Quantitative Membrane Proteomics Reveals New Cellular Targets of Viral Immune Modulators

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-membranespanning 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.

Vpu enhances HIV-1 virus release in the absence of Bst-2 cell surface down-modulation and intracellular depletion

“…Bst-2 expression was inducible by IFN treatment in 293T or HT1080 cells, consistent with the previous observation that cell lines that did not normally require Vpu for efficient virus release became Vpu-dependent upon IFN treatment (22). Although Vpu had no overt effects on ectopically expressed Bst-2 levels and cellular distribution (29), Vpu clearly downregulated cell surface expression of Bst-2 in 293T cells (23) and caused a reduction of endogenous Bst-2 expression in HeLa cells (30). Randomization of the Vpu TM domain abolished the ability of Vpu to overcome the inhibitory effects of Bst-2 on virus release (23), consistent with our previous data on the importance of the Vpu TM domain for this effect (17).…”

“…1 A, lane 4). Of note, Bst-2 appears as multiple bands on the gel with estimated sizes of 25-35 kDa presumably because of glycosylation as reported (30). FACS analysis revealed low but detectable surface expression of Bst-2 in untreated 293T cells that was strongly up-regulated after IFN␣ treatment [supporting information (SI) Fig.…”

“…The recent identification of Bst-2 as an IFN-inducible host factor whose expression renders cells restrictive for vpu-defective viruses (29) and whose intracellular expression and surface presentation are reduced by the HIV-1 Vpu protein (23,30) has spurred new interest in the mechanism of Vpu-mediated virus release. The Vpu TM domain plays a critical function in the process of virus release although its exact function remains a mystery.…”

Autoregulation of MARCH1 Expression by Dimerization and Autoubiquitination

“…Whereas MARCH1 downregulates all isotypes of MHC II but not MHC I, MARCH9 was shown by other groups to act on MHC I and only on the DQ isotype of MHC II (25,49). In our overexpression system, however, we found that MARCH9 was also active on HLA-DR. We reasoned that by introducing portions of MARCH1 of increasing length into MARCH9, one should be able to delineate functional regions.…”

The E3 ubiquitin ligase MARCH8 negatively regulates IL-1β-induced NF-κB activation by targeting the IL1RAP coreceptor for ubiquitination and degradation

“…Ten mammalian MARCH gene products have been recognized, and the majority shares a similar structure, including an N-terminal C4HC3-type RING finger (RING-CH finger) and two or more C-terminal transmembrane spans (22,23). Several members of this novel E3 ubiquitin ligase family have been shown to ubiquitinate and down-regulate transmembrane proteins, such as major histocompatibility complex class I and II, B7-2, CD166, and ICAM-1 (24)(25)(26)(27)(28).…”