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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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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).
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fig2: 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).

Mentions: For higher resolution mapping of the target sequences, we introduced deletions into the cytosolic tail of B7.2 and examined their effects on MIR2-mediated downregulation. Fig. 2 A depicts the deletions employed; as shown in Fig. 2 B, all deletion mutants were stably expressed on the cell surface at normal levels in the absence of MIR2 (pMX-pie control). In the presence of MIR2, mutant B7.2Δ1 was strongly downregulated, but mutant B7.2Δ2 was completely unaffected. Examination of the amino acid sequence of the two mutants revealed the presence of a lysine-rich segment that is present in B7.2Δ1 but absent in B7.2Δ2. Interestingly, mutation of these four contiguous lysine residues to alanine in the context of the intact cytosolic tail (mutant B7.2-7A) did not abolish MIR2-mediated endocytosis (Fig. 2 B). This indicates that there are likely to be multiple redundant signals in the cytosolic tail; in this connection, we note that B7.2-7A retains seven additional lysines in its intracytoplasmic domain.


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)

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).
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Related In: Results  -  Collection

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

fig2: 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).
Mentions: For higher resolution mapping of the target sequences, we introduced deletions into the cytosolic tail of B7.2 and examined their effects on MIR2-mediated downregulation. Fig. 2 A depicts the deletions employed; as shown in Fig. 2 B, all deletion mutants were stably expressed on the cell surface at normal levels in the absence of MIR2 (pMX-pie control). In the presence of MIR2, mutant B7.2Δ1 was strongly downregulated, but mutant B7.2Δ2 was completely unaffected. Examination of the amino acid sequence of the two mutants revealed the presence of a lysine-rich segment that is present in B7.2Δ1 but absent in B7.2Δ2. Interestingly, mutation of these four contiguous lysine residues to alanine in the context of the intact cytosolic tail (mutant B7.2-7A) did not abolish MIR2-mediated endocytosis (Fig. 2 B). This indicates that there are likely to be multiple redundant signals in the cytosolic tail; in this connection, we note that B7.2-7A retains seven additional lysines in its intracytoplasmic domain.

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