Limits...
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.

Show MeSH

Related in: MedlinePlus

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 −.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199318&req=5

fig1: 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 −.

Mentions: To better understand the mechanisms of MIR-mediated regulation, we mapped the determinants on the target host protein that are required for this regulation. For this purpose, we took advantage of the fact that MIR2 can downregulate the cell surface protein B7.2 (Ishido et al., 2000a; Coscoy and Ganem, 2001) (a costimulatory molecule important in T cell activation) but not its closely related homologue B7.1 (Fig. 1 A). (Interestingly, neither protein can be efficiently downregulated by MIR1 [Fig. 1 A; Ishido et al., 2000a; Coscoy and Ganem, 2001].) Accordingly, we constructed chimeras between B7.1 and B7.2 and tested their ability to be downregulated by MIR2 expression (Fig. 1 B). HeLa cells were stably transduced with each of the B7.1/B7.2 chimeras diagrammed in Fig. 1 B. These chimeras were found to be stable and were expressed at normal levels on the cell surface in the absence of MIR2 (unpublished data). Next, HeLa cell expressing each of these chimeras were stably transduced a second time with either an empty vector (pMX-pie) or an expression vector for MIR1 or MIR2 (here expressed as functional GFP fusion proteins with plasmids pB-MIR1/EGFP and pB-MIR2/EGFP, respectively) and levels of the chimera on the cell surface measured by flow cytometry. As summarized in Fig. 1 B, the ability of the chimeras to be downregulated by MIR2 mapped uniquely to the cytosolic tail of B7.2, which was both necessary and sufficient to confer downregulation. Interestingly, several of the chimeras acquired the ability to be downregulated by MIR1, a fact to which we shall return in the Discussion.


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 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 −.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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 −.
Mentions: To better understand the mechanisms of MIR-mediated regulation, we mapped the determinants on the target host protein that are required for this regulation. For this purpose, we took advantage of the fact that MIR2 can downregulate the cell surface protein B7.2 (Ishido et al., 2000a; Coscoy and Ganem, 2001) (a costimulatory molecule important in T cell activation) but not its closely related homologue B7.1 (Fig. 1 A). (Interestingly, neither protein can be efficiently downregulated by MIR1 [Fig. 1 A; Ishido et al., 2000a; Coscoy and Ganem, 2001].) Accordingly, we constructed chimeras between B7.1 and B7.2 and tested their ability to be downregulated by MIR2 expression (Fig. 1 B). HeLa cells were stably transduced with each of the B7.1/B7.2 chimeras diagrammed in Fig. 1 B. These chimeras were found to be stable and were expressed at normal levels on the cell surface in the absence of MIR2 (unpublished data). Next, HeLa cell expressing each of these chimeras were stably transduced a second time with either an empty vector (pMX-pie) or an expression vector for MIR1 or MIR2 (here expressed as functional GFP fusion proteins with plasmids pB-MIR1/EGFP and pB-MIR2/EGFP, respectively) and levels of the chimera on the cell surface measured by flow cytometry. As summarized in Fig. 1 B, the ability of the chimeras to be downregulated by MIR2 mapped uniquely to the cytosolic tail of B7.2, which was both necessary and sufficient to confer downregulation. Interestingly, several of the chimeras acquired the ability to be downregulated by MIR1, a fact to which we shall return in the Discussion.

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
Related in: MedlinePlus