Structure and Function of Viroporins in Lipid Membranes: Insights from Studies by EPR Spectroscopy

Abstract

We report on our studies of human virus-encoded viroporins. These proteins reside and function in viral and cellular membranes. In these environments viroporins act as homo-oligomeric ion-conducting channels or non-specific pores, changing the levels of ions, mostly cations, in the viral interior or cellular compartments. Through these channel or pore activities viroporins contribute to virus adaptation, survival and proliferation. Thus, these proteins are targets for drug development. We apply pulse and continuous-wave EPR spectroscopies as powerful tools to investigate the function and structural aspects of viroporins in lipid bilayers at the conditions representing the native membranes. In our studies we characterized the mechanism by which the M2 protein from influenza a virus assembles into a tetrameric proton channel. We established that the functional channel assembles via a cascade mechanism where dimers come first, followed by the formation of tetramer as a dimer-of-dimers. We also found that amantadine drug stabilized the closed (inhibited) form of M2 tetramer. Our further studies of a different p13II protein from human T-cell leukemia virus type 1 revealed that its purified soluble form oligomerizes upon interaction with lipid membrane mimetics. This behavior is relevant to the p13II function in the inner mitochondrial membrane (IMM) where it leads to increased IMM permeability to ions and depolarization. We also discuss our ongoing applications of EPR spectroscopy to viroporins from coronaviruses and hepatitis C virus, aiming at the effects of variations in the amino acid sequence on viroporin structure and function. Acknowledgements: We thank Haley Norman, Nichita Kulkarni, and Christina Fanouraki for their contributions at different stages of these studies.