UC San Diego

Virus protein 'u' (Vpu) is one of the accessory proteins expressed by the HIV-1 genome. It is a type I integral phosphoprotein which contains a single-helical transmembrane (TM) domain and an amphipathic cytoplasmic domain. In its oligomeric form, the transmembrane domain is responsible for the ion channel activity of Vpu, and the domain itself is the main contributor in the antagonistic function against BST-2, a host cell viral restriction factor. The cytoplasmic domain is responsible for the binding of CD4 receptors at the endoplasmic reticulum (ER) and recruiting other protein complexes that ultimately leads to the CD4 degradation through the ubiquitin-proteasome degradation pathway. In order to structurally characterize this protein by NMR, a lipid environment in the form of either micelle or bicelle was used for the proper solubilization and folding of the protein. Solution NMR was used as a method to quickly examine the stability of the protein within micelles and to identify the secondary structure regions, while solid- state NMR gave helical tilt and rotation information as well as the orientation restraints of individual amide bonds for a protein embedded in a bilayer environment. In this thesis, structural features of Vpu based on NMR results of wildtype and various truncated/mutant forms of the protein are presented. A single site mutation on the TM that enables the inter-conversion of structure and functionality between Vpu and M2, a structural analogue from Influenza A virus, will be discussed. The last section will describe the collaborative effort of examining the protein-protein interaction between Vpu and BST-2 using a combination of spectroscopic, biological, and computational approaches. Residues involved in the interaction between the two proteins' TM domains were identified in solution NMR experiments while the helical tilt angles and interaction faces were determined by solid -state NMR. A model of the complex is presented and shows remarkable agreement with the combination of NMR data, biological results, and computational simulation