Epstein-Barr virus encoded interleukin-10 inhibits HLA-class I, ICAM-1, and B7 expression on human monocytes: implications for immune evasion by EBV.

Monocytes and macrophages play a central role in viral infections, as a target for viruses and in activation of both innate and adaptive immune responses. Epstein-Barr virus (EBV) has evolved elaborate strategies to dampen the immune system and to persist within the host. There is evidence that the product of the BCRF-1 open reading frame of EBV, viral interleukin-10 (vIL-10), inhibits the capacity of monocytes/macrophages to induce T cell activation, but the full mechanism of this effect is unknown. To determine whether this effect might involve modulation of the expression of accessory molecules known to be important in T cell activation, we analyzed by flow cytometry the influence of vIL-10 on the basal as well as on IFN-gamma-induced up-regulation of HLA molecules, ICAM-1, and two members of the B7 family B7.1 (CD80) and B7.2 (CD86) at the surface of human monocytes. Viral IL-10 in a concentration-dependent manner inhibited both basal- and IFN-gamma-induced HLA-class II, ICAM-1 (basal levels of ICAM-2 and ICAM-3 is unaffected), CD80, and CD86 up-regulation when added simultaneously with IFN-gamma. In contrast, complete inhibition of HLA-class I expression on monocytes/macrophages occurred only when vIL-10 was present 2 h prior to the addition of IFN-gamma, implying that vIL-10 affects an early step in the IFN-gamma signaling pathway. As both monocytes and macrophages can be infected by EBV, we propose that vIL-10-mediated impairment of monocyte/macrophage antigen-presenting function could help the virus-infected cells to avoid detection by the host's T cells on one hand and contribute to its immunosuppressive properties on the other.

High level expression and purification of the Epstein-Barr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.

To characterize the structural and functional properties of viral interleukin 10 (vIL-10), its cDNA was cloned into the bacterial expression vector pMAL-c2, which directs the synthesis of the inserted gene as a fusion protein with maltose binding protein (MBP). The MBP-vIL-10 fusion protein was expressed in Escherichia coli and purified from cell lysates using amylose resin chromatography. Viral interleukin 10 (IL-10) was released from the fusion protein by cleavage with the proteolytic enzyme factor Xa. We show that vIL-10 will bind to heparin and use this property to purify vIL-10 from factor Xa cleaved products and trace contaminants using heparin agarose chromatography. A simple one-step procedure is described for the removal of endotoxins from heavily contaminated vIL-10 preparations. The protocol exploits the high binding affinity of MBP for amylose resin or vIL-10 for heparin and the ability of Triton-X114 to dissociate endotoxins from proteins. The biological activity of purified vIL-10 was demonstrated through its ability to inhibit interferon gamma (IFN-gamma) production by mitogen activated peripheral blood mononuclear cells and to down-regulate HLA-class II expression on activated monocytes/macrophages. The availability of an efficient expression and purification strategy for vIL-10 together with appropriate assays will contribute to a greater understanding of how vIL-10 has evolved to retain and modify those activities of cellular IL-10 best suited for Epstein-Barr virus (EBV)'s specialized niche within the host.