Nef-mediated Down-modulation Research Articles

Nef-Mediated CD3-TCR Downmodulation Dampens Acute Inflammation and Promotes SIV Immune Evasion

SUMMARYThe inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However, the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here, we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological, immunological, and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions, however, viral replication and the clinical course of infection are attenuated. Thus, Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion.

Endocytic sorting motif interactions involved in Nef-mediated downmodulation of CD4 and CD3

Lentiviral Nefs recruit assembly polypeptide complexes and target sorting motifs in cellular receptors to induce their internalization. While Nef-mediated CD4 downmodulation is conserved, the ability to internalize CD3 was lost in HIV-1 and its precursors. Although both functions play key roles in lentiviral replication and pathogenicity, the underlying structural requirements are poorly defined. Here, we determine the structure of SIVmac239 Nef bound to the ExxxLM motif of another Nef molecule at 2.5 Å resolution. This provides a basis for a structural model, where a hydrophobic crevice in simian immunodeficiency virus (SIV) Nef targets a dileucine motif in CD4 and a tyrosine-based motif in CD3. Introducing key residues into this crevice of HIV-1 Nef enables CD3 binding but an additional N-terminal tyrosine motif is required for internalization. Our resolution of the CD4/Nef/AP2 complex and generation of HIV-1 Nefs capable of CD3 downregulation provide insights into sorting motif interactions and target discrimination of Nef.

Primate lentiviruses use at least three alternative strategies to suppress NF-κB-mediated immune activation.

Primate lentiviruses have evolved sophisticated strategies to suppress the immune response of their host species. For example, HIV-2 and most simian immunodeficiency viruses (SIVs) use their accessory protein Nef to prevent T cell activation and antiviral gene expression by downmodulating the T cell receptor CD3. This Nef function was lost in HIV-1 and other vpu-encoding viruses suggesting that the acquisition of Vpu-mediated NF-κB inhibition reduced the selection pressure for inhibition of T cell activation by Nef. To obtain further insights into the modulation of NF-κB activity by primate lentiviral accessory factors, we analyzed 32 Vpr proteins from a large panel of divergent primate lentiviruses. We found that those of SIVcol and SIVolc infecting Colobinae monkeys showed the highest efficacy in suppressing NF-κB activation. Vpr-mediated inhibition of NF-κB resulted in decreased IFNβ promoter activity and suppressed type I IFN induction in virally infected primary cells. Interestingly, SIVcol and SIVolc differ from all other primate lentiviruses investigated by the lack of both, a vpu gene and efficient Nef-mediated downmodulation of CD3. Thus, primate lentiviruses have evolved at least three alternative strategies to inhibit NF-κB-dependent immune activation. Functional analyses showed that the inhibitory activity of SIVolc and SIVcol Vprs is independent of DCAF1 and the induction of cell cycle arrest. While both Vprs target the IKK complex or a factor further downstream in the NF-κB signaling cascade, only SIVolc Vpr stabilizes IκBα and inhibits p65 phosphorylation. Notably, only de-novo synthesized but not virion-associated Vpr suppressed the activation of NF-κB, thus enabling NF-κB-dependent initiation of viral gene transcription during early stages of the replication cycle, while minimizing antiviral gene expression at later stages. Our findings highlight the key role of NF-κB in antiviral immunity and demonstrate that primate lentiviruses follow distinct evolutionary paths to modulate NF-κB-dependent expression of viral and antiviral genes.

Lentiviral Nef proteins manipulate T cells in a subset-specific manner.

The role of the accessory viral Nef protein as a multifunctional manipulator of the host cell that is required for effective replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in vivo is well established. It is unknown, however, whether Nef manipulates all or just specific subsets of CD4(+) T cells, which are the main targets of virus infection and differ substantially in their state of activation and importance for a functional immune system. Here, we analyzed the effect of Nef proteins differing in their T cell receptor (TCR)-CD3 downmodulation function in HIV-infected human lymphoid aggregate cultures and peripheral blood mononuclear cells. We found that Nef efficiently downmodulates TCR-CD3 in naive and memory CD4(+) T cells and protects the latter against apoptosis. In contrast, highly proliferative CD45RA(+) CD45RO(+) CD4(+) T cells were main producers of infectious virus but largely refractory to TCR-CD3 downmodulation. Such T cell subset-specific differences were also observed for Nef-mediated modulation of CD4 but not for enhancement of virion infectivity. Our results indicate that Nef predominantly modulates surface receptors on CD4(+) T cell subsets that are not already fully permissive for viral replication. As a consequence, Nef-mediated downmodulation of TCR-CD3, which distinguishes most primate lentiviruses from HIV type 1 (HIV-1) and its vpu-containing simian precursors, may promote a selective preservation of central memory CD4(+) T cells, which are critical for the maintenance of a functional immune system. The Nef proteins of human and simian immunodeficiency viruses manipulate infected CD4(+) T cells in multiple ways to promote viral replication and immune evasion in vivo. Here, we show that some effects of Nef are subset specific. Downmodulation of CD4 and TCR-CD3 is highly effective in central memory CD4(+) T cells, and the latter Nef function protects this T cell subset against apoptosis. In contrast, highly activated/proliferating CD4(+) T cells are largely refractory to receptor downmodulation but are main producers of infectious HIV-1. Nef-mediated enhancement of virion infectivity, however, was observed in all T cell subsets examined. Our results provide new insights into how primate lentiviruses manipulate their target cells and suggest that the TCR-CD3 downmodulation function of Nef may promote a selective preservation of memory CD4(+) T cells, which are critical for immune function, but has little effect on activated/proliferating CD4(+) T cells, which are the main targets for viral replication.

Efficient Nef-Mediated Downmodulation of TCR-CD3 and CD28 Is Associated with High CD4 + T Cell Counts in Viremic HIV-2 Infection

The role of the multifunctional accessory Nef protein in the immunopathogenesis of HIV-2 infection is currently poorly understood. Here, we performed comprehensive functional analyses of 50 nef genes from 21 viremic (plasma viral load, >500 copies/ml) and 16 nonviremic (<500) HIV-2-infected individuals. On average, nef alleles from both groups were equally active in modulating CD4, TCR-CD3, CD28, MHC-I, and Ii cell surface expression and in enhancing virion infectivity. Thus, many HIV-2-infected individuals efficiently control the virus in spite of efficient Nef function. However, the potency of nef alleles in downmodulating TCR-CD3 and CD28 to suppress the activation and apoptosis of T cells correlated with high numbers of CD4(+) T cells in viremic patients. No such correlations were observed in HIV-2-infected individuals with undetectable viral load. Further functional analyses showed that the Nef-mediated downmodulation of TCR-CD3 suppressed the induction of Fas, Fas-L, PD-1, and CTLA-4 cell surface expression as well as the secretion of gamma interferon (IFN-γ) by primary CD4(+) T cells. Moreover, we identified a single naturally occurring amino acid variation (I132T) in the core domain of HIV-2 Nef that selectively disrupts its ability to downmodulate TCR-CD3 and results in functional properties highly reminiscent of HIV-1 Nef proteins. Taken together, our data suggest that the efficient Nef-mediated downmodulation of TCR-CD3 and CD28 help viremic HIV-2-infected individuals to maintain normal CD4(+) T cell homeostasis by preventing T cell activation and by suppressing the induction of death receptors that may affect the functionality and survival of both virally infected and uninfected bystander cells.

Down-Modulation of CD8αβ Is a Fundamental Activity of Primate Lentiviral Nef Proteins

It is well established that the Nef proteins of human and simian immunodeficiency viruses (HIV and SIV) modulate major histocompatibility complex class I (MHC-I) cell surface expression to protect infected cells against lysis by cytotoxic T lymphocytes (CTLs). Recent data supported the observation that Nef also manipulates CTLs directly by down-modulating CD8αβ (J. A. Leonard, T. Filzen, C. C. Carter, M. Schaefer, and K. L. Collins, J. Virol. 85:6867-6881, 2011), but it remained unknown whether this Nef activity is conserved between different lineages of HIV and SIV. In this study, we examined a total of 42 nef alleles from 16 different primate lentiviruses representing most major lineages of primate lentiviruses, as well as nonpandemic HIV-1 strains and the direct precursors of HIV-1 (SIVcpz and SIVgor). We found that the vast majority of these nef alleles strongly down-modulate CD8β in human T cells. Primate lentiviral Nefs generally interacted specifically with the cytoplasmic tail of CD8β, and down-modulation of this receptor was dependent on the conserved dileucine-based motif and two adjacent acidic residues (DD/E) in the C-terminal flexible loop of SIV Nef proteins. Both of these motifs are known to be important for the interaction of HIV-1 Nef with AP-2, and they were also shown to be critical for down-modulation of CD4 and CD28, but not MHC-I, by SIV Nefs. Our results show that down-modulation of CD4, CD8β, and CD28 involves largely overlapping (but not identical) domains and is most likely dependent on conserved interactions of primate lentiviral Nefs with cellular adaptor proteins. Furthermore, our data demonstrate that Nef-mediated down-modulation of CD8αβ is a fundamental property of primate lentiviruses and suggest that direct manipulation of CD8+ T cells plays a relevant role in viral immune evasion.

The Presence of a vpu Gene and the Lack of Nef-Mediated Downmodulation of T Cell Receptor-CD3 Are Not Always Linked in Primate Lentiviruses

Nef is an accessory protein critical for the ability of human and simian immunodeficiency viruses (HIV and SIV) to replicate efficiently in their respective hosts. Previous analyses of members of 15 different primate lentivirus lineages revealed a link between Nef function and the presence of a vpu gene. In particular, Nef proteins of all vpu-containing viruses had lost their ability to downmodulate the T cell (TCR-CD3) receptor. Here we examined Nef proteins from eight additional SIV lineages, including SIVgor, SIVwrc, SIVolc, SIVgri, SIVdrl, SIVlho, SIVden, and SIVasc, from western lowland gorillas, western red colobus monkeys, olive colobus monkeys, grivet monkeys, drills, L'Hoest's monkeys, Dent's mona monkeys, and red-tailed monkeys, respectively. We found that except for the nef gene of SIVdrl, all of them were efficiently expressed and modulated CD4, major histocompatibility complex class I (MHC-I), CD28, CXCR4, and Ii cell surface expression and/or enhanced viral infectivity and replication. Furthermore, the Nef proteins of SIVgri, SIVlho, SIVwrc, SIVolc, and SIVgor antagonized tetherin. As expected, the Nef protein of SIVgor, which carries vpu, failed to downmodulate CD3, whereas those of SIVwrc, SIVgri, SIVlho, and SIVasc, which lack vpu, were capable of performing this function. Surprisingly, however, the Nef protein of the vpu-containing SIVden strain retained the ability to downmodulate TCR-CD3, whereas that of SIVolc, which does not contain vpu, was unable to perform this function. Although the SIVden Vpu is about 20 amino acids shorter than other Vpu proteins, it degrades CD4 and antagonizes tetherin. Our data show that there are exceptions to the link between the presence of a vpu gene and nef alleles deficient in CD3 modulation, indicating that host properties also affect the selective pressure for Nef-mediated disruption of TCR-CD3 signaling. Our results are also further evidence that tetherin antagonism is a common function of primate lentivirus Nef proteins and that the resistance of human tetherin to Nef represents a relevant barrier to cross-species transmission of SIVs to humans.

Counteraction of HLA-C-mediated immune control of HIV-1 by Nef.

A host genetic variant (-35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface, whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However, some individuals with the protective -35CC genotype exhibit high viral loads. Here, we investigated whether the ability of HIV-1 to replicate efficiently in the "protective" high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However, in individuals with the protective -35CC genotype we found a significant association between sVLs and the efficiency of Nef-mediated enhancement of virion infectivity and modulation of CD4, CD28, and the major histocompatibility complex class II (MHC-II)-associated invariant chain (Ii), while this was not observed in subjects with the -35TT genotype. Since the latter Nef functions all influence the stimulation of CD4(+) T helper cells by antigen-presenting cells, they may cooperate to affect both the activation status of infected T cells and the generation of an antiviral cytotoxic T-lymphocyte (CTL) response. In comparison, different levels of viremia in individuals with the common -35TT genotype were not associated with differences in Nef function but with differences in HLA-C mRNA expression levels. Thus, while high HLA-C expression may generally facilitate control of HIV-1, Nef may counteract HLA-C-mediated immune control in some individuals indirectly, by manipulating T-cell function and MHC-II antigen presentation.

Inefficient Nef-Mediated Downmodulation of CD3 and MHC-I Correlates with Loss of CD4+ T Cells in Natural SIV Infection

Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 animals with low (<500) CD4+ T-cells/µl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.

Nef alleles from children with non-progressive HIV-1 infection modulate MHC-II expression more efficiently than those from rapid progressors

It has been established that defective nef genes and differences in the Nef-mediated downmodulation of CD4 and MHC-I cell surface expression can be associated with different rates of HIV-1 disease progression. To evaluate whether nef alleles derived from perinatally HIV-1-infected children showing no, slow or rapid disease progression differ in their abilities to downmodulate mature MHC-II or to upregulate the invariant chain (Ii) associated with immature MHC-II complexes. Nef alleles derived from HIV-1-infected children were cloned into expression vectors and proviral HIV-1 constructs co-expressing Nef and enhanced green fluorescence protein via an internal ribosomal entry site. Nef-mediated modulation of CD4, MHC-I, MHC-II or Ii surface expression was analysed by flow cytometric analysis of Jurkat T cells, monocytic THP-1 cells, CD4 T cells and macrophages transduced with vesicular stomatitis virus G-pseudotyped HIV-1 nef variants or transiently transfected HeLa class II transactivator cells. : Nef alleles derived from HIV-1-infected children with non-progressive infection were significantly more active in the upregulation of Ii and downregulation of MHC-II than those derived from rapid progressors. Nef alleles particularly active in interfering with MHC-II antigen presentation are more frequently found in perinatally HIV-1-infected non-progressors than rapid progressors. Possibly in the context of an immature host immune system, strongly impaired MHC-II function might contribute to lower levels of immune activation and a decelerated loss of CD4 T cells.

Nef interference with HIV-1–specific CTL antiviral activity is epitope specific

AbstractHIV-1 Nef and HIV-1–specific cytotoxic T lymphocytes (CTLs) have important and opposing roles in the immunopathogenesis of HIV-1 infection. Nef-mediated down-modulation of HLA class I on infected cells can confer resistance to CTL clearance, but the factors determining the efficiency of this process are unknown. This study examines the impact of Nef on the antiviral activity of several CTL clones recognizing epitopes from early and late HIV-1 proteins, restricted by HLA-A, -B, and -C molecules. CTL-targeting epitopes in early proteins remained susceptible to the effects of Nef, although possibly to a lesser degree than CTL-targeting late protein epitopes, indicating that significant Nef-mediated HLA down-regulation can precede even the presentation of early protein-derived epitopes. However, HLA-C–restricted CTLs were unaffected by Nef, consistent with down-regulation of cell-surface HLA-A and -B but not HLA-C molecules. Thus, CTLs vary dramatically in their susceptibility to Nef interference, suggesting differences in the relative importance of HLA-A– and HLA-B– versus HLA-C–restricted CTLs in vivo. The data thus indicate that HLA-C–restricted CTLs may have an under-appreciated antiviral role in the setting of Nef in vivo and suggest a benefit of promoting HLA-C–restricted CTLs for immunotherapy or vaccine development.

The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation.

The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.

The Conserved Process of TCR/CD3 Complex Down-Modulation by SIV Nef Is Mediated by the Central Core, Not Endocytic Motifs

The Nef protein of Simian immunodeficiency virus (SIV) associates with multiple T lymphocyte signaling proteins, including the T cell receptor (TCR) ζ chain. We demonstrate here that these interactions are conserved and highly specific. Nefs derived from genetically diverse strains of SIV (SIV mac239, SIV smmPBj, and SIV smmΔB670) all interacted with TCR ζ on two separate domains, referred to as SIV Nef interaction domains (SNIDs), as examined in both yeast two-hybrid and glutathione- S-transferase (GST) fusion protein pull-down assays. Multiple HIV-1 Nefs were examined and none interacted with TCR ζ. In contrast, HIV-2 UC1 Nef, similar to SIV Nef, interacted with TCR ζ on two domains, although only the SIV Nefs potently reduced cell-surface expression of the TCR/CD3 complex in T cells. In addition, we examined the abilities of SIV, HIV-2, and HIV-1 Nefs to interact with the cytoplasmic domains of other signaling molecules including CD3ϵ, CD3γ, and FcϵRIγ, which also contain YxxL motifs, and determined that SIV and HIV-2 Nefs interacted only with TCR ζ, whereas HIV-1 Nef did not interact with any signal-transducing cytoplasmic domain examined. Last, to gain further insight into the mechanism by which Nef down-modulates the TCR/CD3 complex, we mutated or deleted regions on Nef involved in endocytosis, localization of Nef to the plasma membrane, interaction with cellular kinases, or that were conserved among multiple strains of SIV. Mutation of the myristoylation site and a conserved region surrounding a putative PKC phosphorylation site were the only mutations that abrogated Nef-mediated down-modulation of the TCR/CD3 complex. These findings demonstrate there is a spectrum of associations between SIV, HIV-2, and HIV-1 Nefs, and the TCR/CD3 complex, and suggest that down-modulation of the TCR/CD3 complex occurs via association with subsets of cellular proteins that are different from those involved in CD4 and CD28 down-modulation.

HIV-1 Nef Blocks Transport of MHC Class I Molecules to the Cell Surface Via a PI 3-Kinase-Dependent Pathway

HIV causes a chronic infection by evading immune eradication. A key element of HIV immune escape is the HIV-1 Nef protein. Nef causes a reduction in the level of cell surface major histocompatibility complex class I (MHC-I) protein expression, thus protecting HIV-infected cells from anti-HIV cytotoxic T lymphocyte (CTL) recognition and killing. Nef also reduces cell surface levels of the HIV receptor, CD4, by accelerating endocytosis. We show here that endocytosis is not required for Nef-mediated downmodulation of MHC-I molecules. The main effect of Nef is to block transport of MHC-I molecules to the cell surface, leading to accumulation in intracellular organelles. Furthermore, the effect of Nef on MHC-I molecules (but not on CD4) requires phosphoinositide 3-kinase (PI 3-kinase) activity. We propose that Nef diverts MHC-1 proteins into a PI 3-kinase-dependent transport pathway that prevents expression on the cell surface.

Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and degradation of surface CD4.

Human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated protein with a relative molecular mass of 27 kDa, is localized to the cytoplasmic surfaces of cellular membranes, and has been reported to down-modulate CD4 in human T cells. To understand the mechanism of HIV-1 Nef-mediated down-modulation of cell surface CD4, we expressed Nef protein in human T-cell line VB. Expression of HIV-1 Nef protein down-modulated surface CD4 molecules. In pulse-chase experiments, CD4 molecules in Nef-expressing cells were synthesized at normal levels. However, the bulk of newly synthesized CD4 protein was degraded with a half-life of approximately 6 h, compared with the 24-h half-life in control cells. This Nef-induced acceleration of CD4 turnover was inhibited by lysosomotropic agents NH4Cl and chloroquine as well as by the protease inhibitor leupeptin. Surface CD4 biotinylation experiments demonstrated that CD4 molecules in Nef-expressing T cells are transported to the plasma membrane with normal kinetics but are then rapidly internalized. Therefore, HIV-1 Nef-induced down-modulation of CD4 is due to rapid internalization of surface CD4 and subsequent degradation by an acid-dependent process, potentially lysosomal. Additionally, in a Nef-expressing cell, we find accelerated dissociation of the T-cell tyrosine kinase p56lck and CD4 but only after the complex reaches the plasma membrane. This implies that HIV-1 Nef protein might play a role in triggering a series of T-cell activation-like events, which contribute to p56lck dissociation and internalization of surface CD4 molecules.