A novel class of herpesvirus-encoded membrane-bound E3 ubiquitin ligases regulates endocytosis of proteins involved in immune recognition

Abstract

Kaposi's sarcoma-associated herpesvirus encodes two transmembrane proteins (modulator of immune recognition [MIR]1 and MIR2) that downregulate cell surface molecules (MHC-I, B7.2, and ICAM-1) involved in the immune recognition of infected cells. This downregulation results from enhanced endocytosis and subsequent endolysosomal degradation of the target proteins. Here, we show that expression of MIR1 and MIR2 leads to ubiquitination of the cytosolic tail of their target proteins and that ubiquitination is essential for their removal from the cell surface. MIR1 and MIR2 both contain cytosolic zinc fingers of the PHD subfamily, and these structures are required for this activity. In vitro, addition of a MIR2-glutathione S-transferase (GST) fusion protein to purified E1 and E2 enzymes leads to transfer of ubiquitin (Ub) to GST-containing targets in an ATP- and E2-dependent fashion; this reaction is abolished by mutation of the Zn-coordinating residues of the PHD domain. Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

Figure 1.

The intracytoplasmic region of B7.2 is required for MIR2-mediated downregulation. (A) BJAB cells were stably transduced with PMX-pie (the control vector), pBP-MIR1/EGFP, or pBP-MIR2/EGFP–derived retroviral vectors. Cell surface expression of B7.1 and B7.2 was monitored by flow cytometry. (B, left) Schematic representation of chimeras between B7.1 and B7.2. (B, right) HeLa cells stably expressing the indicated chimeric proteins were stably transduced a second time with either PMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. Surface expression of the chimeric proteins was analyzed by flow cytometry. Presence or absence of downregulation is indicated respectively as + or −.

Figure 2.

A small motif within B7.2 intracytoplasmic region is required for MIR2-mediated downregulation. HeLa cells stably expressing truncation mutants of B7.2 (indicated in A) were transduced a second time by either PMX-pie or pBP-MIR2/EGFP. Cell surface expression of the indicated B7.2 constructs was analyzed by flow cytometry (B).

Figure 3.

The presence of lysines in the MHC-I intracytoplasmic region is required for MIR2-mediated downregulation. Lysine 332 and/or lysine 337 within the HLA.B7 cytoplasmic region were mutated to arginine, and the resulting constructs was stably expressed in BJAB cells. Cells were then transiently transduced a second time with either the control vector pMX-pie or pB-MIR2/EGFP, a vector expressing a functional MIR2–GFP fusion protein. Cell surface expression of HLA.B7 wt or mutant molecules was analyzed by flow cytometry.

Figure 4.

MIR1 and MIR2 mediate ubiquitination of their targets. wt HeLa cells or HeLa cells expressing B7.2 were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. B7.2 molecules (right) or MHC-I molecules (left) were immunoprecipitated, and their ubiquitination status was determined by Western blot analysis using an anti-Ub antibody.

Figure 5.

Addition of lysine into B7.1 enable its downregulation. (A) Sequence of the TM and intracytoplasmic regions of B7.1 (top line) and B7.1KKK mutant (bottom line). The mutant residues within the cytoplasmic region of B7.1KKK are indicated in boldface. (B) B7.1 wt and KKK mutant constructs were stably expressed in HeLa cells. Cells were transduced a second time by pBP-MIR2/EGFP, and surface expression of the B7.1 molecules was analyzed by flow cytometry. (C) Cells expressing B7.1 wt or KKK mutant proteins were stably transduced by pMX-pie, pBP-MIR1/EGFP, or pBP-MIR2/EGFP. From each set of transductants, B7.1 molecules were immunoprecipitated and analyzed by Western blot using an anti-Ub antibody.

Figure 6.

The MIR2 PHD domain can mediate ATP/E2-dependent ubiquitination in vitro. In vitro ubiquitination reactions were performed as described in Materials and methods. In the complete reaction mix, 10 μg of GS-bound GST, GST-MIR2 wt PHD domain, and GST-MIR2 mutant PHD domain were added to a cocktail of 10 mM ATP, 2 mg Ub, 40 ng of E1, and 1 μg of the E2 UbcH5a. In the remaining lanes, individual components of the full reaction mix were omitted as indicated above each lane. After the reaction, GS-bound material was recovered by centrifugation, eluted with SDS sample buffer, and examined by Western blotting with anti-Ub antibody.

Figure 7.

A critical domain within the transmembrane region of human MHC-I is required for MIR2-mediated downregulation. (A) Cells stably expressing the control vector or MIR2 were transiently transfected with the human or the mouse MHC-I molecule (HLA.B7 and H2 Dd, respectively). The number of cell expressing the transfected MHC-I at the surface was measured by flow cytometry. (B) Chimeric proteins between human (bold) and mouse (underlined) MHC-I were engineered, and their susceptibility to MIR2-mediated downregulation was analyzed as above.

Figure 8.

A model for MIR2 function as an E3 Ub ligase. MIR2 TM domains mediate recognition of their target molecules TM regions in the plane of the membrane, whereas its cytosolic PHD domain recruits one or more E2 Ub-conjugating enzymes and brings them into proximity with the cytosolic tail of the target protein, facilitating the transfer of Ub to key lysine residues.