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. 2008 Aug;43(2):220-8.
doi: 10.1016/j.cyto.2008.05.016. Epub 2008 Jul 17.

Monkeypox virus viral chemokine inhibitor (MPV vCCI), a potent inhibitor of rhesus macrophage inflammatory protein-1

John M Jones  1 , Ilhem Messauodi, Ryan D Estep, Beata Orzechowska, Scott W Wong
Affiliations

Monkeypox virus viral chemokine inhibitor (MPV vCCI), a potent inhibitor of rhesus macrophage inflammatory protein-1

John M Jones et al. Cytokine. 2008 Aug.

Abstract

Monkeypox virus (MPV) is an orthopoxvirus with considerable homology to variola major, the etiologic agent of smallpox. Although smallpox was eradicated in 1976, the outbreak of MPV in the U.S. highlights the health hazards associated with zoonotic infections. Like other orthopoxviruses, MPV encodes a secreted chemokine binding protein, vCCI that is abundantly expressed and secreted from MPV infected cells. EMSA data shows vCCI efficiently binds rhesus MIP-1alpha (rhMIP-1alpha) at near one to one stoichiometry. In vitro chemotaxis experiments demonstrate that vCCI completely inhibits rhMIP-1alpha mediated chemotaxis, while in vivo recruitment assays in rhesus macaques using chemokine-saturated implants show a decrease in the number of CD14(+) cells responding to rhMIP-1alpha when vCCI is present, suggesting vCCI is effectively inhibiting chemokine function both in vitro and in vivo. More importantly, we demonstrate that vCCI can diminish the severity of the acute phase and completely inhibit the relapsing phase of experimental allergic encephalomyelitis (EAE) disease. These data represent the first in vitro and in vivo characterization of vCCI emphasizing its function as a potent inhibitor of rhMIP-1alpha. Furthermore, the ability of vCCI to inhibit relapsing EAE disease represents a novel therapeutic approach for treating chemokine-mediated diseases.

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Figures

Fig. 1
Fig. 1
Amino acid comparison of MPV vCCI to vCCIs encoded by variola virus (VARV), cowpox virus (CPV), rabbitpox virus (RPV), and vaccinia Copenhagen strain (VV COP). Alignments were preformed with ClustalW using Blosum scoring matrix. Dark shaded boxes indicate either: (1) identical residues, or (2) unique residues to MPV vCCI. Lightly shaded boxes represent similar residues to MPV vCCI.
Fig. 2
Fig. 2
Expression of MPV vCCI during MPV infection. (A) Immunofluorescence analysis on MPV infected (panel A) or mock infected (panel B) BSC40 cells fixed at 24 h.p.i. Cells were stained with a mouse anti-vCCI monoclonal antibody, followed by a biotinylated horse anti-mouse secondary antibody, and visualized using an alexa-488 conjugated to streptavidin. Nuclear staining was preformed using Hoescht stain. All images were taken with 20× objective. (B) Secretion of MPV vCCI during MPV infection. Samples of supernatants and lysates from MPV (lanes 1 and 3) and Mock (lanes 2 and 4) infected BSC40 cells were resolved on 4–12% Bis–Tris NuPAGE® gels and transferred to PVDF. Western blot analysis was preformed using a mouse anti-vCCI monoclonal antibody (3D1) and an HRP-conjugated goat anti-mouse secondary antibody. Purified MPV vCCI was used as a positive control (lane 5).
Fig. 3
Fig. 3
MPV vCCI binds rhesus MIP-1α. (A) Purified MPV vCCI and rhMIP-1α were mixed together at a 1:1 molar ratio and incubated for 10 min at room temperature. Purified MPV vCCI alone and rhMIP-1α alone were used as controls. Reactions were resolved on a 12% native PAGE gel and stained with SimplyBlue™ Safe Stain. (B) MPV vCCI was titrated from limiting to excess, into a reaction mixture with a fixed amount of rhMIP-1α. MPV vCCI alone and rhMIP-1α alone were used as controls. (C) Co-immunoprecipitation of rhMIP-1α with MPV vCCI. Increasing amounts of rhMIP-1α (0.1 μg–2.0 μg—lanes 4–8) were incubated with a fixed amount of MPV vCCI (6 μg), as a result more rhMIP-1α co-elutes with immunoprecipitated MPV vCCI. 3 μg of rhMIP-1α (lane 2) and 6 μg of MPV vCCI (lane 1) were used as positive controls. As a negative control, MPV vCCI immunoprecipitation was preformed on 3 μg of rhMIP-1α alone (lane 3). Proteins were resolved on 4–12% Bis–Tris NuPAGE® gels.
Fig. 4
Fig. 4
Inhibition of rhesus MIP-1α mediated migration of Human THP-1 cells. 5 × 105 THP-1 cells suspended in 100 μL of assay media (RPMI 1640 + 0.5% fetal bovine serum) were placed in 3 μm pore size transwell inserts and placed in 24-well culture plates containing 600 μL assay media with 10−9 M rhMIP-1α plus increasing concentrations of MPV vCCI or 10−7 M heat inactivated MPV vCCI (Δ MPV vCCI). PBS was used as a negative control. Following a 4 h incubation at 37 °C (5% CO2), THP-1 cells migrating through the transwell were counted using a CyQuant® cell proliferation assay kit (Molecular Probes, Eugene, OR). Represented data are the average number of migrated cells of 3 wells (2500×) ± SEM.
Fig. 5
Fig. 5
In vivo inhibition of rhesus MIP-1α mediated chemotaxis. Gelfoam® sponges containing agarose-embedded (A) rhMIP1α or (B) rhMIP1α + MPV vCCI or (C) PBS were implanted s.c. in the back of a rhesus macaque (⩾8 cm apart), where they remained for 7 days before being harvested, sectioned, and stained. CD14 staining shows a clear reduction in CD14+ infiltrates (lack of dark grey/brown coloration) in the Gelfoam® sponges containing rhMIP1α + MPV vCCI, as compared to rhMIP1α. An isotopically matched primary antibody was used on a section of rhMIP1α-containing Gelfoam® as an antibody control (D). Quantification of CD14+ infiltrates (E) was preformed by comparing the number of DAB+ pixels in each image and normalizing to PBS and represented as a migration index ± SEM. All images (A–D) were taken using a 20× objective and are the same size, 345,000 pixels.
Fig. 6
Fig. 6
MPV vCCI inhibits relapsing EAE. Following induction of EAE by administration of PLP139–151 peptide ± MPV vCCI, mice (n = 4) were observed on a daily basis and scored for disease using the following scale: 0—normal, 0.5—partially limp tail, 1.0—paralyzed tail, 2.0—hind limb paresis, 2.5—one hind limb paralyzed, 3.0—both hind limbs paralyzed, 3.5—hind limbs paralyzed; fore limbs weak, 4.0—fore limbs paralyzed, 5.0—moribund. Represented values are the average scores for all mice within each group. Symbols represent the four groups: ♦—PLP139–151 peptide; ■—PLP139–151 peptide + MPV vCCI; ▴—MPV vCCI alone control; ●—buffer alone control.
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