Specific and distinct determinants mediate membrane binding and lipid raft incorporation of HIV-1SF2 Nef

Abstract

Membrane association is believed to be a prerequisite for the biological activity of the HIV-1 pathogenicity factor Nef. Attachment to cellular membranes as well as incorporation into detergent-insoluble microdomains (lipid rafts) require the N-terminal myristoylation of Nef. However, this modification is not sufficient for sustained membrane association and a specific raft-targeting signal for Nef has not yet been identified. Using live cell confocal microscopy and membrane fractionation analyses, we found that the N-terminal anchor domain (aa 1-61) is necessary and sufficient for efficient membrane binding of Nef from HIV-1(SF2). Within this domain, highly conserved lysine and arginine residues significantly contributed to Nef's membrane association and localization. Plasma membrane localization of Nef was also governed by an additional membrane-targeting motif between residues 40 and 61. Importantly, two lysines at positions 4 and 7 were not essential for the overall membrane association but critically contributed to Nef's incorporation into lipid raft domains. Cell surface receptor downmodulation was largely unaffected by mutations of all N-terminal basic residues, while the association of Nef with Pak2 kinase activity and its ability to augment virion infectivity correlated with its lysine-mediated raft incorporation. In contrast, all basic residues were required for efficient HIV-1 replication in primary human T lymphocytes but did not contribute to the incorporation of Nef into HIV-1 virions. Together, these results unravel that Nef's membrane association is governed by a complex pattern of signature motifs that differentially contribute to individual Nef activities. The identification of a critical raft targeting determinant and the functional characterization of a membrane-bound, non-raft-associated Nef variant indicate raft incorporation as a regulatory mechanism that determines the biological activity of distinct subpopulations of Nef in HIV-infected cells.

Role of Membrane Rafts in Viral Infection

“…The role of membrane rafts in the assembly and budding of enveloped viruses has been investigated for influenza virus [59,63,[150][151][152][153][154][155][156][157][158][159][160], HIV-1 [63,[161][162][163][164][165][166][167][168][169][170][171][172][173], HTLV-1 [174], measles virus (Paramyxoviridae) [63,175,176], Sendai virus [Paramyxoviridae] [177,178], Newcastle disease virus (NDV; Paramyxoviridae) [179,180], RSV [147,148,[181][182][183][184], HSV [185,186], murine cytomegalovirus (MCMV; Herpesviridae) [187], Ebola virus [70,188], Marburg virus [70], and vesicular stomatitis virus (VSV; Rhabdoviridae) …”

“…Nef also binds to both the plasma membrane and viral structural proteins and participates directly in formation of the budding scaffold, leading to incorporation of Nef into virions, concomitantly with viral structural proteins [162,163,166]. The N-terminus of Nef determines the differential membrane avidities and selective incorporation into a specific membrane raft for surface membranes or for subcellular membranes [170]. Moreover, Nef modulates the lipid composition of virions and host cell membrane rafts through activation of lipid kinases such as PI3K [171].…”

Viruses, lipid rafts and signal transduction

“…Based on biochemical and microscopic evidence, a subpopulation of membrane-associated Nef is incorporated into lipid microdomains in cell lines but also primary resting T lymphocytes [105][106][107]. This raft association depends on Nef's myristoylation and is specifically mediated by a di-lysine motif at the Nef N-terminus [107,108]. The use of experimental enrichment of raft association (by virtue of an additional palmitoylation) or abrogation of its raft incorporation (by mutating the di-lysine motif) revealed that raft-association is dispensable for the receptor transport activities of Nef [108,109].…”

“…This raft association depends on Nef's myristoylation and is specifically mediated by a di-lysine motif at the Nef N-terminus [107,108]. The use of experimental enrichment of raft association (by virtue of an additional palmitoylation) or abrogation of its raft incorporation (by mutating the di-lysine motif) revealed that raft-association is dispensable for the receptor transport activities of Nef [108,109]. In contrast, signal transduction properties of Nef appear to be governed by the raft-associated Nef subpopulation.…”

“…Nef associates with a highly active subpopulation of cellular Pak2, a function that is well conserved among different Nef variants and that might correlate with some of its pathological properties [118][119][120]. This association of Nef with Pak2 kinase activity strictly depends on Nef's raft association and the integrity of these microdomains [106,108,121]. Combined imaging and flotation analysis suggested that this effect is mediated by recruiting the involved TCR machinery, including the Rac and Cdc42 GTPases as well as the Pak2 and Lck kinases into plasma membrane lipid raft domains [106,122].…”