Biochemical Indication for Myristoylation-Dependent Conformational Changes in HIV-1 Nef

Abstract

The accessory HIV-1 Nef protein is essential for viral replication, high virus load, and progression to AIDS. These functions are mediated by the alteration of signaling and trafficking pathways and require the membrane association of Nef by its N-terminal myristoylation. However, a large portion of Nef is also found in the cytosol, in line with the observation that myristoylation is only a weak lipidation anchor for membrane attachment. We performed biochemical studies to analyze the implications of myristoylation on the conformation of Nef in aqueous solution. To establish an in vivo myristoylation assay, we first optimized the codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold higher protein yield. Myristoylation was achieved by coexpression with the N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated protein was soluble, and proton NMR spectra confirmed proper folding. Size exclusion chromatography revealed that myristoylated Nef appeared of smaller size than the unmodified form but not as small as an N-terminally truncated from of Nef that omits the anchor domain. Western blot stainings and limited proteolysis of both forms showed different recognition profiles and degradation pattern. Analytical ultracentrifugation revealed that myristoylated Nef prevails in a monomeric state while the unmodified form exists in an oligomeric equilibrium of monomer, dimer, and trimer associations. Finally, fluorescence correlation spectroscopy using multiphoton excitation revealed a shorter diffusion time for the lipidated protein compared to the unmodified form. Taken together, our data indicated myristoylation-dependent conformational changes in Nef, suggesting a rather compact and monomeric form for the lipidated protein in solution.

Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef

“…The interaction depends on a well conserved surface-exposed hydrophobic patch, reported to form an interface between Nef molecules in crystals of the Nef core domain (45). However, myristylated Nef is predominantly monomeric in solution (46,47), and the binding site for Dyn2 would thus be expected to be accessible on native Nef. Mutations that inhibited Dyn2 binding also impaired the infectivity enhancement function of Nef, as did the siRNA-mediated depletion of Dyn2.…”

N‐Lauroylation during the Expression of Recombinant N‐Myristoylated Proteins: Implications and Solutions

“…However, the factors leading to lauroylation remain unclear, and the degree of lauroylation seems to be highly variable and inconsistent. Indeed, other studies based on this myristoylation strategy (both in rich and in minimal media) did not report the formation of a lauroylated form 15, 17, 18…”

Functions of the Lentiviral Accessory Protein Nef During the Distinct Steps of HIV and SIV Replication Cycle

“…After membrane targeting, Nef is rapidly internalized though interaction of its C-terminal flexible loop and cell proteins involved in the endocytic pathways. It has been shown that while Nef is in its membrane-bound form it appears more compactly folded, suggesting that membrane-bound and cytossolic Nef shown distinct accessible interaction domains (Breuer et al, 2006), which could explain the distinct phenotypes of Nef. Nef's ability to dimerize or even oligomerize can also function as a mechanism of regulating Nef's effects (Breuer et al, 2006;Dennis et al, 2005).…”

“…It has been shown that while Nef is in its membrane-bound form it appears more compactly folded, suggesting that membrane-bound and cytossolic Nef shown distinct accessible interaction domains (Breuer et al, 2006), which could explain the distinct phenotypes of Nef. Nef's ability to dimerize or even oligomerize can also function as a mechanism of regulating Nef's effects (Breuer et al, 2006;Dennis et al, 2005). Exogenous Nef have been shown to enter monocytes/macrophages, B cells and Dendritic Cells (DC) by adsorptive endocytosis.…”