IL-18 binding and inhibition of interferon gamma induction by human poxvirus-encoded proteins

Abstract

Molluscum contagiosum virus (MCV) is a common, human poxvirus that causes small papular skin lesions that persist for long periods without signs of inflammation. Previous studies revealed that MCV encodes a family of proteins with homology to mammalian IL-18 binding proteins. IL-18 is a proinflammatory cytokine that induces synthesis of interferon gamma, activates NK cells, and is required for a T-lymphocyte helper type 1 response. We expressed and purified the proteins encoded by the MC53L and MC54L genes of MCV, as well as their human and murine homologs. All four recombinant proteins were able to bind with high affinity to human and murine IL-18 molecules and inhibited IL-18 mediated interferon gamma production in a dose-dependent manner. The pirating of IL-18 binding proteins by poxviruses and their use as decoy receptors is consistent with the critical role of IL-18 in defense against virus infections and provides a mechanism for evasion of the immune system by MCV.

Figure 1

Purification of recombinant proteins. Recombinant MC53L, MC54L, huIL-18BP, and muIL-18BP were purified by metal affinity chromatography, were analyzed by SDS/PAGE, and were detected by Coomassie blue staining (Left) or by chemiluminescence after Western blotting with a mAb to the polyhistidine tag (Right). Hu, huIL-18BP; Mu, muIL-18BP; 53, MC53L; 54, MC54L. The positions and masses in kilodaltons of protein markers are shown on the extreme left.

Figure 2

Binding of IL-18 to SDS/PAGE-purified recombinant proteins. Metal affinity resin-purified recombinant proteins were treated with SDS in the presence or absence of 2-mercaptoethanol and were analyzed by SDS/PAGE. The proteins were transferred to a membrane and were incubated successively with rhIL-18 and an hIL-18 mAb. Bound mAb was detected by chemiluminescence (Left). The membranes then were stripped and incubated with a mAb to the histidine tag (Right). His-tag, 6-histidine tag; other abbreviations are as in Fig. 1.

Figure 3

Cross-linking of IL-18 to native recombinant proteins. Metal affinity resin-purified recombinant proteins were incubated with rhIL-18 in the presence of bis(sulfosuccinimidyl) suberate and then were analyzed by SDS/PAGE. The proteins were transferred to a membrane and then were incubated with polyclonal Ab to hIL-18. The Ab was detected by chemiluminescence. SA, bovine serum albumin; OA, ovalbumin; other abbreviations are as in Fig. 1.

Figure 4

Detection of IL-18 binding to recombinant proteins by using surface plasmon resonance. Metal affinity resin-purified recombinant proteins were individually immobilized on a CM5 chip with the standard amine coupling procedure. Injection of rhIL-18 started at 0 seconds and stopped at 750 seconds. For huIL-18BP, rhIL-18 concentrations used were 0.35, 0.7, 3.5, 7, 10.5, 17.5, 21, and 24.5 nM. For MC54L, rhIL-18 concentrations used were 3.5, 7, 10.5, 14, 17.5, 21, 24.5, and 28 nM. For MC53L, rhIL-18 concentrations used were 7,14, 28, 42, 56, 70, 84, 112, and 140 nM. The colored and black lines are the actual responses in RU and globally fitted curves, respectively. The residual responses, below each set of curves, represent deviations of the actual responses from the fitted curves. The rms deviations were 0.749, 0.369, and 0.236 for huIL-18BP, MC54L, and MC54L, respectively.

Figure 5

Inhibition of IFN-γ induction. KG-1 cells were incubated with 20 ng/ml TNF-α and either 10 ng/ml of rhIL-18 or rhIL-18 premixed with the indicated amounts of metal affinity resin-purified recombinant protein. After 24 h at 37°C, clarified supernatants were assayed for human IFN-γ.