The export of major histocompatibility complex class I molecules from the endoplasmic reticulum of rat brown adipose cells is acutely stimulated by insulin

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase-dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.     

Tmp21, a novel MHC-I interacting protein, preferentially binds to Β2-microglobulin-free MHC-I heavy chains

MHC-I molecules play a critical role in immune surveillance against viruses by presenting peptides to cytotoxic T lymphocytes. Although the mechanisms by which MHC-I molecules assemble and acquire peptides in the ER are well characterized, how MHC-I molecules traffic to the cell surface remains poorly understood. To identify novel proteins that regulate the intracellular transport of MHC-I molecules, MHC-I-interacting proteins were isolated by affinity purification, and their identity was determined by mass spectrometry. Among the identified MHC-I-associated proteins was Tmp21, the human ortholog of yeast Emp24p, which mediates the ER-Golgi trafficking of a subset of proteins. Here, we show that Tmp21 binds to human classical and non-classical MHC-I molecules. The Tmp21-MHC-I complex lacks Β(2)-microglobulin, and the number of the complexes is increased when free MHC-I heavy chains are more abundant. Taken together, these results suggest that Tmp21 is a novel protein that preferentially binds to Β(2)-microglobulin-free MHC-I heavy chains.     

Selective export of HLA-F by its cytoplasmic tail

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.     

The C-Terminal Amino Acid of the MHC-I Heavy Chain Is Critical for Binding to Derlin-1 in Human Cytomegalovirus US11-Induced MHC-I Degradation

Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.     

Adaptor Protein 1 Promotes Cross-Presentation through the Same Tyrosine Signal in Major Histocompatibility Complex Class I as That Targeted by HIV-1

Certain antigen-presenting cells (APCs) process and present extracellular antigen with major histocompatibility complex class I (MHC-I) molecules to activate naive CD8⁺ T cells in a process termed cross-presentation. We used insights gained from HIV immune evasion strategies to demonstrate that the clathrin adaptor protein adaptor protein 1 (AP-1) is necessary for cross-presentation by MHC-I molecules containing a cytoplasmic tail tyrosine signal (murine MHC-I molecules, human MHC-I HLA-A and HLA-B allotypes). In contrast, AP-1 activity was not needed for cross-presentation by MHC-I molecules containing a human MHC-I HLA-C cytoplasmic tail, which does not contain a tyrosine signal. AP-1 activity was also dispensable for presentation of endogenous antigens by MHC-I via the classical pathway. In APCs, we show that HIV Nef disrupts cross-presentation by MHC-I containing the tyrosine signal but does not affect cross-presentation by MHC-I containing the HLA-C cytoplasmic tail. Thus, we provide evidence for two separable cross-presentation pathways, only one of which is targeted by HIV.     

Equine herpesvirus type 4 UL56 and UL49.5 proteins downregulate cell surface major histocompatibility complex class I expression independently of each other

Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.     

Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation

Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation.     

The putative natural killer decoy early gene m04 (gp34) of murine cytomegalovirus encodes an antigenic peptide recognized by protective antiviral CD8 T cells

Several early genes of murine cytomegalovirus (MCMV) encode proteins that mediate immune evasion by interference with the major histocompatibility complex class I (MHC-I) pathway of antigen presentation to cytolytic T lymphocytes (CTL). Specifically, the m152 gene product gp37/40 causes retention of MHC-I molecules in the endoplasmic reticulum (ER)-Golgi intermediate compartment. Lack of MHC-I on the cell surface should activate natural killer (NK) cells recognizing the "missing self." The retention, however, is counteracted by the m04 early gene product gp34, which binds to folded MHC-I molecules in the ER and directs the complex to the cell surface. It was thus speculated that gp34 might serve to silence NK cells and thereby complete the immune evasion of MCMV. In light of these current views, we provide here results demonstrating an in vivo role for gp34 in protective antiviral immunity. We have identified an antigenic nonapeptide derived from gp34 and presented by the MHC-I molecule D(d). Besides the immunodominant immediate-early nonapeptide consisting of IE1 amino acids 168-176 (IE1(168-176)), the early nonapeptide m04(243-251) is the second antigenic peptide described for MCMV. The primary immune response to MCMV generates significant m04-specific CD8 T-cell memory. Upon adoptive transfer into immunodeficient recipients, an m04-specific CTL line controls MCMV infection with an efficacy comparable to that of an IE1-specific CTL line. Thus, gp34 is the first noted early protein of MCMV that escapes viral immune evasion mechanisms. These data document that MCMV is held in check by a redundance of protective CD8 T cells recognizing antigenic peptides in different phases of viral gene expression.     

A specific endoplasmic reticulum export signal drives transport of stem cell factor (Kitl) to the cell surface

Stem cell factor, also known as Kit ligand (Kitl), belongs to the family of dimeric transmembrane growth factors. Efficient cell surface presentation of Kitl is essential for the migration, proliferation, and survival of melanocytes, germ cells, hemopoietic stem cells, and mastocytes. Here we demonstrate that intracellular transport of Kitl to the cell surface is driven by a motif in the cytoplasmic tail that acts independently of the previously described basolateral sorting signal. Transport of Kitl to the cell surface is controlled at the level of the endoplasmic reticulum (ER) and requires a C-terminal valine residue positioned at a distance of 19-36 amino acids from the border between the transmembrane and cytoplasmic domains. Deletion or substitution of the valine with other hydrophobic amino acids results in ER accumulation and reduced cell surface transport of Kitl at physiological expression levels. When these mutant proteins are overexpressed in the ER, they are transported by bulk flow to the cell surface albeit at lower efficiency. A fusion construct between Kitl and the green fluorescent protein-labeled extracellular domain of a temperature-sensitive mutant of vesicular stomatitis virus G protein revealed the valine-dependent recruitment into coat protein complex II-coated ER exit sites and vesicular ER to Golgi transport in living cells. Thus the C-terminal valine defines a specific ER export signal in Kitl. It is responsible for the capture of Kitl at coat protein complex II-coated ER exit sites, leading to subsequent cell surface transport under physiological conditions.     

Multiple residues in the transmembrane helix and connecting peptide of mouse tapasin stabilize the transporter associated with the antigen-processing TAP2 subunit

The type I endoplasmic reticulum (ER) glycoprotein tapasin (Tpn) is essential for loading of major histocompatibility complex class I (MHC-I) molecules with an optimal spectrum of antigenic peptides and for stable expression of the heterodimeric, polytopic TAP peptide transporter. In a detailed mutational analysis, the transmembrane domain (TMD) and ER-luminal connecting peptide (CP) of mouse Tpn were analyzed for their capacity to stabilize the TAP2 subunit. Replacement of the TMD of Tpn by TMDs from calnexin or the Tpn-related protein, respectively, completely abolished TAP2 stabilization after transfection of Tpn-deficient cells, whereas TMDs derived from distantly related Tpn molecules (chicken and fish) were functional. A detailed mutational analysis of the TMD and adjacent residues in the ER-luminal CP of mouse Tpn was performed to elucidate amino acids that control the stabilization of TAP2. Single amino acid substitutions, including a conserved Lys residue in the center of the putative TMD, did not affect TAP2 expression levels. Mutation of this Lys plus four additional residues, predicted to be neighbors in an assumed alpha-helical TMD arrangement, abrogated the TAP2-stabilizing capacity of Tpn. In the presence of a wild-type TMD, also the substitution of a highly conserved Glu residue in the CP of Tpn strongly affected TAP2 stabilization. Defective TAP2 stabilization resulted in impaired cell surface expression of MHC-I molecules. This study thus defines a novel, spatially arranged motif in the TMD of Tpn essential for stable expression of the TAP2 protein and a novel protein interaction mode involving an ER-luminal Glu residue close to the membrane.     

Direct binding of human immunodeficiency virus type 1 Nef to the major histocompatibility complex class I (MHC-I) cytoplasmic tail disrupts MHC-I trafficking

Nef, an essential pathogenic determinant for human immunodeficiency virus type 1, has multiple functions that include disruption of major histocompatibility complex class I molecules (MHC-I) and CD4 and CD28 cell surface expression. The effects of Nef on MHC-I have been shown to protect infected cells from cytotoxic T-lymphocyte recognition by downmodulation of a subset of MHC-I (HLA-A and -B). The remaining HLA-C and -E molecules prevent recognition by natural killer (NK) cells, which would otherwise lyse cells expressing small amounts of MHC-I. Specific amino acid residues in the MHC-I cytoplasmic tail confer sensitivity to Nef, but their function is unknown. Here we show that purified Nef binds directly to the HLA-A2 cytoplasmic tail in vitro and that Nef forms complexes with MHC-I that can be isolated from human cells. The interaction between Nef and MHC-I appears to be weak, indicating that it may be transient or stabilized by other factors. Supporting the fact that these molecules interact in vivo, we found that Nef colocalizes with HLA-A2 molecules in a perinuclear distribution inside cells. In addition, we demonstrated that Nef fails to bind the HLA-E tail and also fails to bind HLA-A2 tails with deletions of amino acids necessary for MHC-I downmodulation. These data provide an explanation for differential downmodulation of MHC-I allotypes by Nef. In addition, they provide the first direct evidence indicating that Nef functions as an adaptor molecule able to link MHC-I to cellular trafficking proteins.     

Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine phosphorylation

Knowledge of the origin and biochemical status of beta(2)-microglobulin-free or misfolded major histocompatibility complex (MHC)-I molecules is essential for understanding their pleiotropic properties. Here we show that in normal human T cells, misfolding of MHC-I molecules is turned on upon activation and cell division and is proportional to the level of proliferation. Immunoprecipitation showed that a number of proteins are associated with MHC-I heavy chains at the surface of activated T cells, including the CD8alphabeta receptor and the chaperone tandem calreticulin/ERp57, associations that rely upon the existence of a pool of HC-10-reactive molecules. Biochemical analysis showed that misfolded MHC-I molecules present at the cell surface are fully glycosylated mature molecules. Importantly, misfolded MHC-I molecules are tyrosine phosphorylated and are associated with kinase activity. In vitro kinase assays followed by reprecipitation indicated that tyrosine phosphorylation of the class I heavy chain is probably mediated by a Src tyrosine kinase because Lck was found associated with HC-10 immunocomplexes. Finally, we show that inhibition of tyrosine phosphorylation by using the Src-family tyrosine kinase inhibitor PP2 resulted in enhanced release of MHC-I heavy chains from the cell surface of activated T cells and a slight down-regulation of cell surface W6/32-reactive molecules. This study provides new insights into the biology of MHC-I molecules and suggests that tyrosine phosphorylation may be involved in the regulation of MHC-I misfolding and expression.     

Myxoma virus leukemia-associated protein is responsible for major histocompatibility complex class I and Fas-CD95 down-regulation and defines scrapins, a new group of surface cellular receptor abductor proteins

Down-modulation of major histocompatibility class I (MHC-I) molecules is a viral strategy for survival in the host. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, can down-modulate the expression of MHC-I molecules, but the viral factor(s) has not been described. We cloned and characterized a gene coding for an endoplasmic reticulum (ER)-resident protein containing an atypical zinc finger and two transmembrane domains, which we called myxoma virus leukemia-associated protein (MV-LAP). MV-LAP down-regulated surface MHC-I and Fas-CD95 molecules upon transfection; the mechanism probably involves an exacerbation of endocytosis and was lost when the ER retention signal was removed. In addition, the lytic activity of MHC-I-restricted antigen-specific cytolytic T lymphocytes (CTL) against myxoma virus-infected antigen-presenting target cells was significantly reduced, revealing a strong correlation between MHC-I down-regulation by MV-LAP and CTL killing in vitro. In vivo experiments with a knockout virus showed that MV-LAP is a virulence factor, potentially involved in the immunosuppression characteristic of myxomatosis. Data bank analysis revealed that MV-LAP has homologs in herpesviruses and other poxviruses. We propose the name "scrapins" to define a new group of ER-resident surface cellular receptor abductor proteins. The down-regulation of cell surface molecules by scrapins probably helps protect infected cells during viral infections.     

HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail

To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the mu1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef-MHC-I complex is an important step required for inhibition of antigen presentation by HIV.     

A New Class of Endoplasmic Reticulum Export Signal ΦXΦXΦ for Transmembrane Proteins and Its Selective Interaction with Sec24C

Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), ²⁶VSIPM³⁰ in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues ⁸⁹⁵LIL⁸⁹⁷ (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to ⁸⁹⁵AAA⁸⁹⁷, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.     

A novel trafficking signal within the HLA-C cytoplasmic tail allows regulated expression upon differentiation of macrophages

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.     

Natural splice variant of MHC class I cytoplasmic tail enhances dendritic cell-induced CD8+ T-cell responses and boosts anti-tumor immunity

Dendritic cell (DC)-mediated presentation of MHC class I (MHC-I)/peptide complexes is a crucial first step in the priming of CTL responses, and the cytoplasmic tail of MHC-I plays an important role in modulating this process. Several species express a splice variant of the MHC-I tail that deletes exon 7-encoding amino acids (Δ7), including a conserved serine phosphorylation site. Previously, it has been shown that Δ7 MHC-I molecules demonstrate extended DC surface half-lives, and that mice expressing Δ7-K(b) generate significantly augmented CTL responses to viral challenge. Herein, we show that Δ7-D(b)-expressing DCs stimulated significantly more proliferation and much higher cytokine secretion by melanoma antigen-specific (Pmel-1) T cells. Moreover, in combination with adoptive Pmel-1 T-cell transfer, Δ7-D(b) DCs were superior to WT-D(b) DCs at stimulating anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival. Human DCs engineered to express Δ7-HLA-A*0201 showed similarly enhanced CTL stimulatory capacity. Further studies demonstrated impaired lateral membrane movement and clustering of human Δ7-MHC-I/peptide complexes, resulting in significantly increased bioavailability of MHC-I/peptide complexes for specific CD8+ T cells. Collectively, these data suggest that targeting exon 7-encoded MHC-I cytoplasmic determinants in DC vaccines has the potential to increase CD8+ T-cell stimulatory capacity and substantially improve their clinical efficacy.     

VIPL,a VIP36-like membrane protein with a putative function in the export of glycoproteins from the endoplasmic reticulum

Subsets of glycoproteins are thought to require lectin-like membrane receptors for efficient export out of the endoplasmic reticulum (ER). To identify new members related to two previously characterized intracellular lectins ERGIC-53/p58 and VIP36, we carried out an extensive database search using the conserved carbohydrate recognition domain (CRD) as a search string. A gene, more closely related to VIP36 than to ERGIC-53/p58, and hence called VIPL (VIP36-Like), was identified. VIPL has been conserved through evolution from zebra fish to man. The 2.4-kb VIPL mRNA was widely expressed to varying levels in different tissues. Using an antiserum prepared against the CRD, the 32-kDa VIPL protein was detected in various cell lines. The single N-linked glycan of VIPL remained endoglycosidase H-sensitive during a 2-h pulse-chase, even when the protein was overexpressed or mutated to allow export to the plasma membrane. VIPL localized primarily to the ER and partly to the Golgi complex. Like VIP36, the cytoplasmic tail of VIPL terminates in the sequence KRFY, a motif characteristic for proteins recycling between the ER and ERGIC/cis-Golgi. Mutating the retrograde transport signal KR to AA resulted in transport of VIPL to the cell surface. Finally, knock-down of VIPL mRNA using siRNA significantly slowed down the secretion of two glycoproteins (M(R) 35 and 250 kDa) to the medium, suggesting that VIPL may also function as an ER export receptor.     

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 CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.