Abstract 304: Mutational Analysis of M-T7, a Viral Anti-inflammatory Protein, Provides Insight into Its Activity
Viruses express an arsenal of anti-inflammatory proteins that are being developed as immunotherapeutic drugs to treat cardiovascular disease. M-T7, a myxoma viral protein, has been shown to significantly block cell invasion and plaque growth after balloon angioplasty injury and transplantation. The binding target of M-T7 during a myxoma infection is IFNγ, however in vitro, M-T7 has been shown to bind a broad class of chemokines, C, CC, and CXC. To understand the mechanism by which M-T7 disrupts chemokine interactions and inflammatory cell responses, M-T7 mutants were generated and characterized in vitro for changes in cell activation and migration by the chemokine RANTES. Experiments with a balloon angioplasty model monitored changes in vivo. M-T7 mutants were generated to characterize the interactions with chemokines. Protein was expressed in a baculovirus mediated expression system and purified with a Cobalt affinity column. Changes in membrane fluidity were measured with BPP after RANTES activation. As expected, wildtype M-T7 decreased the Iex/Imon fluorescence ratio when compared to RANTES activated saline controls. However, mutants F137D, R171E, and E209I did not alter membrane fluidity. Interestingly, when cells were activated with PMA, F137D had a decreased Iex/Imon ratio (P<0.04). M-T7 and mutants decreased cell migration of THP-1 cells (P<0.001) in an in vitro cell migration assay using RANTES as the chemoattractant. Treatment with heparin increased cell migration of M-T7 (P<0.001) and R171E (P<0.029) treated cells, suggesting inhibition of RANTES binding by heparin, but no change for F137D and E209I. With PMA activated cells, migration was disrupted with R171E and E209I (P<0.001) treatment, but not with M-T7 and F137D. Balloon angioplasty was performed on ApoE-/- mice, and treated with 100ng/g of M-T7/mutant. Change in the vascular architecture was monitored at the site of injury by measuring plaque size and the Internal Elastic Lamina. F137D and wildtype decreased plaque, but R171E treated mice had significantly increased plaque (P<0.018) and increased IEL (P<0.024). E209I treated mice trended toward an increase in plaque and IEL size.
Of the three mutants, F137D was the most effective in vivo. It differed from the other mutants by reducing the Iex/Imon ratio of PMA activated cells and binding to RANTES was also inhibited when treated with heparin suggesting these two characteristics are important for its in vivo activity. In contrast, R171E, which does not reduce plaque formation, does not reduce PMA induced cell activation and activity is not altered by the addition of heparin.
- © 2012 by American Heart Association, Inc.