Comparative NMR studies demonstrate profound differences between two viroporins: p7 of HCV and Vpu of HIV-1

The p7 protein from hepatitis C virus and the Vpu protein from HIV-1 are members of the viroporin family of small viral membrane proteins. It is essential to determine their structures in order to obtain an understanding of their molecular mechanisms and to develop new classes of anti-viral drugs. Because they are membrane proteins, it is challenging to study them in their native phospholipid bilayer environments by most experimental methods. Here we describe applications of NMR spectroscopy to both p7 and Vpu. Isotopically labeled p7 and Vpu samples were prepared by heterologous expression in bacteria, initial isolation as fusion proteins, and final purification by chromatography. The purified proteins were studied in the model membrane environments of micelles by solution NMR spectroscopy and in aligned phospholipid bilayers by solid-state NMR spectroscopy. The resulting structural findings enable comparisons to be made between the two proteins, demonstrating that they have quite different architectures. Most notably, Vpu has one trans-membrane helix and p7 has two trans-membrane helices; in addition, there are significant differences in the structures and dynamics of their internal loop and terminal regions.

Research Highlights
► It is feasible to obtain high resolution NMR spectra of p7 and Vpu in both micelle and bilayer environments.
► Both p7 and Vpu have complex architectures with both highly structured and mobile regions.
► p7 and Vpu have different architectures, since p7 has two trans-membrane helices and Vpu has one trans-membrane helix. There are other differences as well.