HIV Nef increasing viral particle infectivity may be through affecting the protein transport of HIV factors or host cellular factors which promote viral assembly or budding

Human immunodeficiency virus type 1 (HIV 1) has evolved to encode multifunctional accessory proteins to promote the viral life cycle. Nef, a HIV 1 encoded accessory protein that originally thought to be a negative factor that inhibited viral replications, has been reported increasing HIV1 viral particle infectivity through a still unknown mechanism. Recently, lots of experimental evidences showed that Nef could extensively interact with multiple key factors of protein intracellular trafficking pathways, such as adaptor protein families (APs), to promote the HIV pathogenesis through down-regulation of the membrane localization of MHC1 and CD4 molecules.Taking together with the current progresses of the biological nature of Nef in recent years, here, we proposed that the Nef also could increase the infectivity of viral particle possibly through affecting the protein transport pathways of HIV1 factors or other host cellular factors that promote viral assembly or budding. If true, this will let us better understand how Nef manipulate the host cell environment to promote the HIV pathogenicity and will also provide more choices for developing novel therapeutic strategies.     

Modulation of HIV pathogenesis and T-cell signaling by HIV-1 Nef

HIV-1 Nef protein is an approximately 27-kDa myristoylated protein that is a virulence factor essential for efficient viral replication and infection in CD4(+) T cells. The functions of CD4(+) T cells are directly impeded after HIV infection. HIV-1 Nef plays a crucial role in manipulating host cellular machinery and in HIV pathogenesis by reducing the ability of infected lymphocytes to form immunological synapses by promoting virological synapses with APCs, and by affecting T-cell stimulation. This article reviews the current status of the efficient Nef-mediated spread of virus in the unreceptive environment of the immune system by altering CD4(+) T-lymphocyte signaling, intracellular trafficking, cell migration and apoptotic pathways.     

The HIV Nef protein alters Ca(2+) signaling in myelomonocytic cells through SH3-mediated protein-protein interactions

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca(2+) signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef((PXXP)4(-))). In differentiated HL60 cells, Nef markedly altered cellular Ca(2+) signaling; the amount of intracellularly stored Ca(2+) was increased, and as a consequence, store-operated Ca(2+)-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca(2+) signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59(hck) co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca(2+) stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca(2+) stores through SH3-mediated interactions in myelomonocytic cells.     

Nef enhances human immunodeficiency virus type 1 infectivity and replication independently of viral coreceptor tropism

We investigated the infectivities and replicative capacities of a large panel of variants of the molecular human immunodeficiency virus type 1 (HIV-1) NL4-3 clone that differ exclusively in the V3 region of the viral envelope glycoprotein and the nef gene. Our results demonstrate that Nef enhances virion infectivity and HIV-1 replication independently of the viral coreceptor tropism.     

Dynamic Nef and Nef dynamics: how structure could explain the complex activities of this small HIV protein.

The Nef protein of the human immunodeficiency virus is as important for disease progression as it is perplexing in its plethora of target molecules and functions. In this article, it is proposed that the complex biology of Nef is regulated through conformational changes of the protein that are triggered by cellular location and specific interactions as Nef traffics through the infected cell.     

Sequence- and interactome-based prediction of viral protein hotspots targeting host proteins: a case study for HIV Nef

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk.     

HIV type 1 Nef protein is a viral factor for leukocyte recruitment into the central nervous system.

Recombinant HIV-1 Nef protein, but not Tat, gp120, and gp160, provoked leukocyte recruitment into the CNS in a rat model. The strong reduction of bioactivity by heat treatment of Nef, and the blocking effect of the mAb 2H12, which recognizes the carboxy-terminal amino acid (aa) residues 171-190 (but not of mAb 3E6, an anti-Nef Ab of the same isotype, which maps the aa sequence 168-175, as well as a mixture of mAbs to CD4) provided evidence for the specificity of the observed Nef effects. Using a modified Boyden chamber technique, Nef exhibited chemotactic activity on mononuclear cells in vitro. Coadministration of the anti-Nef mAb 2H12, as well as treatment of Nef by heat inhibited Nef-induced chemotaxis. Besides soluble Nef, chemotaxis was also induced by a Nef-expressing human astrocytoma cell line, but not by control cells. These data suggest a direct chemotactic activity of soluble Nef. The detection of elevated levels of IL-6, TNF-alpha, and IFN-gamma in rat cerebrospinal fluid 6 h after intracisternal Nef injection hint at the additional involvement of indirect mechanisms in Nef-induced leukocyte migration into rat CNS. These data propose a mechanism by which HIV-1 Nef protein may be essential for AIDS neuropathogenesis, as a mediator of the recruitment of leukocytes that may serve as vehicles of the virus and perpetrators for disease through their production of neurotoxins.     

The Nef protein of human immunodeficiency virus type 1 enhances serine phosphorylation of the viral matrix.

The human immunodeficiency virus type 1 matrix (MA) protein is phosphorylated during virion maturation on its C-terminal tyrosine and on several serine residues. Whereas MA tyrosine phosphorylation facilitates viral nuclear import, the significance of MA serine phosphorylation remains unclear. Here, we report that MA serine but not tyrosine phosphorylation is strongly enhanced by Nef. Mutations that abrogated the membrane association of Nef and its ability to bind a cellular serine/threonine kinase greatly diminished the extent of virion MA serine phosphorylation. Correspondingly, a protein kinase coimmunoprecipitated with Nef could phosphorylate MA on serine in vitro, producing a phosphopeptide pattern reminiscent of that of virion MA. Recombinant p21-activated kinase hPAK65, a recently proposed relative of the Nef-associated kinase, achieved a comparable result. Taken together, these data suggest that MA is a target of the Nef-associated serine kinase.     

Glucose enhances leptin signaling through modulation of AMPK activity

Leptin exerts its action by binding to and activating the long form of leptin receptors (LEPRb). LEPRb activates JAK2 that subsequently phosphorylates and activates STAT3. The JAK2/STAT3 pathway is required for leptin control of energy balance and body weight. Defects in leptin signaling lead to leptin resistance, a primary risk factor for obesity. Body weight is also regulated by nutrients, including glucose. Defects in glucose sensing also contribute to obesity. Here we report crosstalk between leptin and glucose. Glucose starvation blocked the ability of leptin to stimulate tyrosyl phosphorylation and activation of JAK2 and STAT3 in a variety of cell types. Glucose dose-dependently enhanced leptin signaling. In contrast, glucose did not enhance growth hormone-stimulated phosphorylation of JAK2 and STAT5. Glucose starvation or 2-deoxyglucose-induced inhibition of glycolysis activated AMPK and inhibited leptin signaling; pharmacological inhibition of AMPK restored the ability of leptin to stimulate STAT3 phosphorylation. Conversely, pharmacological activation of AMPK was sufficient to inhibit leptin signaling and to block the ability of glucose to enhance leptin signaling. These results suggest that glucose and/or its metabolites play a permissive role in leptin signaling, and that glucose enhances leptin sensitivity at least in part by attenuating the ability of AMPK to inhibit leptin signaling.     

HIV: a new role for Nef in the spread of HIV.

The HIV Nef protein downregulates the cell-surface expression of the HIV receptor glycoprotein CD4, but the significance of this event has remained obscure. Recent data suggest that Nef reduces cell-surface CD4 to promote the efficient spread of the virus.     

HIV-1 Nef enhances dendritic cell-mediated viral transmission to CD4+ T cells and promotes T-cell activation

HIV-1 Nef enhances dendritic cell (DC)-mediated viral transmission to CD4(+) T cells, but the underlying mechanism is not fully understood. It is also unknown whether HIV-1 infected DCs play a role in activating CD4(+) T cells and enhancing DC-mediated viral transmission. Here we investigated the role of HIV-1 Nef in DC-mediated viral transmission and HIV-1 infection of primary CD4(+) T cells using wild-type HIV-1 and Nef-mutated viruses. We show that HIV-1 Nef facilitated DC-mediated viral transmission to activated CD4(+) T cells. HIV-1 expressing wild-type Nef enhanced the activation and proliferation of primary resting CD4(+) T cells. However, when co-cultured with HIV-1-infected autologous DCs, there was no significant trend for infection- or Nef-dependent proliferation of resting CD4(+) T cells. Our results suggest an important role of Nef in DC-mediated transmission of HIV-1 to activated CD4(+) T cells and in the activation and proliferation of resting CD4(+) T cells, which likely contribute to viral pathogenesis.