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. 2008 Feb 5;105(5):1656-61.
doi: 10.1073/pnas.0707883105. Epub 2008 Jan 29.

Down-regulation of NKG2D and NKp80 ligands by Kaposi's sarcoma-associated herpesvirus K5 protects against NK cell cytotoxicity

Mair Thomas  1 , Jessica M Boname, Sarah Field, Sergey Nejentsev, Mariolina Salio, Vincenzo Cerundolo, Mark Wills, Paul J Lehner
Affiliations

Down-regulation of NKG2D and NKp80 ligands by Kaposi's sarcoma-associated herpesvirus K5 protects against NK cell cytotoxicity

Mair Thomas et al. Proc Natl Acad Sci U S A. .

Abstract

Natural killer (NK) cells are important early mediators of host immunity to viral infections. The NK activatory receptors NKG2D and NKp80, both C-type lectin-like homodimeric receptors, stimulate NK cell cytotoxicity toward target cells. Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV) down-regulates MHC class I molecules to avoid detection by cytotoxic T lymphocytes but renders cells susceptible to NK cell cytotoxicity. We now show that the KSHV immune evasion gene, K5, reduces cell surface expression of the NKG2D ligands MHC class I-related chain A (MICA), MICB, and the newly defined ligand for NKp80, activation-induced C-type lectin (AICL). Down-regulation of both MICA and AICL requires the ubiquitin E3 ligase activity of K5 to target substrate cytoplasmic tail lysine residues. The common MICA *008 allele has a frameshift mutation leading to a premature stop codon and is resistant to down-regulation because of the loss of lysine residues. K5-mediated ubiquitylation signals internalization but not degradation of MICA and causes a potent reduction in NK cell-mediated cytotoxicity. The down-regulation of ligands for both the NKG2D and NKp80 activation pathways provides KSHV with a powerful mechanism for evasion of NK cell antiviral functions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
K5 down-regulates specific MICA alleles, MICB and AICL. (A) U373, Jurkat, and HeLa cells were stained with anti-MICA (2C10), anti-MHC I (w6/32) and isotype control (dotted lines), after transduction with K5 GFP lentivirus (gray) or GFP lentivirus (black). (B) C1R-MICB stable cell lines were transduced as above and stained with anti-MICA/B (6D4). (C) Sequence alignment of part of the transmembrane (gray) and cytoplasmic tails of MICA alleles, MICB, and AICL. (D) U937 AICL-myc stable cell lines were transduced as above and stained with anti-c-myc (9E10) and anti-MHC I (w6/32) mAb.
Fig. 2.
Fig. 2.
Down-regulation of MICA and AICL by K5 is RING- and lysine-dependent. (A) C1R-MICA (Left and Center) or U937 AICL-myc cells (Right) were stained with anti-MHC I (w6/32), anti-MICA (2C10), anti-c-myc (9E10), and isotype control (dotted lines), after transduction with K5 W/I GFP (gray) or control GFP lentivirus (black). (B) MICA cytoplasmic tail mutants stably expressed in C1R cells were transduced with K5 GFP lentivirus to give ≈50% transduction levels. Cells were stained with anti-MICA (2C10) mAb. Sequences show cysteine and lysine residues of the MICA cytoplasmic tail, with mutations underlined. Lysine (K) was mutated to arginine (R); cysteine (C) to serine (S). (C) U937 expressing AICL lacking cytoplasmic lysines (AICL-Kless-myc) were stained with anti-c-myc (9E10) and anti-MHC I (w6/32) mAb after transduction with K5 GFP (gray) or control GFP lentivirus (black).
Fig. 3.
Fig. 3.
K5 ubiquitylates MICA and targets AICL for endolysosomal degradation. (A) K5 promotes ubiquitylation of MICA. The indicated constructs were transiently expressed in COS-7 cells. Cell lysates were immunoprecipitated with anti-GFP mAb, proteins were separated, and membranes were probed with anti-ubiquitin (P4D1) mAb. First lane is 2% of cell lysate from MICA transfection. Ten percent of cell lysates were run on SDS/PAGE and probed for GFP-myc-MICA (9E10) mAb. (B and C) Pulse–chase analysis of MICA in the presence of K5. Cells were radiolabeled for 30 min and chased for the indicated time periods. MICA protein (B) or tagged and untagged MICA proteins (C) were immunoprecipitated with anti-MICA (2C10) mAb from 1% Triton X-100 lysates and resolved by SDS/PAGE. (D) Immunofluoresence of GFP-MICA in HeLa cells in the presence or absence of K5. (Scale bar: 10 μm.) (E) Myc-tagged AICL-expressing U937 cells (U937-AlCL-myc) were transduced with lentiviral K5 or K5W/I, and 72 h later they were treated with 6 μM MG132 or 50 nM concanamycin A (CCM A). Solubilized lysates were PNGase F-treated, and separated proteins were probed with anti-c-myc (8E10) or anti-calnexin (CNX) (AF8) mAb.
Fig. 4.
Fig. 4.
Lytic KSHV infection decreases cell surface MICA levels in BC3 cells. (A) Induction of lytic KSHV replication. (Left) RT-PCR of KSHV K5 expression after 2 mM sodium butyrate (NaBu) treatment of BC3 cells. (Right) Acetone/methanol-permeabilized BC3 cells treated as in the Left were stained with anti-ORF-59 mAb. (B and C) BC3 stably transudced with lentiviral wild-type MICA (B) or MICA lacking cytoplasmic tail lysine residues (MICA-Kless) (C) were stained with anti-MICA (2C10), anti-MHC I (w6/32), anti-ICAM-1 (15.2), and isotype control (dotted lines) before (solid lines) or after (gray) NaBu treatment. (D) Cytofluorometric analysis of MHC I and ICAM-1 after sodium butyrate treatment of the KSHV negative C1R cells, treated as in A.
Fig. 5.
Fig. 5.
K5 protects cells from NK cytotoxicity by down-regulation of MICA and AICL. (A) Cytofluorometric analysis of C1R clones used in NK cytotoxicity assays. Cell surface staining (as Fig. 1) of C1R (bold line), stable C1R clones expressing MICA or MICA lacking cytoplasmic tail lysine residues (MICA-Kless) transduced with K5 GFP (gray) or GFP lentivirus (black). Dotted lines show isotype control. (B) 51Cr release assay (percentage specific lysis) with polyclonal NK cells against C1R clones. Error bars give the SE of the six replicates at each point. The results are representative of three independent experiments with polyclonal NK cells from unrelated donors. (C) 51Cr release assay using IgG1- or NKp80-specific mAb blocked NK cell line against control or K5-expressing U937 cells. Error bars give the SE of the six replicates at each point.
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