Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef

The human immunodeficiency virus type 1 (HIV-1) Nef protein upregulates the expression of the invariant chain (Ii)/major histocompatibility complex class II (MHC-II) complex at the cell surface. This complex appears to reach the antigen-loading endosomal compartment at least in part via an indirect pathway in which it is internalized from the cell surface via the adaptor protein 2 (AP-2) complex. Here we provide evidence for a competition model to explain how Nef upregulates the expression of Ii at the cell surface. In this model, Nef and Ii compete for binding to AP-2. In support of this model, Nef decreased the rate of internalization of Ii from the cell surface. The AP-binding dileucine motif in Nef, ENTSLL(165), was necessary and sufficient for the upregulation of Ii. In addition, two leucine-based AP-binding motifs in the Ii cytoplasmic tail, DDQRDLI(8) and EQLPML(17), were critical for the efficient upregulation of Ii by Nef. Experiments using Nef variants in which the native dileucine-based sorting motif was replaced with similar motifs from cellular transmembrane proteins allowed modulation of AP-binding specificity. Analysis of these variants suggested that the binding of Nef to AP-2 is sufficient to upregulate Ii at the plasma membrane. Finally, interference with the expression of AP-2 caused an upregulation of Ii at the plasma membrane, and this decreased the effect of Nef. These data indicate that Nef usurps AP-2 complexes to dysregulate Ii trafficking and potentially interfere with antigen presentation in the context of MHC-II.     

Down-modulation of mature major histocompatibility complex class II and up-regulation of invariant chain cell surface expression are well-conserved functions of human and simian immunodeficiency virus nef alleles

Recently, it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Nef from laboratory strains down-modulates cell surface expression of mature major histocompatibility complex class II (MHC-II) molecules, while up-regulating surface expression of the invariant chain (Ii) associated with immature MHC-II (P. Stumptner-Cuvelette, S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, O. Schwartz, and P. Benaroch, Proc. Natl. Acad. Sci. USA 98:12144-12149, 2001). These Nef functions could contribute to impaired CD4(+)-T-helper-cell responses found in HIV-1-infected patients with progressive disease. However, it is currently unknown whether nef alleles derived from HIV-1-infected individuals or from other primate lentiviruses also modulate MHC-II and Ii. In the present study, we demonstrate that both activities are conserved among primary HIV-1 nef alleles, as well as among HIV-2 and simian immunodeficiency virus (SIV) nef alleles. Down-modulation of mature MHC-II required high levels of Nef expression. In contrast, surface expression of Ii was already strongly increased at low to medium levels of Nef expression. Notably, nef genes derived from two of four HIV-1-infected long-term nonprogressors did not up-regulate Ii, whereas nef alleles derived from 10 individuals with progressive disease were active in this assay. Unlike other in vitro Nef functions, the average activity of Nef in modulating MHC-II and Ii surface expression did not change significantly during the course of infection. Mutational analysis confirmed that MHC-II down- and Ii up-regulation are functionally separable from each other and from other Nef functions and identified acidic residues, located at the base of the flexible C-proximal loop of Nef, that are critical for increased Ii expression. Overall, our results suggest that the ability of Nef to interfere with MHC-II antigen presentation might play a role in AIDS pathogenesis.     

CD4 and major histocompatibility complex class I downregulation by the human immunodeficiency virus type 1 nef protein in pediatric AIDS progression

The human immunodeficiency virus type 1 (HIV-1) nef gene is a crucial determinant in AIDS disease progression. Although several in vitro activities have been attributed to the Nef protein, identifying the one critical for in vivo pathogenicity remains elusive. In this study, we examined a large number of nef alleles derived at various time points from 13 perinatally infected children showing different progression rates: six nonprogressors (NPs), three slow progressors (SPs), and four rapid progressors (RPs). The patient-derived nef alleles were analyzed for their steady-state expression of a Nef protein, for their relative ability to downregulate cell surface expression of CD4 and major histocompatibility complex class I (MHC-I) and for their capacity to bind the clathrin adaptor AP-1 complex. We found that NP-derived nef alleles, compared to nef alleles isolated from SPs and RPs, had reduced CD4 and MHC-I downregulation activities. In contrast, SP- and RP-derived nef alleles did not differ and efficiently downregulated both CD4 and MHC-I. AP-1 binding was a conserved function of primary nef alleles not correlated with clinical progression. Defective Nef proteins from NPs, rather than sharing common specific changes in their sequences, accumulated various amino acid substitutions, mainly located outside the conserved domains previously associated with Nef biological properties. Our data indicate that Nef-mediated downregulation of cell surface CD4 and MHC-I significantly contributes to the expression of the pathogenic potential of HIV-1.     

Human major histocompatibility complex class II invariant chain is expressed on the cell surface.

Class II major histocompatibility complex antigens are intracellularly associated with a nonpolymorphic polypeptide referred to as the invariant chain. Before the class II heterodimer appears on the cell surface, the invariant chain dissociates but it has so far been unclear as to whether or not a proportion of the invariant chain also appears on the plasma membrane. We describe a study with three monoclonal antibodies which recognize an extracytoplasmic determinant present on all forms of the invariant chain and use them to demonstrate its presence on the surface of the intact cells. The determinants recognized by two of the antibodies were found to be located within the 60 amino acids at the extreme C-terminal (extracytoplasmic) end of the invariant chain. The invariant chain-specific monoclonal antibody, VIC-Y1, was found to bind a determinant located between amino acids 1 and 73, which correspond to mainly cytoplasmic residues. Using the C-terminal specific antibodies, the number of antibody binding sites on the surface of two B lymphoma lines was estimated to be 10(5) per cell. The results of this study appear to resolve the highly disputed question of whether or not the invariant chain can appear as a plasma membrane protein. The results are discussed in the context of a possible role for the invariant chain in antigen processing and presentation.     

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.     

Human immunodeficiency virus type 1 Vpu protein interacts with CD74 and modulates major histocompatibility complex class II presentation

The human immunodeficiency virus type 1 (HIV-1) Vpu accessory protein is a transmembrane protein that down regulates CD4 expression and promotes the release of new virions. We screened a human leukocyte-specific yeast two-hybrid expression library to discover novel Vpu-interacting cellular proteins. The major histocompatibility complex class II (MHC II) invariant chain, also called Ii or CD74, was found to be one such protein. We show direct binding of Vpu and CD74 by using a yeast two-hybrid assay and coimmunoprecipitation from HIV-1-infected cells. The cytoplasmic region of Vpu was found to interact with the 30-amino-acid cytoplasmic tail of CD74. Human monocytic U937 cells infected with wild-type or Vpu-defective HIV-1 and transfected cells showed that Vpu down modulated the surface expression of mature MHC II molecules. The reduction in cell surface mature MHC II molecules correlated with decreased antigen presentation to T cells in culture. Thus, the Vpu protein also contributes to viral persistence by attenuating immune responses during HIV infection. This report further exemplifies the rich diversity and redundancy shown by HIV in immune evasion.     

Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit

Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the mu but not the gamma/sigma subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical mu-binding motif. These data identify the mu subunit of AP-1 (mu1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with mu1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based mu-binding signal in the MHC-I CD by AP-1.     

Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element.

Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.     

Human immunodeficiency virus type 1 nef quasispecies in pathological tissue.

The role of the nef gene in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood. To provide a basis for studies on the role of nef in AIDS, we used targeted polymerase chain reaction amplification and DNA sequencing to determine the structure of nef genes in pathologic tissue from HIV-1-infected children and adults. We find that the nef reading frame is open in 92% of clones derived from both brain and lymphocytic tissue of children, suggesting that nef is expressed in these tissues. One HIV-1 clone, BRVA, obtained by coculture from the brain of an adult AIDS patient with progressive dementia, was previously shown to contain a duplicated region in nef. We show here that similar duplications are widespread in both adults and children with AIDS. However, coculture strongly selects against the broad spectrum of nef quasispecies found in tissue. These findings suggest functional selection for nef quasispecies in pathologic tissues during HIV-1 infection of the human host.     

Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain

The intracellular transport and location of major histocompatibility complex (MHC) class II molecules and associated invariant chain (Ii) were investigated in a human melanoma cell line. In contrast to the class II molecules, which remain stable for greater than 4 h after synthesis, the associated Ii is proteolytically processed within 2 h. During or shortly after synthesis the NH2-terminal cytoplasmic and membrane-spanning segment is in some of the Ii molecules cleaved off; during intracellular transport, class II associated and membrane integrated Ii is processed from its COOH terminus in distinct steps in endocytic compartments. Immunocytochemical studies at the light and electron microscopic level revealed the presence of class II molecules, but not of Ii on the cell surface. Intracellularly both Ii and class II molecules were localized in three morphologically and kinetically distinct compartments, early endosomes, multivesicular bodies, and prelysosomes. This localization in several distinct endosomal compartments contrasts with the localization of class II molecules in mainly one endocytic compartment in B lymphoblastoid cell lines. As in these lymphoblastoid cell lines Ii is known to be rapidly degraded it is conceivable that the rate of proteolysis of the class II associated Ii and its dissociation from class II molecules modulates the retention of the oligomeric complex in endocytic compartments, and as a consequence the steady-state distribution of these molecules within the endosomal system.     

Human T-cell leukemia virus type I trans activator induces class I major histocompatibility complex antigen expression in glial cells

Transfection of the tax gene encoding the trans activator of human T-cell leukemia virus type I into glial line cells induced class I major histocompatibility complex (MHC) antigens on these cells. This occurred through the interaction of tax protein with the gene encoding class I MHC antigens but not through any soluble factors, such as interferons, or factors from glial cells. Since neural cells do not usually express MHC antigens, this novel mechanism may be an intermediate event between viral infection and subsequent immune-mediated pathology in the central nervous system.     

Human immunodeficiency virus type 1 Nef domains required for disruption of major histocompatibility complex class I trafficking are also necessary for coprecipitation of Nef with HLA-A2

Human immunodeficiency virus type 1 (HIV-1) Nef is a critical protein that is necessary for HIV pathogenesis. Its roles include the disruption of major histocompatibility complex class I (MHC-I) and CD4 trafficking to promote immune evasion and viral spread. Mutational analyses have revealed that separate domains of Nef are required to affect these two molecules. To further elucidate how Nef disrupts MHC-I trafficking in T cells, we examined the role of protein domains that are required for this function (N-terminal alpha helix, polyproline, acidic, and oligomerization domains). We found that each of these regions was required for Nef to disrupt the transport of HLA-A2 to the cell surface and for Nef to coprecipitate with HLA-A2.     

Efficient endosomal localization of major histocompatibility complex class II-invariant chain complexes requires multimerization of the invariant chain targeting sequence

During biosynthesis, MHC class II-invariant chain complexes are transported into endosomal compartments where invariant chain (Ii) is degraded and class II encounters antigenic peptides. One of the signals that determines this intracellular transport route has been localized to the cytosolic domain of Ii. Deletion of this signal disrupts endosomal targeting and results in the stable expression of class II-Ii complexes at the surface. In this paper we have examined the role of Ii trimerization on the generation of this endosomal localization signal. In L cell transfectants expressing class II and both wild type Ii and a truncated form of Ii that lacks this endosomal localization signal, Ii was found to form multimers which could contain both wild type and truncated Ii. The multimers were not large aggregates but were found to be discrete complexes, probably the nine molecule class II-Ii complex that has been observed in human B cells. The co-expression of truncated Ii allowed for cell surface expression of a subset of wild type Ii. This surface-expressed wild type Ii associated with truncated Ii in multimers at a 2:1 ratio, indicating that these trimers contain two truncated and one wild type Ii molecule. These data suggest a division in trafficking of Ii trimers: if two wild type Ii molecules are present, the complex is transported to and rapidly degraded in endosomes, whereas the presence of only one wild type Ii results in trafficking and expression of the heterotrimer on the cell surface. Following surface arrival, complexes containing only a single wild type Ii molecule are internalized more rapidly and have a shorter half-life than complexes containing only truncated Ii molecules. These data suggest that although a single Ii cytosolic domain can function as a plasma membrane internalization signal, multimerization of Ii is required for efficient Golgi complex to endosome targeting of class II- Ii complexes.     

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.     

Interaction of human immunodeficiency virus type 2 Vpx and invariant chain

Vpx is a virion-associated protein of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency viruses. The yeast two-hybrid system was used to identify invariant chain (Ii) as a cellular protein that interacts with HIV-2 Vpx. Vpx-Ii interaction was confirmed in cell-free reactions using bacterially expressed glutathione S-transferase fusion proteins and by coimmunoprecipitation in transfected and infected cells. In chronically infected cells expressing Vpx, Ii levels were markedly decreased, presumably due to enhanced degradation. These findings suggest that Vpx may disrupt major histocompatibility complex class II antigen presentation.     

Simian immunodeficiency virus induces expression of class II major histocompatibility complex structures on infected target cells in vitro

The human immunodeficiency virus (HIV) and the closely related simian immunodeficiency virus (SIV) induce profound immune dysfunction in primate species. The present studies show that cell populations infected in vitro with SIV exhibit increases in major histocompatibility complex (MHC) class II antigen expression. Cell lines chronically infected with both the monkey and human viruses express substantially more MHC class II but not more lineage-restricted or activation antigens on their membranes than do uninfected cell lines. Furthermore, 2'-deoxy-5-iodouridine increased MHC class II antigen expression on SIV-infected cell lines in parallel with increased expression of viral antigens. MHC class II induction does not appear to be mediated through the production of a soluble factor, such as gamma interferon, by SIV-infected cells. Interestingly, studies of the kinetics of antigen expression by cell lines after SIV infection indicate that the induction of MHC class II structures is a late event. Immunoelectron microscopy revealed that MHC class II antigen is expressed not only on the surfaces of the SIV-infected cells but also on the envelope of virus particles derived from those cells. MHC antigen expression on virus-infected cells and the expression of those determinants by the virus may play a role in the pathogenesis of acquired immunodeficiency syndrome and the autoimmune abnormalities observed in HIV-infected individuals.     

Effect of invariant chain on major histocompatibility complex class I molecule expression and stability on human breast tumor cell lines

Invariant chain (Ii) binds to the human leukocyte antigen (HLA) class II molecule and assists it in the process of peptide acquisition. In addition, Ii binds to the HLA class I molecule, although there has been little study of its effects on the HLA class I molecule. In addition to its normal expression on antigen-presenting cells, Ii expression is up regulated in a variety of tumors. By flow cytometric analysis, we found that expression of Ii resulted in an increase in the number of cell surface HLA class I molecules and in the proportion of unstable HLA class I molecules at the surface of breast tumor cell lines. These data suggest that the expression of Ii by tumor cells may quantitatively and qualitatively alter the presentation of antigens on those cells.     

Selective downregulation of rhesus macaque and sooty mangabey major histocompatibility complex class I molecules by Nef alleles of simian immunodeficiency virus and human immunodeficiency virus type 2

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIV(smm/mac) and HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys.     

Maintenance of Nef-mediated modulation of major histocompatibility complex class I and CD4 after sexual transmission of human immunodeficiency virus type 1

Viruses encounter changing selective pressures during transmission between hosts, including host-specific immune responses and potentially varying functional demands on specific proteins. The human immunodeficiency virus type 1 Nef protein performs several functions potentially important for successful infection, including immune escape via down-regulation of class I major histocompatibility complex (MHC-I) and direct enhancement of viral infectivity and replication. Nef is also a major target of the host cytotoxic T-lymphocyte (CTL) response. To examine the impact of changing selective pressures on Nef functions following sexual transmission, we analyzed genetic and functional changes in nef clones from six transmission events. Phylogenetic analyses indicated that the diversity of nef was similar in both sources and acutely infected recipients, the patterns of selection across transmission were variable, and regions of Nef associated with distinct functions evolved similarly in sources and recipients. These results weighed against the selection of specific Nef functions by transmission or during acute infection. Measurement of Nef function provided no evidence that the down-regulation of either CD4 or MHC-I was optimized by transmission or during acute infection, although rare nef clones from sources that were impaired in these activities were not detected in recipients. Nef-specific CTL activity was detected as early as 3 weeks after infection and appeared to be an evolutionary force driving the diversification of nef. Despite the change in selective pressure between the source and recipient immune systems and concomitant genetic diversity, the majority of Nef proteins maintained robust abilities to down-regulate MHC-I and CD4. These data suggest that both functions are important for the successful establishment of infection in a new host.