Quantitative Membrane Proteomics Reveals New Cellular Targets of Viral Immune Modulators

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Abstract
Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma–associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif–deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins. Viral immune modulators often target multiple cellular proteins for destruction. Presumably, this strategy enables viral pathogens to optimize evasion of multiple immune responses. To systematically identify such host cell targets in an unbiased fashion, Bartee et al. applied recently developed quantitative proteomics methods to identify novel targets for K5. K5 belongs to a family of viral ubiquitin ligases found in gamma-herpesviruses and poxviruses that target multiple cellular transmembrane proteins for destruction. Using stable isotope labeling combined with tandem mass spectrometry, the authors compared the abundance of proteins in membrane preparations from cells that expressed K5 to that in cells without K5. In their experiments, three novel membrane proteins (BST-2, Syntaxin-4, and ALCAM) were consistently found in lower abundance in K5-expressing cells. Importantly, the authors were able to confirm the K5-dependent downregulation of all of these proteins in independent experiments and by independent methods. ALCAM was chosen for a more in-depth analysis to firmly demonstrate that this protein is downregulated by K5 in a manner similar to other known targets. This proof-of-principle study demonstrates that novel targets of viral immune modulators can be identified with quantitative proteomics.