Zoonotic orthopoxviruses encode a high-affinity antagonist of NKG2D

Abstract

NK and T lymphocytes express both activating and inhibiting receptors for various members of the major histocompatibility complex class I superfamily (MHCISF). To evade immunologic cytotoxicity, many viruses interfere with the function of these receptors, generally by altering the displayed profile of MHCISF proteins on host cells. Using a structurally constrained hidden Markov model, we discovered an orthopoxvirus protein, itself distantly class I-like, that acts as a competitive antagonist of the NKG2D activating receptor. This orthopoxvirus MHC class I-like protein (OMCP) is conserved among cowpox and monkeypox viruses, secreted by infected cells, and bound with high affinity by NKG2D of rodents and humans (K(D) approximately 30 and 0.2 nM, respectively). OMCP blocks recognition of host-encoded ligands and inhibits NKG2D-dependent killing by NK cells. This finding represents a novel mechanism for viral interference with NKG2D and sheds light on intercellular recognition events underlying innate immunity against emerging orthopoxviruses.

Figure 1.

OMCP is likely a secreted class I–like protein. (A) Multiple alignment of the four OMCP variants. Cysteines (C) and potential N-linked glycosylation sites (closed circles) are indicated. (B) Backbone model of OMCP_BR derived by threading through the RAE1β structure (closed squares, cysteines). Similar results were obtained for OMCP_MPX. (C) Immunoblot of culture media conditioned by mock-infected and CPXV-BR–infected B-SC-1 cells suggests secretion of OMCP. (D) Immunoblot showing that brefeldin A, but not its ethanol vehicle, prevents the appearance of OMCP_BR in supernatants from infected OP9 cells (far right lane vs. second lane from right). Brefeldin A also causes the accumulation of OMCP in lysates of treated cells (third lane from left).

Figure 2.

OMCP binds huNKG2D and muNKG2D. (A) Ba/F3 cells expressing EGFP only (top), huNKG2D (middle), or muNKG2D (bottom) were treated with nothing, SA-PE, or fluorescent tetramers. (B) Ba/F3 cells transduced with huNKG2D or muNKG2D were incubated with anti-NKG2D mAb, isotype-matched mAb, irrelevant soluble protein, or soluble ligand before staining with OMCP_BR (rows 1 and 3) or OMCP_MPX (rows 2 and 4) tetramers. (C) Ba/F3 cells transduced either with Thy1.1 (top) or OMCP-Thy1.1 (middle and bottom) were treated with SA-APC or fluorescent tetramers. Untransduced (nongreen) Ba/F3 cells were included as internal standards in A–C. Transduction vectors are dicistronic, with EGFP in the second cistron. (D, 1) SPR assay of OMCP_BR (0, 2, 5, 10, 20, 50, 100, 200, 500, and 1,000 nM) binding to immobilized muNKG2D. The horizontal bar represents the OMCP injection, and the star represents the time point used for K_D fitting. (D, 2) Nonlinear curve fitting of steady-state SPR data reveals high affinity for muNKG2D and a good coefficient of determination for the fit. (D, 3) One representative experiment out of four showing kinetic data (black lines) and global fit (gray lines) of OMCP_BR (5, 10, 20, 50, and 100 nM) binding to muNKG2D. (D, 4) One representative experiment out of six showing kinetic data (black lines) and global fit (gray lines) of OMCP_BR (0.32, 0.63, 1.25, and 2.5 nM) binding to huNKG2D. Note the very different dissociation kinetics between the data in 3 and 4.

Figure 3.

OMCP binds to mouse and human leukocyte subpopulations. (A) B6 splenocytes were stained with mAbs against NK markers and OMCP_BR tetramers. OMCP_BR staining of cells in each gate is indicated in histograms. Each histogram group shows unstained cells (lines 1), cells stained with SA-APC alone (lines 2), irrelevant tetramers (lines 3), OMCP_BR tetramers (lines 4), or OMCP_BR tetramers after pretreatment with 50 μg/ml of irrelevant hamster IgG (lines 5), equimolar anti-muNKG2D (lines 6), 9.5 μM of irrelevant protein (lines 7), or equimolar MULT1 (lines 8). (B) Human PBMCs were stained with NK or T cell marker mAbs and were tested for OMCP binding, as in A, for lines 1–4. Pretreatments with 50 μg/ml of mouse IgG1 (lines 5) or equimolar anti-huNKG2D mAb (lines 6) were also performed before staining with tetramers. Representative experiments are shown (n = 4). Vertical lines in histograms represent the 99th percentile of staining for irrelevant tetramer- stained cells.

Figure 4.

OMCP inhibits muNKG2D-dependent killing by activated NK cells but does not down-regulate NKG2D. (A) B6 IL2-activated NK cells were used as cytotoxic effectors against transduced RMA cells in 4-h ⁵¹Cr release assays at effector/target ratios of 1:1, 3:1, or 10:1. There was minimal killing of EGFP-only transductants (open squares). There was effective NKG2D-dependent killing of H60 transductants (circles), but 1 μM of irrelevant protein failed to inhibit NKG2D-dependent killing (X). However, OMCP_BR at 1 (closed squares), 0.5 (diamond), 0.25 (closed triangle), and 0.1 μM (open triangle) did effectively inhibit NKG2D-dependent killing. Error bars were omitted to avoid crowding; coefficients of variation for all data points were <10%. (B) B6 splenocytes were cultured for 24 h with 1 μM of irrelevant protein (lines 1–3) or 1 μM OMCP_BR (lines 4–6) and stained with nothing (lines 1 and 4), isotype-matched mAb (lines 2 and 5), or anti-muNKG2D (lines 3 and 6). All histograms are gated on live, CD3⁻, NK1.1⁺ cells. No differences in the scatter properties or viability of cells was noted (not depicted). (C) Immunoblotting of OMCP was performed with known amounts of recombinant protein (left blot) or CPXV-infected OP9 supernatants (right blot). The former was used to quantitate the latter using a linear fit of OMCP densitometry to quantity (plot), as detailed in Materials and methods. One experiment out of three is shown. Xs represent mean calibration data, dots represent supernatant data, the continuous line represents linear fit, and the dotted lines bound the 95% confidence interval. (D) Ba/F3 cells were treated either with mock viral (left) or CPXV-BR (right) preparations for 24 h. Cells were stained with nothing (lines 1), SA-PE (rows 2), control tetramers (rows 3), or muNKG2D tetramers alone (rows 4), as well as with 7-amino-actinomycin D. NKG2D binds infected cells, indicating up-regulation of NKG2D ligands by CPXV-BR infection. The percentage of positive cells corresponds to the percentage of productively infected cells (not depicted). Vertical lines in the histograms (B and D) represent the 99th percentile of staining for irrelevant tetramer-stained cells.