The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, ...Vincendeau‐Scherrer C, Raposo G, Bonnerot C. Exosomal‐like vesicles are present in human blood plasma. Int Immunol 2005;17:879–887). Extracellular Nef protein has deleterious effects on CD4+ T cells (James CO, Huang MB, Khan M, Garcia‐Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4+ T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099–3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40‐dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302–310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV‐1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation‐induced cell death of resting PBLs in vitro. Thus, HIV‐infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4+ T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS).
The negative factor (Nef) of human immunodeficiency virus (HIV) is an accessory protein that is thought to be integral to HIV-associated immune- and neuroimmune pathogenesis. Here, we show that ...nef-transfected microglia-released Nef+ exosome (exNef) disrupts the apical blood–brain barrier (BBB) and that only nef-transfected microglia release Nef in exosomes. nef–gfp-transduced neurons and astrocytes release exosomes but did not release exNef in the extracellular space. Apical administration of exNef derived from nef-transfected 293T cells reduced transendothelial electrical resistance (TEER) and increased permeability of the BBB. Microglia-derived exNef applied to either the apical/basal BBB significantly reduced expression of the tight junction protein, ZO-1, suggesting a mechanism of exNef-mediated neuropathogenesis. Microglia exposed to exNef release elevated levels of Toll-like receptor-induced cytokines and chemokines IL-12, IL-8, IL-6, RANTES, and IL-17A. Magnetic nanoparticle delivery of Nef peptides containing the Nef myrisolation site across an in vitro BBB ultimately reduced nef-transfected microglia release of Nef exosomes and prevented the loss of BBB integrity and permeability as measured by TEER and dextran-FITC transport studies, respectively. Overall, we show that exNef is released from nef–gfp-transfected microglia; exNef disrupts integrity and permeability, and tight junctions of the BBB, and induces microglial cytokine/chemokine secretion. These exNef-mediated effects were significantly restricted by Nef peptides. Taken together, this study provides preliminary evidence of the role of exNef in HIV neuroimmune pathogenesis and the feasibility of a nanomedicine-based therapeutics targeting exNef to treat HIV-associated neuropathogenesis.
HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the ...intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.
HIV‐associated neurocognitive disorders (HAND) is a term describing a complex set of cognitive impairments accompanying HIV infection. Successful antiretroviral therapy (ART) reduces the most severe ...forms of HAND, but milder forms affect over 50% of people living with HIV (PLWH). Pathogenesis of HAND in the ART era remains unknown. A variety of pathogenic factors, such as persistent HIV replication in the brain reservoir, HIV proteins released from infected brain cells, HIV‐induced neuroinflammation, and some components of ART, have been implicated in driving HAND pathogenesis in ART‐treated individuals. Here, we propose another factor—impairment of cholesterol homeostasis and lipid rafts by HIV‐1 protein Nef—as a possible contributor to HAND pathogenesis. These effects of Nef on cholesterol may also underlie the effects of other pathogenic factors that constitute the multifactorial nature of HAND pathogenesis. The proposed Nef‐ and cholesterol‐focused mechanism may provide a long‐sought unified explanation of HAND pathogenesis that takes into account all contributing factors. Evidence for the impairment by Nef of cellular cholesterol balance, potential effects of this impairment on brain cells, and opportunities to therapeutically target this element of HAND pathogenesis are discussed.
HIV infection (blue circle) by definition is the key contributor to the pathogenesis of HAND with many other contributing factors greatly influencing the progression of the disease. These factors are grouped according to their common features; HAND is at the intersection of these groups (dash‐line circle). The Nef‐cholesterol‐lipid raft axis is marked in red as it is proposed to be the main causative factor and underlie the activity of some other contributing factors.
Identification of CD8+ T lymphocyte (CTL) escape mutations that compromise the pathogenic functions of the Nef protein may be relevant for an HIV‐1 attenuation‐based vaccine. Previously, ...HLA‐associated mutations 102H, 105R, 108D, and 199Y were individually statistically associated with decreased Nef‐mediated HLA‐I downregulation ability in a cohort of 298 HIV‐1 subtype C infected individuals. In the present study, these mutations were introduced by site‐directed mutagenesis into different patient‐derived Nef sequence backgrounds of high similarity to the consensus C Nef sequence, and their ability to downregulate HLA‐I was measured by flow cytometry in a CEM‐derived T cell line. A substantial negative effect of 199Y on HLA‐I downregulation and Nef expression was observed, while 102H and 105R displayed negative effects on HLA‐I downregulation ability and Nef expression to a lesser extent. The total magnitude of CTL responses in individuals harboring the 199Y mutation was lower than those without the mutation, although this was not statistically significant. Overall, a modest positive relationship between Nef‐mediated HLA‐I downregulation ability and total magnitude of CTL responses was observed, suggesting that there is a higher requirement for HLA‐I downregulation with increased CTL pressure. These results highlight a region of Nef that could be targeted by vaccine‐induced CTL to reduce HLA‐I downregulation and maximize CTL efficacy.
Highlights
Mutant HIV‐1 Nef clones encoding immune‐driven were constructed.
Mutation 199Y had a substantial negative effect on Nef‐mediated HLA‐I down‐regulation ability, a cytotoxic T cell (CTL) evasion mechanism.
Findings highlight a region of Nef that could be targeted by vaccine‐induced immune responses to reduce Nef‐mediated HLA‐I down‐regulation and maximise CTL efficacy.
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•HIV-1 Nef is essential for viral pathogenesis and often functions as a homodimer.•Neutron reflectometry revealed myristoylated Nef homodimers at lipid bilayers.•Dimeric Nef core ...displaced ∼90 Å from bilayer center for partner protein binding.•Control studies of monomeric Nef mutant validate helical dimerization interface.
The HIV-1 Nef protein plays a critical role in viral infectivity, high-titer replication in vivo, and immune escape of HIV-infected cells. Nef lacks intrinsic biochemical activity, functioning instead through interactions with diverse host cell signaling proteins and intracellular trafficking pathways. Previous studies have established an essential role for Nef homodimer formation at the plasma membrane for most if not all its functions. Here we combined neutron reflectometry of full-length myristoylated Nef bound to model lipid bilayers with molecular simulations based on previous X-ray crystal structures of Nef homodimers. This integrated approach provides direct evidence that Nef associates with the membrane as a homodimer with its structured core region displaced from the membrane for partner protein engagement. Parallel studies of a dimerization-defective mutant, Nef-L112D, demonstrate that the helical dimerization interface present in previous crystal structures stabilizes the membrane-bound dimer. X-ray crystallography of the Nef-L112D mutant in complex with the SH3 domain of the Nef-associated host cell kinase Hck revealed a monomeric 1:1 complex instead of the 2:2 dimer complex formed with wild-type Nef. Importantly, the crystal structure of the Nef-L112D core and SH3 interface are virtually identical to the wild-type complex, indicating that this mutation does not affect the overall Nef fold. These findings support the intrinsic capacity of Nef to homodimerize at lipid bilayers using structural features present in X-ray crystal structures of dimeric complexes.
Autophagy is a conserved eukaryotic process with metabolic, immune, and general homeostatic functions in mammalian cells. Mammalian autophagosomes fuse with lysosomes in a SNARE-driven process that ...includes syntaxin 17 (Stx17). How Stx17 translocates to autophagosomes is unknown. In this study, we show that the mechanism of Stx17 recruitment to autophagosomes in human cells entails the small guanosine triphosphatase IRGM. Stx17 directly interacts with IRGM, and efficient Stx17 recruitment to autophagosomes requires IRGM. Both IRGM and Stx17 directly interact with mammalian Atg8 proteins, thus being guided to autophagosomes. We also show that Stx17 is significant in defense against infectious agents and that Stx17-IRGM interaction is targeted by an HIV virulence factor Nef.
Serine incorporator 5 (SERINC5) reduces the infectivity of progeny HIV-1 virions by incorporating into the outer host-derived viral membrane during egress. To counter SERINC5, the HIV-1 accessory ...protein Nef triggers SERINC5 internalization by engaging the adaptor protein 2 (AP-2) complex using the D/ExxxLL/I
Nef dileucine motif. Nef also engages AP-2 via its dileucine motif to downregulate the CD4 receptor. Although these two Nef functions are related, the mechanisms governing SERINC5 downregulation are incompletely understood. Here, we demonstrate that two primary Nef isolates, referred to as 2410 and 2391 Nef, acquired from acutely HIV-1 infected women from Zimbabwe, both downregulate CD4 from the cell surface. However, only 2410 Nef retains the ability to downregulate cell surface SERINC5. Using a series of Nef chimeras, we mapped the region of 2391 Nef responsible for the functional uncoupling of these two antagonistic pathways to the dileucine motif. Modifications of the first and second x positions of the 2410 Nef dileucine motif to asparagine and aspartic acid residues, respectively (ND
), impaired cell surface SERINC5 downregulation, which resulted in reduced infectious virus yield in the presence of SERINC5. The ND
mutation additionally partially impaired, but did not completely abrogate, Nef-mediated cell surface CD4 downregulation. Furthermore, the patient infected with HIV-1 encoding 2391 Nef had stable CD4
T cell counts, whereas infection with HIV-1 encoding 2410 Nef resulted in CD4
T cell decline and disease progression.
A contributing factor to HIV-1 persistence is evasion of the host immune response. HIV-1 uses the Nef accessory protein to evade the antiviral roles of the adaptive and intrinsic innate immune responses. Nef targets SERINC5, a restriction factor which potently impairs HIV-1 infection by triggering SERINC5 removal from the cell surface. The molecular determinants underlying this Nef function remain incompletely understood. Recent studies have found a correlation between the extent of Nef-mediated SERINC5 downregulation and the rate of disease progression. Furthermore, single-residue polymorphisms outside the known Nef functional motifs can modulate SERINC5 downregulation. The identification of a naturally occurring Nef polymorphism impairing SERINC5 downregulation in this study supports a link between Nef downregulation of SERINC5 and the rate of plasma CD4
T cell decline. Moreover, the observed functional impairments of this polymorphism could provide clues to further elucidate unknown aspects of the SERINC5 antagonistic pathway via Nef.
Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is ...complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant Nef
protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1β, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.