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J Skowronski

Publications and source records attributed to J Skowronski.

At least 19 recordsLinked to original sources
PubMed2003

Circulating tumour necrosis factor alpha and soluble tumour necrosis factor receptors in patients with different patterns of rheumatoid synovitis.

OBJECTIVE: To examine the relation between the serum levels of tumour necrosis factor alpha (TNFalpha), soluble tumour necrosis factor receptors (sTNF-R), and the histological pattern of rheumatoid synovitis. METHODS: An enzyme linked immunosorbent assay (ELISA) was used to measure TNFalpha, p55 sTNF-R, and p75 sTNF-R concentrations in the serum of 43 patients with rheumatoid arthritis (RA) and 34 patients with osteoarthritis (OA). RESULTS: Upon histological analysis two variants of rheumatoid synovitis emerged. Twenty six RA specimens presented only diffuse infiltrates of mononuclear cells. In the remaining 17 samples the formation of lymphocytic follicles with germinal centre-like structures was found. Serum concentrations of TNFalpha, p55 and p75 sTNF-R were raised in patients with RA compared with the OA control group (p<0.001 for all comparisons). Levels of TNFalpha, p55 and p75 sTNF-R were higher in the serum of patients with RA with follicular synovitis than in patients with diffuse synovitis (p<0.001, p<0.01, and p<0.05, respectively). Serum concentrations of TNFalpha, p55 and p75 sTNF-R correlated with markers of disease activity. CONCLUSION: Different histological types of rheumatoid synovitis associated with distinct serum levels of TNFalpha and sTNF-R reflect varying clinical activity of the disease and support the concept of RA heterogeneity.

Aged↗
PubMed2002

Serum matrix metalloproteinases and tissue inhibitors of metalloproteinases in different histological variants of rheumatoid synovitis.

OBJECTIVE: Rheumatoid synovitis is characterized by an invasive and tissue-destructive infiltrate of lymphocytes, macrophages and synoviocytes. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) produced by these cells are important in the remodelling of the articular tissues in rheumatoid arthritis (RA). The aim of this study was to explore whether the serum concentrations of MMPs and their inhibitors were correlated with the histological appearance of the disease. METHODS: Tissue and serum samples were obtained from 37 patients with clinically active RA and 30 with osteoarthritis (OA). Morphological analysis allowed the division of RA synovial specimens into two distinct types. In 22 samples only diffuse infiltrates of mononuclear cells without further microanatomical organization were found. In 15 specimens we observed lymphocytic conglomerates with germinal centre-like structures. Serum concentrations of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase B (MMP-9), TIMP-1 and TIMP-2 were measured with an ELISA technique. RESULTS: Unique serum profiles of MMPs and TIMPs were identified in each of the two histological types of RA synovitis. The serum concentrations of MMP-1, MMP-3 and MMP-9 were higher in RA patients than in OA patients used as a control group (P<0.001 for all comparisons). These three MMPs dominated in the serum of RA patients with follicular synovitis compared with those with diffuse synovitis (P<0.05, P<0.01 and P<0.001 respectively). The analysis of the serum concentrations of TIMP-1 and TIMP-2 showed that their levels were also elevated in RA patients compared with OA patients (P<0.001 and P<0.01 respectively). Only TIMP-1 was found in a significantly higher concentration in the serum of RA patients with follicular synovitis than in those with diffuse synovitis (P<0.05). The serum concentrations of MMPs and TIMP-1 clearly identified patients with two different histological types of rheumatoid synovitis and with OA. Additionally, the analysis of clinical data showed that the rheumatoid disease in patients with follicular synovitis seemed to be more active than in those with diffuse synovitis. CONCLUSION: The morphological appearance of rheumatoid synovitis and the serum MMP and TIMP-1 profile were correlated with the clinical activity of the disease, confirming the heterogeneity of RA. These associations also suggest that patients with different histological forms of RA might require different treatment regimens.

Adult↗
PubMed2002

Soluble adhesion molecules (ICAM-1, VCAM-1, and E-selectin) and vascular endothelial growth factor (VEGF) in patients with distinct variants of rheumatoid synovitis.

BACKGROUND: Cell adhesion molecules and endothelial growth factors have an important role in the infiltrating of rheumatoid synovium with mononuclear cells, leading to the initiation and progression of the disease. OBJECTIVE: To investigate whether the serum profile of soluble adhesion molecules and of vascular endothelial growth factor (VEGF) is associated with the histological appearance of rheumatoid arthritis (RA). METHODS: Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sE-selectin), and VEGF were assessed by enzyme linked immunosorbent assay (ELISA) in 40 patients with RA and 32 patients with osteoarthritis (OA). RESULTS: Histological analysis of synovium specimens distinguished two types of rheumatoid synovitis. Twenty four RA samples presented diffuse infiltrates of mononuclear cells without any further microanatomical organisation, whereas in the remaining 16 samples lymphocytic follicles with germinal centre-like structures were identified. In comparison with patients with OA, constituting a control group, higher serum concentrations of sICAM-1 (p<0.001), sVCAM-1 (p<0.001), sE-selectin (p<0.01), and VEGF (p<0.001) were detected in patients with RA. Raised concentrations of sICAM-1, sVCAM-1, and VEGF dominated in the serum of patients with RA with follicular synovitis compared with those with diffuse synovitis (p<0.01 for all comparisons). The serum concentrations of sICAM-1, sVCAM-1, and VEGF correlated with markers of disease activity such as the erythrocyte sedimentation rate and C reactive protein levels. Furthermore, the clinical data analysed in our study indicated that patients with RA with follicular synovitis tend to have more severe disease. CONCLUSIONS: The distinct histological appearances of rheumatoid synovitis associated with different serum profiles of sICAM-1, sVCAM-1, and VEGF reflect varied clinical activity of the disease and confirm RA heterogeneity. Patients with different histological forms of synovitis may respond differently to the treatment regimens.

Arthritis, Rheumatoid↗
PubMed2001

Mechanism for down-regulation of CD28 by Nef.

SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

Modulation of different human immunodeficiency virus type 1 Nef functions during progression to AIDS.

The human immunodeficiency virus type 1 (HIV-1) Nef protein has several independent functions that might contribute to efficient viral replication in vivo. Since HIV-1 adapts rapidly to its host environment, we investigated if different Nef properties are associated with disease progression. Functional analysis revealed that nef alleles obtained during late stages of infection did not efficiently downmodulate class I major histocompatibility complex but were highly active in the stimulation of viral replication. In comparison, functional activity in downregulation of CD4 and enhancement of HIV-1 infectivity were maintained or enhanced after AIDS progression. Our results demonstrate that various Nef activities are modulated during the course of HIV-1 infection to maintain high viral loads at different stages of disease progression. These findings suggest that all in vitro Nef functions investigated contribute to AIDS pathogenesis and indicate that nef variants with increased pathogenicity emerge in a significant number of HIV-1-infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Serum cytokines in different histological variants of rheumatoid arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by an invasive and tissue destructive infiltrate of lymphocytes, macrophages, and synoviocytes formed in the joints. Its etiopathogenesis and the role of the particular morphological components of synovitis remain unclear. There is evidence that its histological heterogeneity is correlated with synovium cytokine transcription. We investigated whether the serum cytokine profile is associated with the morphological appearance of the disease. METHODS: Tissue and serum samples were collected from 25 patients with clinically active RA and 25 with osteoarthritis (OA) as a control group. After histological analysis RA synovial biopsies were divided into 2 distinct types; 16 samples were characterized by diffuse lymphocyte infiltrates with no additional microanatomical organization. Lymphocytic aggregates with germinal center-like structures were found in 9 specimens. Serum concentrations of interferon-gamma (IFN-gamma), interleukin 12 (IL-12, p70 heterodimer), tumor necrosis factor-alpha (TNF-alpha), and IL-15 were measured by ELISA. RESULTS: Low concentrations of IFN-gamma (p < 0.01) and IL-12 (NS) were found in RA patients' serum compared with OA controls. RA patients with follicular synovitis had lower serum concentration of IFN-gamma (p < 0.05) and IL-12 (p < 0.05) than patients with diffuse infiltrates. High concentration of TNF-alpha and IL-15 characterized RA patient serum in comparison with controls (respectively, p < 0.001 and p < 0.01). In the serum of RA patients with follicular synovitis TNF-alpha was a dominant cytokine (p < 0.01) compared to patients with diffuse disease. At TNF-alpha level > or = 44 pg/ml, 5 (56%) of 9 patients with follicular RA had such elevated values vs one of 16 diffuse patients (< 10%; p < 0.02). Only serum concentrations of TNF-alpha could effectively differentiate between patients with OA and subgroups of RA. Analysis of clinical data suggested that activity of rheumatoid disease in patients with follicular synovitis was more severe than in those with diffuse infiltrates. CONCLUSION: The association between distinct histological appearance of rheumatoid synovitis and serum cytokine profile and diverse clinical activity of disease seems to confirm its heterogeneity.

Adult↗
PubMed2000

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.

Adult↗
PubMed2000

Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression.

Simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins are related regulatory proteins that share several functions, including the ability to downregulate class I major histocompatibility complex (MHC) and CD4 expression on the cell surface and to alter T-cell-receptor-initiated signal transduction in T cells. We compared the mechanisms used by SIV mac239 Nef and HIV-1 Nef to downregulate class I MHC and found that the ability of SIV Nef to downregulate class I MHC requires a unique C-terminal region of the SIV mac239 Nef molecule which is not found in HIV-1 Nef. Interestingly, mutation of the PxxP motif in SIV Nef, unlike in HIV-1 Nef, does not affect class I MHC downregulation. We also found that downregulation of class I MHC by SIV Nef requires a conserved tyrosine in the cytoplasmic domain of the class I MHC heavy chain and involves accelerated endocytosis of class I complexes, as previously found with HIV-1 Nef. Thus, while SIV and HIV-1 Nef proteins use a similar mechanism to downregulate class I MHC expression, they have evolved different surfaces for molecular interactions with cell factors that regulate class I MHC traffic. Mutations in the C-terminal domain of SIV mac239 Nef selectively disrupt class I MHC downregulation, having no detectable effect on other functions of Nef, such as the downregulation of CD4 and CD3 surface expression, the stimulation of SIV virion infectivity, and the induction of SIV replication from T cells infected in the absence of stimulation. The resulting mutants will be useful reagents for studying the importance of class I MHC downregulation for SIV replication and AIDS pathogenesis in infected rhesus macaques.

Adaptor Protein Complex alpha Subunits↗
PubMed2000

Disrupting surfaces of nef required for downregulation of CD4 and for enhancement of virion infectivity attenuates simian immunodeficiency virus replication in vivo.

The multifunctional simian and human immunodeficiency virus (SIV and HIV) Nef proteins are important for virulence. We studied the importance of selected Nef functions using an SIV Nef with mutations in two regions that are required for CD4 downregulation. This Nef mutant is defective for downregulating CD4 and, in addition, for enhancing SIV infectivity and induction of SIV replication from infected quiescent peripheral blood mononuclear cells, but not for other known functions, including downregulation of class I major histocompatibility complex (MHC) cell surface expression. Replication of SIV containing this Nef variant in rhesus monkeys was attenuated early during infection. Subsequent increases in viral load coincided with selection of reversions and second-site compensatory changes in Nef. Our results indicate that the surfaces of Nef that mediate CD4 downregulation and the enhancement of virion infectivity are critical for SIV replication in vivo. Furthermore, these findings indicate that class I MHC downregulation by Nef is not sufficient for SIV virulence early in infection.

Amino Acid Substitution↗
PubMed2000

Simian immunodeficiency virus containing mutations in N-terminal tyrosine residues and in the PxxP motif in Nef replicates efficiently in rhesus macaques.

SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P(104)xxP(107)). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y(28)F, Y(39)F, P(104)A, and P(107)A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A(107)-->P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y(28)/Y(39)-->F(28)/F(39) substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.

Animals↗
PubMed1999

Two elements target SIV Nef to the AP-2 clathrin adaptor complex, but only one is required for the induction of CD4 endocytosis.

The simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) Nef proteins induce the endocytosis of CD4 and class I MHC molecules. Here we show that SIV Nef interacts with the AP-2 adaptor complex via two elements located in the N-terminal region of the Nef molecule, but only the N-distal element is required to induce CD4 endocytosis. This N-distal AP-2 targeting element contains no canonical endocytic signals and probably contacts the AP-2 complex via a novel interaction surface. The data support a model where SIV Nef induces CD4 endocytosis by promoting the normal interactions between the di-leucine sorting signal in the CD4 cytoplasmic domain and AP-2, but does not substitute for the CD4-AP-2 adaptor interaction. Neither element is important for the induction of class I MHC endocytosis, thus indicating that different mechanisms underlie the induction of class I MHC and CD4 endocytosis by Nef. In contrast to SIV Nef, HIV-1 Nef interacts with AP-2 via a surface containing a di-leucine endocytosis signal in the C-terminal disordered loop of Nef. The fact that genetic selection maintains similar molecular interactions via different surfaces in SIV and HIV-1 Nef proteins indicates that these interactions have critical roles for the viral life cycle in vivo.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

The acidic region and conserved putative protein kinase C phosphorylation site in Nef are important for SIV replication in rhesus macaques.

Variants of the pathogenic SIVmac239 clone with changes in Nef were analyzed to assess the functional relevance of two highly conserved regions in Nef in vitro and in vivo. Changes in a region with an acidic charge (Aci-Nef), or a potential protein kinase C phosphorylation site (PKC-Nef), impaired the ability of Nef to down-regulate CD4 and MHC class I surface expression and to alter CD3-initiated signal transduction in Jurkat T cells. The Aci-Nef, but not the PKC-Nef, associated with the previously described p65 phosphoprotein. SIV containing Aci-Nef, but not SIV containing PKC-Nef, showed reduced infectivity and replication in cell culture systems. One of two rhesus macaques infected with the PKC-Nef mutant virus showed rapid reversion and progressed to disease. In the second animal no reversions and nonprogressive infection was observed. In one of two macaques infected with the Aci-Nef variant, the mutations were stable during the first 40 weeks after infection. Thereafter, variants evolved in which up to six of the eight mutated positions in Nef were reverted and functional activity in vitro was partially restored. These changes occurred concomitantly with increasing viral load and disease progression. The second animal infected with the Aci-Nef variant showed no reversions and remained asymptomatic. Our study suggests that the acidic region and conserved PKC phosphorylation site in Nef are important for SIV replication in rhesus macaques and for several in vitro Nef functions. An almost wild-type activity in in vitro infectivity and replication assays seems insufficient to confer a full nef-positive phenotype in vivo.

Amino Acid Sequence↗
PubMed1999

The human immunodeficiency virus type 1 nef gene can to a large extent replace simian immunodeficiency virus nef in vivo.

The nef gene of the pathogenic simian immunodeficiency virus (SIV) 239 clone was replaced with primary human immunodeficiency virus type 1 (HIV-1) nef alleles to investigate whether HIV-1 Nef can substitute for SIV Nef in vivo. Initially, two rhesus macaques were infected with the chimeric viruses (Nef-SHIVs). Most of the nef alleles obtained from both animals predicted intact open reading frames. Furthermore, forms containing upstream nucleotide substitutions that enhanced expression of the inserted gene became predominant. One animal maintained high viral loads and slowly progressed to immunodeficiency. nef long terminal repeat sequences amplified from this animal were used to generate a second generation of Nef-SHIVs. Two macaques, which were subsequently infected with a mixture of cloned chimeric viruses, showed high viral loads and progressed to fatal immunodeficiency. Five macaques received a single molecular clone, named SHIV-40K6. The SHIV-40K6 nef allele was active in CD4 and class I major histocompatibility complex downregulation and enhanced viral infectivity and replication. Notably, all of the macaques inoculated with SHIV-40K6 showed high levels of viral replication early in infection. During later stages, however, the course of infection was variable. Three animals maintained high viral loads and developed immunodeficiency. Of the remaining two macaques, which showed decreasing viral loads after the acute phase of infection, only one efficiently controlled viral replication and remained asymptomatic during 1.5 years of follow-up. The other animal showed an increasing viral load and developed signs of progressive infection during later stages. Our data demonstrate that HIV-1 nef can, to a large extent, functionally replace SIVmac nef in vivo.

Alleles↗
PubMed1999

Effect of the attenuating deletion and of sequence alterations evolving in vivo on simian immunodeficiency virus C8-Nef function.

The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8 nef-long terminal repeat region encompasses only 12 bp and is "repaired" in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmac nef variants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in the nef gene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.

Alleles↗
PubMed1998

A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4.

Nef, a approximately 200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1-7]. Nef contains a globular core domain and two disordered segments--a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the beta-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes.

Amino Acid Sequence↗
PubMed1998

The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes.

Nef, a regulatory protein of human and simian immunodeficiency viruses, downregulates cell surface expression of both class I MHC and CD4 molecules in T cells by accelerating their endocytosis. Fibroblasts were used to study alterations in the traffic of class I MHC complexes induced by Nef. We found that Nef downregulates class I MHC complexes by a novel mechanism involving the accumulation of endocytosed class I MHC in the trans-Golgi, where it colocalizes with the adaptor protein-1 complex (AP-1). This effect of Nef on class I MHC traffic requires the SH3 domain-binding surface and a cluster of acidic amino acid residues in Nef, both of which are also required for Nef to downregulate class I MHC surface expression and to alter signal transduction in T cells. Downregulation of class I MHC complexes from the surface of T cells also requires a tyrosine residue in the cytoplasmic domain of the class I MHC heavy chain molecule. The requirement of the same surfaces of the Nef molecule for downregulation of surface class I MHC complexes in T cells and for their accumulation in the trans-Golgi of fibroblasts indicates that the two effects of Nef involve similar interactions with the host cell machinery and involve a molecular mechanism regulating class I MHC traffic that is common for both of these cell types. Interestingly, the downregulation of class I MHC does not require the ability of Nef to colocalize with the adaptor protein-2 complex (AP-2). We showed previously that the ability of Nef to colocalize with AP-2 correlates with the ability of Nef to downregulate CD4 expression. Our observations indicate that Nef downregulates class I MHC and CD4 surface expression via different interactions with the protein sorting machinery, and link the sorting and signal transduction machineries in the regulation of class I MHC surface expression by Nef.

Adaptor Protein Complex alpha Subunits↗
PubMed1997

Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation.

The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.

Adaptor Protein Complex alpha Subunits↗