Virotrap eliminates the need for cell lysis altogether by making use of the vesicle-forming properties of the human immunodeficiency virus type 1 (HIV-1) GAG protein (55 kDa) [97]. Expression of an N-terminally GAG-tagged bait results in the budding of so-called virus-like particles (VLPs)-wherein interacting proteins are "trapped".…”

“…In this context, it is noteworthy that VLPs have successfully been isolated from plant tissue expressing full-length HIV-1 GAG [100], so application of Virotrap in plants could be an interesting subject of future investigation. Virotrap has fruitfully assisted in selecting candidate interactions for human ring finger protein 41 (RNF41) and proved to be orthogonal to BioID and AP-MS in that regard [97,99]. Furthermore, we recently obtained proof-of-concept data that Virotrap can be used for studying EH-PPIs by fusing the C-terminus of GAG to Salmonella T3Es, by the identification of known host interactors (unpublished data).…”

Targeting Persistent Human Papillomavirus Infection

“…Additional studies illustrate that targeting the interaction between E2 and other identified host cellular proteins could be an efficient therapeutic strategy to eliminate persistent infection [ 47 , 48 , 64 , 65 , 66 ], providing a framework with which to approach developing HPV-specific therapeutics. Highly specific visualization/detection techniques such as bimolecular fluorescence complementation and Mammalian Protein–Protein Interaction Trap (MAPPIT) will likewise prove integral to identifying these synthetic and naturally occurring small molecule inhibitors [ 57 , 90 , 91 , 92 ].…”