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Functional and Structural Study of Vpu from HIV-1 by Nuclear Magnetic Resonance Spectroscopy

Abstract

Viral protein U (Vpu) is an 81-residue membrane protein encoded by HIV-1. It has two distinct domains, a hydrophobic transmembrane domain, which has been well studied, and a cytoplasmic helical domain with two conserved phosphorylation sites, which has proven to be more difficult to study in large part because of its dynamics. The two domains are associated with different biological activities that contribute to the pathogenicity of HIV-1 infections in humans. Vpu removes CD4 receptor from ER and causes its subsequent degradation to enhance viral infectivity. Vpu enhances release of newly formed virus particles from infected cells by antagonize human immune restriction factor BST-2. Recently, Vpu has also been discovered to interact with NK cell receptor NK-cell, T-cell and B-cell antigen (NTB-A) to induce down- modulation of NTB-A, and prevent HIV-Infected cells from degranulation and lysis by NK cells. A combination of solution and solid-state NMR experiments are used to determine the structure of cytoplasmic domain (VpuCyto) and full-length of Vpu in micelles and liposomes environment. Both constructs have a U-shape cytoplasmic domain that associated with the lipid environment. The second section describes the membrane anchoring property of W76 in the C-terminus of Vpu. Biological study found that W76 is specifically important for the enhancement of virion release, and NMR data suggests that W76 might function by interacting directly with the lipid bilayer. Significant intensity change and chemical shift perturbation were observed for W76 upon addition of liposomes to VpuCyto; in addition, paramagnetic relaxation enhancement (PRE) indicates that the residue is embedded in the interior of micelles. The last section describes the interaction between transmembrane domains of Vpu and NTB-A base on a combination of biological, NMR spectroscopic and computational methods. The knowledge about the transmembrane domain of Vpu (VpuTM) enables detailed studies of its interactions with NTB-A. NMR spectral changes demonstrate that specific residues in VpuTM interact with specific residues in the transmembrane domains of NTB-A. A model of the complex is generated by docking, with the input of interaction faces base on NMR and biological results

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