HIV-1 Infection of T Cells and Macrophages Are Differentially Modulated by Virion-Associated Hck: A Nef-Dependent Phenomenon

The proline repeat motif (PxxP) of Nef is required for interaction with the SH3 domains of macrophage-specific Src kinase Hck. However, the implication of this interaction for viral replication and infectivity in macrophages and T lymphocytes remains unclear. Experiments in HIV-1 infected macrophages confirmed the presence of a Nef:Hck complex which was dependent on the Nef proline repeat motif. The proline repeat motif of Nef also enhanced both HIV-1 infection and replication in macrophages, and was required for incorporation of Hck into viral particles. Unexpectedly, wild-type Hck inhibited infection of macrophages, but Hck was shown to enhance infection of primary T lymphocytes. These results indicate that the interaction between Nef and Hck is important for Nef-dependent modulation of viral infectivity. Hck-dependent enhancement of HIV-1 infection of T cells suggests that Nef-Hck interaction may contribute to the spread of HIV-1 infection from macrophages to T cells by modulating events in the producer cell, virion and target cell.     

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.     

Spotlight on HIV-1 Nef: SERINC3 and SERINC5 Identified as Restriction Factors Antagonized by the Pathogenesis Factor

The Nef protein is an accessory gene product encoded by human immunodeficiency virus types 1 and 2 (HIV-1/-2) and simian immunodeficiency virus (SIV) that boosts virus replication in the infected host and accelerates disease progression. Unlike the HIV-1 accessory proteins Vif, Vpr and Vpu, Nef was, until recently, not known to antagonize the antiviral activity of a host cell restriction factor. Two recent reports now describe the host cell proteins serine incorporator 3 and 5 (SERINC3 and SERINC5) as potent inhibitors of HIV-1 particle infectivity and demonstrate that Nef counteracts these effects. These findings establish SERINC3/5 as restrictions to HIV replication in human cells and define a novel activity for the HIV pathogenesis factor Nef.     

Extracellular Nef protein activates signal transduction pathway from Ras to mitogen-activated protein kinase cascades that leads to activation of human immunodeficiency virus from latency

We previously reported that viral antigen expression was markedly up-regulated by stimulation with extracellular Nef, similar to the effects of tumor necrosis factor (TNF)-alpha and phorbol myristate acetate, in model cells for HIV-1 latency. In this study, we examined the molecular mechanism of this novel Nef function. Flow cytometry revealed specific binding of Nef on the surface of latently infected cells. Furthermore, activation of Ras in the cells was detected after treatment with Nef, indicating the involvement of Ras in Nef-mediated activation of HIV-1 from latency. This was also confirmed by the observations that HIV-1 long-terminal repeat-luciferase (LTR-Luc) activity was significantly up-regulated by introduction of the active Ras into uninfected cells, and that LTR-Luc activity observed in Nef-treated cells was specifically inhibited by introduction of a dominant negative Ras. In addition, PD98059 inhibited the activation of HIV-1 by Nef, but not by TNF-alpha. Thus, Nef-mediated reactivation of HIV-1 in latent model cells occurs by signal transduction from Ras to mitogen-activated protein kinase cascades.     

Partial "repair" of defective NEF genes in a long-term nonprogressor with human immunodeficiency virus type 1 infection

A 36-bp deletion close to the 5' end of NEF that impaired Nef function was found in a long-term nonprogressor with human immunodeficiency virus type 1 (HIV-1) infection. Forms containing an adjacent duplication of 33 bp were also frequently observed. The duplication showed no homology to the deleted region but restored the overall length of the first variable loop of Nef. NEF alleles carrying the duplication were active in class I major histocompatibility complex (MHC-I) down-modulation and enhancement of virus infectivity. However, they showed little activity in CD4 down-regulation and were unable to stimulate viral replication in human peripheral blood mononuclear cells. Our study indicates that the enhancement of virion infectivity and the stimulation of HIV-1 replication in lymphocytes are distinct functions of Nef. Our findings also illustrate the capacity for repair of attenuating deletions in HIV-1 infection and suggest that a selective pressure for Nef-mediated MHC-I down-modulation and/or enhancement of virion infectivity exists.     

Substitution of HIV Type 1 Nef with HTLV-1 p12

Human retroviruses, such as HTLV-1 and HIV-1, encode accessory proteins, which regulate viral pathogenesis. The p12 protein of HTLV-1 is encoded from the pX-I open reading frame, and is critical for efficient virus replication in rabbits. Although dispensable for infection, replication, and immortalization of activated lymphocytes in culture, p12 expression is important for infection of quiescent lymphocytes. Similar to HTLV-1 p12, Nef is important for virus infectivity in SIV animal models. We questioned whether p12 could replace Nef in HIV-1, and reconstitute virus replication in culture. We found that p12 could complement for effects of Nef on HIV-1 infection of Magi-CCR5 cells or macrophages.     

Nef Obtained from Individuals with HIV-1 Vary in Their Ability to Antagonize SERINC3- and SERINC5-Mediated HIV-1 Restriction

Nef is a multifunctional viral protein that has the ability to downregulate cell surface molecules, including CD4 and major histocompatibility complex class I (MHC-I) and, as recently shown, also members of the serine incorporator family (SERINC). Here, we analyzed the impact of naturally occurring mutations in HIV-1 Nef on its ability to counteract SERINC restriction and the clinical course of infection. HIV-1 Nef sequences were obtained from 123 participants of the Amsterdam Cohort Studies and showed multiple amino acid variations and mutations. Most of the primary Nef proteins showed increased activity to counteract SERINC3 and SERINC5 as compared to NL4-3 Nef. Several mutations in Nef were associated with either an increased or decreased infectivity of Bal26-pseudotyped HIV-1 produced in the presence of SERINC3 or SERINC5. The 8R, 157N and R178G Nef mutations were shown to have an effect on disease progression. Survival analysis showed an accelerated disease progression of individuals infected with HIV-1 carrying arginine or asparagine at position 8 or 157 in Nef, respectively, or the R178G Nef mutation. Here, we observed that naturally occurring mutations in Nef affect the ability of Nef to counteract SERINC3- and SERINC5-mediated inhibition of viral infectivity. The majority of these Nef mutations had no significant effect on HIV-1 pathogenesis and only the 8R, 157N and R178G mutations were associated with disease course.     

Role of the CD4 down-modulation activity of Nef in HIV-1 infectivity

The regulatory Nef protein of HIV-1/2 and SIV is required for high viral replication and disease progression, thus represents a very attractive therapeutic target. Because of the multi-functional nature of the Nef protein, it is unclear which of the several Nef activities are most crucial in vivo for the outcome of viral infection. Some findings indicate that the CD4 down-regulation activity of Nef is critical for viral infectivity as well as for progression to immunodeficiency. On the other hand, more recent evidences suggest that CD4 targeting and stimulation of infectivity are two separate functions of Nef. This controversial issue will be discussed here in the light of the latest findings.     

The Nef-infectivity enigma: mechanisms of enhanced lentiviral infection

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.     

HIV Type 1 Nef is released from infected cells in CD45(+) microvesicles and is present in the plasma of HIV-infected individuals

HIV-1 Nef has been demonstrated to be integral for viral persistence, infectivity, and the acceleration of disease pathogenesis (AIDS) in humans. Nef has also been detected in the plasma of HIV-infected individuals and is released from infected cells. The form in which Nef is released from infected cells is unknown. However, Nef is a myristoylated protein and has been shown to interact with the intracellular vesicular trafficking network. Here we show that Nef is released in CD45-containing microvesicles. This microvesicular Nef (mvNef) is detected in the plasma of HIV-infected individuals at relatively high concentrations (10?ng/ml). It is also present in tissue culture supernatants of Jurkat cells infected with HIV(MN). Interestingly, plasma mvNef levels in HIV(+) patients did not significantly correlate with viral load or CD4 count. Microvesicular Nef levels persisted in the plasma of HIV-infected individuals despite the use of antiretroviral therapy, even in individuals with undetectable viral loads. Using cell lines, we found Nef microvesicles induce apoptosis in Jurkat T-lymphocytes but had no observed effect on the U937 monocytic cell line. Given the large amount of mvNef present in the plasma of HIV-infected individuals, the apoptotic effect of mvNef on T cells, and the observed functions of extracellular soluble Nef in vitro, it seems likely that in vivo mvNef may play a significant role in the pathogenesis of AIDS.     

Interleukin 10 is induced by recombinant HIV-1 Nef protein involving the calcium/calmodulin-dependent phosphodiesterase signal transduction pathway

HIV-1 Nef protein shares a significant homology with the immunosuppressive and highly conserved retroviral transmembrane protein p15E. In the present study, extracellular Nef protein is shown to induce interleukin (IL)-10 mRNA expression in human peripheral blood mononuclear cells as well as in cells of H9 T and U937 promonocytic human cell lines. Release of IL-10 protein into supernatants of peripheral blood mononuclear cells stimulated with Nef is dose-dependent. Expression of cytokines IL-2, IL-4, IL-5, IL-12 p40, IL-13, and interferon γ is not affected by Nef stimulation. IL-10 protein production induced by Nef is inhibited by the calcium/calmodulin phosphodiesterase inhibitor W-7 but not by the protein kinase A inhibitor H-89 nor the protein kinase C inhibitors staurosporine and calphostin C. The calcium chelating agent EGTA also inhibits the IL-10 production induced by Nef, and this inhibition is reversed by the addition of calcium along with Nef. These findings indicate that extracellular Nef may contribute to the immunopathogenesis of HIV infection by inducing IL-10.     

Inhibition of HIV-1 infection by alkylureas.

The risk of infection by HIV-1 through transfusion of contaminated blood products has been markedly decreased but not eliminated by serological screening of donors. Methods are required to further minimize or eliminate the risk of infection of blood product recipients. We therefore examined the capacity of alkylureas to inhibit infectivity of HIV-1. Incubation of free HIV-1 virions with alkylureas suppressed their infectivity, and the minimal inhibitory concentration of the alkylureas was related to the length of the alkyl chain. Butylurea, the most potent inhibitor of HIV-1, inhibited the infectivity of 10(5) median tissue culture infective dose (TCID)50 of HIV-1, chronically HIV-1-infected H9 cells and mononuclear cells from two HIV-1-infected patients. Size fractionation of HIV-1 following incubation with butylurea indicated that the structure of the virus was disrupted by butylurea. This study demonstrates that butylurea, at a concentration that has been shown not to affect red blood cell function, can inhibit infectivity of extracellular and intracellular HIV-1. Since the HIV-1 inhibitory capacity of the alkylureas increases with the length of the alkyl side chain, it is likely that hydrophobic interactions between the alkylureas and HIV-1 are responsible for the observed effect.     

Nef surfaces: where to interfere with function

The HIV-1 Nef protein is an accessory protein of 24-27 kDa mass that mediates a multitude of effector functions in infected cells. Although not essentially required for viral replication, HIV-1 Nef exhibits stimulating potential towards disease progression to AIDS and is therefore considered a pathogenic factor in retroviridae. Here we correlate sequence conservation in HIV-1 Nef with surface hydrophobicity and functionality in protein-protein interaction to identify accessible substructures on the surface of Nef that might be suitable as pharmacological target sites. Recent advances in targeting of Nef by small molecular compounds that interfere with SH3 domain binding or MHC class I down-regulation are discussed. Similarly, approaches for the use of larger molecules are introduced, such as tailored fusion proteins that simultaneously interact with multiple highly conserved sequence motifs of Nef. In addition, the design of a single domain antibody from llama that interferes with CD4 down-regulation activity and PAK2 binding is discussed. The flexibility in binding recognition is exemplarily shown for the modulation of RT-loop binding using engineered SH3 domains. The various considerations corroborate the potential of HIV-1 Nef as a promising target for the development of potent Nef inhibitors.     

Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages

Although successful attempts to inhibit HIV-1 replication in T cells using RNAi have been reported, the effect of HIV-specific RNAi on macrophages is not well known. Macrophages are key targets for anti-HIV-1 therapy because they are able to survive long after the initial infection with HIV and can spread the virus to T cells. In this study, we identified a putative RNAi target of HIV, consisting of the portion of the nef gene overlapping the U3 region (Nef366), and generated a lentivirus-based short hairpin RNA (shRNA) expression vector (Lenti shNef366). We show that Lenti shNef366 inhibits (1) HIV-1 replication in a monocytic cell line and in primary monocyte-derived macrophages (MDMs), (2) reactivation of latent HIV-1 infection, and (3) the production of secondary HIV-1 from MDMs harboring a genomic copy of Nef366. Moreover, we found that the up-regulated production of macrophage inflammatory protein 1beta (MIP-1beta), but not MIP-1alpha, in MDMs by Nef expression was considerably suppressed by Lenti shNef366, which suggests that HIV-1 dissemination to T cells through its interaction with HIV-1-infected MDMs can also be controlled by Lenti shNef366. Thus, lentivirus-mediated shRNA expression targeting the U3-overlapping region of HIV nef represents a feasible approach to genetic vaccine therapy for HIV-1.     

nef-deleted HIV-1 inhibits phagocytosis by monocyte-derived macrophages in vitro but not by peripheral blood monocytes in vivo

OBJECTIVE: HIV-1 infection impairs a number of macrophage effector functions, but the mechanism is unknown. We studied the role of HIV-1 Nef in modulating phagocytosis by human monocytes and monocyte-derived macrophages (MDM). DESIGN AND METHODS: Using a flow cytometric assay, phagocytosis of Mycobacterium avium complex (MAC) by monocytes in whole blood of Sydney Blood Bank Cohort (SBBC) members infected with a nef-deleted (Delta nef) strain of HIV-1 was compared with that of monocytes from uninfected or wild-type (WT) HIV-infected subjects. The specific impact of Nef on phagocytosis by MDM was determined by either infecting cells in vitro with Delta nef strains of HIV-1 or electroporating Nef into uninfected MDM. RESULTS: MAC phagocytic capacity of monocytes from SBBC members was equivalent to that of cells from uninfected individuals (P = 0.81); it was greater than that of cells from individuals infected with WT HIV-1 (P < 0.0001), irrespective of CD4 counts and HIV viral load. In contrast, in vitro infection of MDM with either Delta nef or WT strains of HIV-1 resulted in similar levels of HIV replication and equivalent impairment of phagocytosis via Fc gamma and complement receptors. Electroporation of Nef into MDM did not alter phagocytic capacity. CONCLUSIONS: This study provides evidence demonstrating the complex indirect effect of Nef on phagocytosis by peripheral blood monocytes (infrequently infected with HIV-1) in vivo. Conversely, the fact that MDM infected with either Delta nef or WT HIV-1 in vitro (high multiplicity of infection) show comparably impaired phagocytosis, indicates that HIV-1 infection of macrophages can directly impair function, independent of Nef.     

Epitope targeting and viral inoculum are determinants of Nef-mediated immune evasion of HIV-1 from cytotoxic T lymphocytes

The impact of HIV-1 Nef-mediated HLA-I down-regulation on CD8(+) cytotoxic T lymphocytes (CTLs) varies by epitope, but the determining factors have not been elucidated. In the present study, we investigated the impact of Nef on the antiviral efficiency of HIV-1-specific CTLs targeting 17 different epitopes to define properties that determine susceptibility to Nef. The impact of Nef was not correlated with the presenting HLA-I type or functional avidity of CTLs, but instead was related directly to the kinetics of infected cell clearance. Whereas Gag-specific CTLs generally were less susceptible to Nef than those targeting other proteins, this was determined by the ability to eliminate infected cells before de novo synthesis of viral proteins, which was also observed for CTLs targeting a Nef epitope. This very early clearance of infected cells depended on virus inoculum, and the required inoculum varied by epitope. These results suggest that whereas Gag-specific CTLs are more likely to recognize infected cells before Nef-mediated HLA-I down-regulation, this varies depending on the specific epitope and virus inoculum. Reduced susceptibility to Nef therefore may contribute to the overall association of Gag-specific CTL responses to better immune control if a sufficient multiplicity of infection is attained in vivo, but this property is not unique to Gag.     

HIV type 1 Nef increases the association of T cell receptor (TCR)-signaling molecules with T cell rafts and promotes activation-induced raft fusion

HIV-1 Nef (Nef) is a myristoylated protein that contributes to HIV disease pathogenesis. Nef has a modulatory effect on T cell receptor (TCR) signaling, resulting in up-regulation of interleukin-2 (IL-2) production in stimulated T cells. Recent studies have shown that efficient TCR signaling requires enhanced association of TCR-signaling molecules with plasma membrane microdomains (lipid rafts) and fusion of rafts into larger structures. We utilized Jurkat T cell lines expressing wild-type Nef (Nef(wt)) and a myristoylation-deficient form of Nef (Nef(G)2(A)), from an inducible promoter, to determine the effects of Nef on the association of TCR-signaling molecules with rafts in nonstimulated T cells. In addition, the effect of Nef on raft size, before and after TCR activation by CD3 cross-linking, was also examined. Following induction, Nef(wt) was associated with both rafts and nonrafts, while Nef(G)2(A) was almost exclusively cytosolic. Induction of Nef(wt), but not Nef(G)2(A), coincided with an increased association of the src family tyrosine kinase, Lck, and TCRzeta with rafts, but not with nonrafts. Further, rafts were found to be significantly larger in CD3-activated T cells in the presence of Nef(wt) when compared to nonexpressing cells. We propose that myristoylated, raft-localized Nef primes resting T cells for activation by increasing the levels of signaling molecules within rafts, and that TCR activation is enhanced by the capacity of Nef to promote raft fusion.     

HIV-1 Nef impairs MHC class II antigen presentation and surface expression

HIV-1-infected cells can avoid cytotoxic T lymphocyte killing by Nef-mediated down-regulation of surface MHC I. Here, we show that HIV-1 Nef inhibits MHC II restricted peptide presentation to specific T cells and thus may affect the induction of antiviral immune responses. Nef mediates this effect by reducing the surface level of mature (i.e., peptide-loaded) MHC II while increasing levels of immature MHC II, which are functionally incompetent because of their association with the invariant chain. Nef was the only HIV-1 gene product to possess this capacity, which was also observed in the context of the whole HIV-1 genome. Other proteins of the endocytic pathway were not affected by Nef expression, suggesting that Nef effects on MHC II did not result from a general alteration of the endocytic pathway. Response patterns to previously characterized mutations of Nef differed for Nef-induced modulation of mature and immature MHC II. Furthermore, the doses of Nef required to observe each of the two effects were clearly different, suggesting that Nef could affect MHC II peptide presentation through distinct mechanisms. Cooperation between those mechanisms may enable Nef to efficiently inhibit MHC II function.