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

Coscoy L, Sanchez DJ, Ganem D - J. Cell Biol. (2001)

Bottom Line: 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.Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143, USA.

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.

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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.
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fig3: 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.

Mentions: To better define the contribution of lysine residues to MIR2-mediated endocytosis, we examined the regulation of the class I MHC allele HLA-B7 whose cytosolic tail harbors only two lysines (at positions 332 and 337). Each of these was singly mutated to arginine (to minimize changes in charge or basicity); in addition, we constructed the corresponding double mutant. Each construct was first transduced into BJAB cells; the resulting neomycin-resistant cell population was then transduced a second time with either pMX-pie (vector control) or pB-MIR2/EGFP, a vector expressing a functional MIR2-GFP fusion protein. 2 d later, cells were examined by flow cytometry for GFP and surface MHC-I expression. As shown in Fig. 3, cells expressing MIR2-GFP strongly downregulated surface expression of wild-type (wt) MHC-I and each of the single lys/arg mutants; however, the double mutant was completely refractory to MIR2 regulation


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

Coscoy L, Sanchez DJ, Ganem D - J. Cell Biol. (2001)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199318&req=5

fig3: 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.
Mentions: To better define the contribution of lysine residues to MIR2-mediated endocytosis, we examined the regulation of the class I MHC allele HLA-B7 whose cytosolic tail harbors only two lysines (at positions 332 and 337). Each of these was singly mutated to arginine (to minimize changes in charge or basicity); in addition, we constructed the corresponding double mutant. Each construct was first transduced into BJAB cells; the resulting neomycin-resistant cell population was then transduced a second time with either pMX-pie (vector control) or pB-MIR2/EGFP, a vector expressing a functional MIR2-GFP fusion protein. 2 d later, cells were examined by flow cytometry for GFP and surface MHC-I expression. As shown in Fig. 3, cells expressing MIR2-GFP strongly downregulated surface expression of wild-type (wt) MHC-I and each of the single lys/arg mutants; however, the double mutant was completely refractory to MIR2 regulation

Bottom Line: 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.Thus, MIR2 defines a novel class of membrane-bound E3 Ub ligases that modulates the trafficking of host cell membrane proteins.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Microbiology, University of California Medical Center, San Francisco, CA 94143, USA.

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.

Show MeSH