Human Immunodeficiency Virus Type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains

BACKGROUND: The Nef protein of Human Immunodeficiency Viruses optimizes viral spread in the infected host by manipulating cellular transport and signal transduction machineries. Nef also boosts the infectivity of HIV particles by an unknown mechanism. Recent studies suggested a correlation between the association of Nef with lipid raft microdomains and its positive effects on virion infectivity. Furthermore, the lipidome analysis of HIV-1 particles revealed a marked enrichment of classical raft lipids and thus identified HIV-1 virions as an example for naturally occurring membrane microdomains. Since Nef modulates the protein composition and function of membrane microdomains we tested here if Nef also has the propensity to alter microdomain lipid composition. RESULTS: Quantitative mass spectrometric lipidome analysis of highly purified HIV-1 particles revealed that the presence of Nef during virus production from T lymphocytes enforced their raft character via a significant reduction of polyunsaturated phosphatidylcholine species and a specific enrichment of sphingomyelin. In contrast, Nef did not significantly affect virion levels of phosphoglycerolipids or cholesterol. The observed alterations in virion lipid composition were insufficient to mediate Nef's effect on particle infectivity and Nef augmented virion infectivity independently of whether virus entry was targeted to or excluded from membrane microdomains. However, altered lipid compositions similar to those observed in virions were also detected in detergent-resistant membrane preparations of virus producing cells. CONCLUSION: Nef alters not only the proteome but also the lipid composition of host cell microdomains. This novel activity represents a previously unrecognized mechanism by which Nef could manipulate HIV-1 target cells to facilitate virus propagation in vivo

HIV-1 Vpu's lipid raft association is dispensable for counteraction of the particle release restriction imposed by CD317/Tetherin.

HIV-1 Vpu antagonizes the block to particle release mediated by CD317 (BST-2/HM1.24/Tetherin) via incompletely understood mechanisms. Vpu and CD317 partially reside in cholesterol-rich lipid rafts where HIV-1 budding preferentially occurs. Here we find that lipid raft association of ectopically expressed or endogenous CD317 was unaltered upon co-expression with Vpu or following HIV-1 infection. Similarly, Vpu's lipid raft association remained unchanged upon expression of CD317. We identify amino acids V25 and Y29 of Vpu as crucial for microdomain partitioning and single substitution of these amino acids resulted in Vpu variants with markedly reduced or undetectable lipid raft association. These mutations did not affect Vpu's subcellular distribution and binding capacity to CD317, nor its ability to downmodulate cell surface CD317 and promote HIV-1 release from CD317-positive cells. We conclude that (i) lipid raft incorporation is dispensable for Vpu-mediated CD317 antagonism and (ii) Vpu does not antagonize CD317 by extraction from lipid rafts

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

AbstractMembrane 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-1SF2. 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

Determinants in HIV-1 Nef for enhancement of virus replication and depletion of CD4⁺T lymphocytes in human lymphoid tissue <it>ex vivo</it>

Abstract Background HIV-1 Nef critically contributes to AIDS in part by augmenting virus titers in infected individuals. Analyzing which of Nef's activities contribute to HIV pathogenesis has been hampered by the lack of a cell culture model in which Nef exerts pronounced effects on HIV replication. The human lymphoid aggregate culture (HLAC) from tonsil maintains the cell populations and cytokine milieu found in vivo, supports a productive infection without exogenous stimulation, and Nef contributes to efficient HIV-1 replication as well as CD4+ T cell depletion in this experimental ex vivo-model. Results To identify determinants in Nef that mediate these activities, we infected HLAC with a panel of isogenic HIV-1NL4-3 strains that encode for well-characterized mutants of HIV-1SF2 Nef. Determination of HIV-1 replication revealed that enhancement of the virus spread by Nef is governed by a complex set of protein interaction surfaces. In contrast, increased CD4+ T lymphocyte depletion depended on only two protein interaction surfaces in Nef that mediate either downregulation of cell surface CD4 or interaction with the NAKC signalosome. Consistently, in HLAC from 9 out of 14 donors, Nef enhanced CD4+ T cell depletion in the absence of a significant effect on virus replication. Moreover, our results suggest that this Nef-dependent enhancement in depletion occurred predominately in uninfected bystander CD4+ T cells. Conclusion Our findings suggest that Nef facilitates depletion of CD4+ T lymphocytes in HIV-1-infected lymphoid tissue ex vivo by increasing the pool of productively infected cells and by sensitizing bystander cells for killing. This ability might contribute to Nef's pathogenic potential in vivo.</p

Antagonism of CD317 Restriction of Human Immunodeficiency Virus Type 1 (HIV-1) Particle Release and Depletion of CD317 Are Separable Activities of HIV-1 Vpu▿

Vpu antagonizes human immunodeficiency virus type 1 (HIV-1) particle release inhibition by CD317/BST-2/Tetherin. Whether this Vpu activity strictly requires cellular depletion of the restriction factor is unclear. Here, we characterized CD317 variants with mutations in putative sorting or ubiquitination motifs. All mutants still potently impaired release of Vpu-defective HIV-1 and remained sensitive to Vpu-mediated release enhancement. Importantly, this virological antagonism correlated with surface downregulation of CD317 mutants by Vpu, while intracellular pools of these mutants, which were consistently depleted of the wild-type protein, were highly variable or even enhanced. Thus, Vpu can efficiently antagonize virion tethering in the absence of CD317 degradation

Functional characterization of HIV-1 Nef mutants in the context of viral infection.

International audienceNef is an important pathogenesis factor of HIV-1 with a multitude of effector functions. We have designed a broad panel of isogenic viruses encoding defined mutants of HIV-1(SF2) Nef and analyzed their biological activity in the context of productive HIV-1 infection. Analysis of subcellular localization, virion incorporation, downregulation of cell surface CD4 and MHC-I, enhancement of virion infectivity and facilitation of HIV replication in primary human T lymphocytes mostly confirmed the mapping of Nef determinants previously reported upon isolated expression of Nef. However, reduced activity in downregulation of CD4, infectivity enhancement and virion incorporation of a Nef variant (Delta12-39) lacking an amphipatic helix required for binding of a cellular kinase complex and the association of Nef with MHC-I/AP-1 suggested a novel role of this N-terminal motif. The SH3 binding motif of Nef was partially required for infectivity enhancement and replication but not for receptor downmodulation. In contrast to previous results obtained using other Nef alleles, non-myristoylated SF2-Nef was only partly defective when expressed during HIV infection and was present in HIV-1 particles. Importantly, incorporation of Nef into HIV-1 virions was not required for any of the tested Nef activities. Altogether, this study provides a broad characterization and mapping of multiple Nef activities in HIV-infected cells. The results emphasize that multiple activities govern Nef's effects on HIV replication and argue against a role of virion incorporation for Nef's activity as pathogenicity factor

HIV Nef- and Notch1-dependent Endocytosis of ADAM17 Induces Vesicular TNF Secretion in Chronic HIV Infection

Tumor necrosis factor (TNF) is a key cytokine in HIV replication and pathogenesis. For reasons that are not entirely clear, the cytokine remains upregulated despite anti-retroviral therapy (ART). Here we demonstrate that HIV Nef induces an alternative TNF secretion mechanism that remains active in chronic infection. Ingestion of Nef-containing plasma extracellular vesicles (pEV) from ART patients by primary immune cells, but also Nef expression, induced intracellular proTNF cleavage and secretion of vesicular TNF endosomes. Key event was the Nef-mediated routing of the TNF-converting enzyme ADAM17 into Rab4+ early endosomes and the Rab27+ secretory pathway. Analysis of lymph-node tissue by multi-epitope-ligand-cartography (MELC) confirmed a vesicular TNF secretion phenotype that co-localized with persistent Nef expression, and implicated Notch1 as an essential co-factor. Surprisingly Notch1 had no transcriptional effect but was required for the endosomal trafficking of ADAM17. We conclude that Nef expression and Nef-containing pEV mobilize TNF from endosomal compartments in acute and chronic infection