Yaba monkey tumor virus encodes a functional inhibitor of interleukin-18

Abstract

Interleukin-18 (IL-18) is a critical proinflammatory cytokine whose extracellular bioactivity is regulated by a cellular IL-18 binding protein (IL-18BP). Many poxviruses have acquired variants of this IL-18BP gene, some of which have been shown to act as viral virulence factors. Yaba monkey tumor virus (YMTV) encodes a related family member, 14L, which is similar to the orthopoxvirus IL-18BPs. YMTV 14L was expressed from a baculovirus system and tested for its ability to bind and inhibit IL-18. We found that YMTV 14L bound both human IL-18 (hIL-18) and murine IL-18 with high affinity, at 4.1 nM and 6.5 nM, respectively. YMTV 14L was able to fully sequester hIL-18 but could only partially inhibit the biological activity of hIL-18 as measured by gamma interferon secretion from KG-1 cells. Additionally, 17 hIL-18 point mutants were tested by surface plasmon resonance for their ability to bind to YMTV 14L. Two clusters of hIL-18 surface residues were found to be important for the hIL-18-YMTV 14L interaction, in contrast to results for the Variola virus IL-18BP, which has been shown to primarily interact with a single cluster of three amino acids. The altered binding specificity of YMTV 14L most likely represents an adaptation resulting in increased fitness of the virus and affirms the plasticity of poxviral inhibitor domains that target cytokines like IL-18.

FIG. 1.

YMTV 14L binds hIL-18 (A) and mIL-18 (B) with nanomolar affinity. YMTV 14L was immobilized to a CM5 chip and was analyzed on a BIAcore2000. (A) hIL-18 was injected over the chip at indicated concentrations for 120 s. (B) mIL-18 was injected over the chip at indicated concentrations for 120 s. Both sets of curves were globally fitted to a 1:1 binding model (BIAevaluation).

FIG. 2.

Production of IFN-γ is inhibited by AcY14L. AcY14L was added at various concentrations (nanograms/milliliter) to wells containing TNF-α (5 ng/ml) and IL-18 (10 ng/ml). KG-1 cells were added, and 24 h later, IFN-γ was assayed in duplicate by ELISA. +, present; −, absent.

FIG. 3.

Sequestration of hIL-18 with AcY14L-M/H. Protein A/G beads were incubated with anti-penta-His antibody and supernatants from insect cells infected with either AcY14L-M/H or AcNPVpolh⁻ (negative control). Beads were then mixed at the ratios (AcY14L-M/H/control) indicated below the sixth, seventh, and eighth bars, and hIL-18 (100 ng/ml) was added to the beads. TNF (5 ng/ml) and supernatants at a 1 in 10 dilution were added to KG-1 cells. IFN-γ was assayed by ELISA. Error bars show standard deviations.

FIG. 4.

The IL-18 binding site for YMTV IL-18BP overlaps with both hIL-18BP and hIL-18Rα. YMTV 14L was immobilized to a CM5 chip, 100 nM hIL-18 was incubated with the indicated concentrations of either hIL-18BP or hIL-18Rα for 30 min, and the solution was then injected over the sensor chip surface. The maximum level of binding is shown in relative units (RU).

FIG. 5.

YMTV 14L binding is influenced by several residues located on one face of hIL-18. Mutated residues are displayed in spacefill. Residues are colored based on the decrease (n-fold) in affinity of the mutant compared to that of wild-type hIL-18. Mutations R13A, D17A, D35A, and M33A are located on residues in site I; all other residues shown belong to site II. Residues in site III are not shown.

FIG. 6.

YMTV 14L binds to hIL-18 in a more promiscuous manner than the VARV IL-18BP. Values for the graph were taken from reference and from the current study. The change (n-fold) with respect to the affinity of the wild-type IL-18 is shown.