Hetero-Oligomeric Complexes Formed by the Homo-Oligomeric Transmembrane Domains of HIV-1 Vpu and Human Tetherin

Abstract

The function of many membrane proteins is dependent on their ability to form higher order homo or hetero oligomers. The host cell restriction factor tetherin, a homo-dimeric transmembrane protein, is responsible for tethering nascent viral particles to the host cell membrane. These restricted virions are later internalized and degraded by the cell. During HIV-1 infection, tetherin activity is antagonized by the direct association of the HIV-1 transmembrane protein Vpu, which sequesters tetherin within the cell. This association occurs between the transmembrane domains of Vpu and tetherin, which form a hetero-complex of unknown size. A homo-pentamer, Vpu was originally proposed as an ion channel with weak, cation specific, activity. This was supported by its susceptibility to amiloride-based channel blocking drugs in vitro. This class of molecules functioned to block channel activity and restored Vpu mediated viral release- implying that the drugs functioned to directly inhibit the Vpu/tetherin association, or reduced the availability of Vpu, possibly by affecting its oligomer/monomer equilibrium. To discern between these possible mechanisms we must first clarify the specifics of the respective homo oligomerization of Vpu and tetherin as well as the formation of the hetero-oligomeric complex. We hypothesize that the proposed Vpu pentamer dissociates in order to interact with tetherin. Here we elucidate the nature of the transmembrane domain interactions occurring within the Vpu-tetherin system in vitro. Using Forster resonance energy transfer we have measured the homo and hetero oligomerization of fluorophore labeled transmembrane domains of Vpu and tetherin in liposomes. Our results confirm the homo-oligomerization of both proteins and support our hypothesis that the hetero complex is a higher order oligomer. This provides the necessary foundation for determining how Vpu channel blockers prevent antagonism of tetherin.