Academic literature on the topic 'Proteomic;HIV Receptors'

The human leukocyte antigen (HLA)-Ib molecule, HLA-F, is known as a CD4+ T-cell protein and mediator of HIV progression. While HLA-Ia molecules do not have the chance to select and present viral peptides for immune recognition due to protein downregulation, HLA-F is upregulated. Post HIV infection, HLA-F loses the affinity to its activating receptor KIR3DS1 on NK cells leading to progression of the HIV infection. Several studies aimed to solve the question of the biophysical interface between HLA ligands and their cognate receptors. It became clear that even an invariant HLA molecule can be structurally modified by the variability of the bound peptide. We recently discovered the ability of HLA-F to select and present peptides and the HLA-F allele-specific peptide selection from the proteomic content using soluble HLA (sHLA) technology and a sophisticated MS method. We established recombinant K562 cells that express membrane-bound HLA-F*01:01, 01:03 or 01:04 complexes. While a recombinant soluble form of KIR3DS1 did not bind to the peptide-HLA-F complexes, acid elution of the peptides resulted in the presentation of HLA-F open conformers, and the binding of the soluble KIR3DS1 receptor increased. We used CD4+/HIV− and CD4+/HIV+ cells and performed an MS proteome analysis. We could detect hemoglobin as significantly upregulated in CD4+ T-cells post HIV infection. The expression of cellular hemoglobin in nonerythroid cells has been described, yet HLA-Ib presentation of hemoglobin-derived peptides is novel. Peptide sequence analysis from HLA-F allelic variants featured hemoglobin peptides as dominant and shared. The reciprocal experiment of binding hemoglobin peptide fractions to the HLA-F open conformers resulted in significantly diminished receptor recognition. These results underpin the molecular involvement of HLA-F and its designated peptide ligand in HIV immune escape.

Peptide-dependent engagement between human leucocyte antigens class I (HLA-I) molecules and their cognate receptors has been extensively analyzed. HLA-F belongs to the non-classical HLA-Ib molecules with marginal polymorphic nature and tissue restricted distribution. The three common allelic variants HLA-F*01:01/01:03/01:04 are distinguished by polymorphism outside the peptide binding pockets (residue 50, α1 or residue 251, α3) and are therefore not considered relevant for attention. However, peptide selection and presentation undergoes a most elaborated extraction from the whole available proteome. It is known that HLA-F confers a beneficial effect on disease outcome during HIV-1 infections. The interaction with the NK cell receptor initiates an antiviral downstream immune response and lead to delayed disease progression. During the time of HIV infection, HLA-F expression is upregulated, while its interaction with KIR3DS1 is diminished. The non-polymorphic nature of HLA-F facilitates the conclusion that understanding HLA-F peptide selection and presentation is essential to a comprehensive understanding of this dynamic immune response. Utilizing soluble HLA technology we recovered stable pHLA-F*01:01, 01:03 and 01:04 complexes from K562 cells and analyzed the peptides presented. Utilizing a sophisticated LC-MS-method, we analyzed the complete K562 proteome and matched the peptides presented by the respective HLA-F subtypes with detected proteins. All peptides featured a length of 8 to 24 amino acids and are not N-terminally anchored; the C-terminus is preferably anchored by Lys. To comprehend the alteration of the pHLA-F surface we structurally compared HLA-F variants bound to selected peptides. The peptides were selected from the same cellular content; however, no overlap between the proteomic source of F*01:01, 01:03 or 01:04 selected peptides could be observed. Recognizing the balance between HLA-F expression, HLA-F polymorphism and peptide selection will support to understand the role of HLA-F in viral pathogenesis.

DC-SIGN (dendriticcellspecificintracellular adhesion molecule 3grabbingnon-integrin) or CD209 is a type II transmembrane protein and one of several C-type lectin receptors expressed by dendritic cell subsets, which bind to high mannose glycoproteins promoting their endocytosis and potential degradation. DC-SIGN also mediates attachment of HIV to dendritic cells and binding to this receptor can subsequently lead to endocytosis or enhancement of CD4/CCR5-dependent infection. The latter was proposed to be facilitated by an interaction between DC-SIGN and CD4. Endocytosis of HIV virions does not necessarily lead to their complete degradation. A proportion of the virions remain infective and can be later presented to T cells mediating their infectionin trans. Previously, the extracellular domain of recombinant DC-SIGN has been shown to assemble as tetramers and in the current study we use a short range covalent cross-linker and show that DC-SIGN exists as tetramers on the surface of immature monocyte-derived dendritic cells. There was no evidence of direct binding between DC-SIGN and CD4 either by cross-linking or by fluorescence resonance energy transfer measurements suggesting that there is no constitutive association of the majority of these proteins in the membrane. Importantly we also show that the tetrameric complexes, in contrast to DC-SIGN monomers, bind with high affinity to high mannose glycoproteins such as mannan or HIV gp120 suggesting that such an assembly is required for high affinity binding of glycoproteins to DC-SIGN, providing the first direct evidence that DC-SIGN tetramers are essential for high affinity interactions with pathogens like HIV.

Background: The cell-of-origin (COO) concept, based on gene expression profiling (GEP), dividing diffuse large B-cell lymphoma (DLBCL) patients into germinal center B cell (GCB) or activated B cell (ABC) subtypes, is a well-established subclassification where ABC patients have an inferior survival. The hallmark the ABC-type is constitutive activation of nuclear factor kappa B (NF-κB), often due to mutations in the B-cell receptor (BCR) signaling pathway. This has been the underlying rationale for adding newer drugs, such as bortezomib, ibrutinib or lenalidomide, to R-CHOP for ABC patients. However, none of these combinations studied in phase III trials have shown any clinical benefit. So, the complexity of ABC DLBCL is probably not only explained by genetic alterations and transcriptional changes as gene expression not necessarily correlate with protein expression, and protein action and dynamics are not caught by genomics-based techniques. Instead, using methods to measure global protein expression and interactions could offer new insights into the ABC subtype and possibly aid in the identification of novel drug targets. Aim: To study possible differences in global protein expression between ABC and GCB DLBCL subtypes using quantitative proteomics. Patients: A total of 213 adult DLBCL patients in western Sweden diagnosed between 1/1 2004 and 31/12 2016, were included. All patients received immunochemotherapy (R-CHOP). Primary mediastinal large B-cell lymphoma, primary CNS lymphoma, HIV-related lymphoma and transformed lymphoma were excluded. From archived formalin-fixed, paraffin-embedded (FFPE) tissue sections, from the time of diagnosis, a core biopsy (1 mm diameter) were obtained from each patient sample. Methods: COO was determined using the Hans immunohistochemistry algorithm. For 92 of the 213 patients, COO was also determined using the gene expression Lymph2cx chip: 14% changed subtype group from either non-GCB to GCB (n=8), GCB to ABC (n=4) or GCB to unclassified (n=1). From the FFPE samples a proteomic analysis was performed. In short, peptides were labelled using tandem mass tag (TMT) according to the manufacturer instructions and samples were analysed on an Orbitrap Fusion Tribrid mass spectrometer. The data files were merged for identification and relative quantification using Proteome Discoverer version 1.4.The search used the Human Swissprot Database version August 2016 using Mascot 2.3 as a search engine. The differentially expressed proteins were analysed using STRING version 10.0, for pathway analysis we used the Reactome database resource, and for potentially druggable proteins we used the Human Protein Atlas website which holds protein information of the current FDA approved drugs directed to 672 separate human proteins. Results: In all, 3078 proteins could be identified in all patients and 793 proteins were differentially expressed (p<0.05 adjusted for mass significance according to Benjamin-Hochberg) between ABC and GCB patients. Of these, 410 proteins were overexpressed in the ABC group. Among the most expressed proteins were several well-known ABC-associated proteins (such as IRF4/MUM1, HSP90B1, CCDC50 and STAT3) in addition to a large number of proteins previously not described in ABC DLBCL, e.g. neudesin, BLNK, MPST, BPGM, SUB1, SP140, PCK2, PARP4, SRP54, SRP68, SRP72, TRPV2, IGF2R and FGD2. A majority of the 410 proteins were closely linked with an enrichment p-value < 1x10-16(Fig. 1) and the most enriched pathways were immune system (FDR rate 3.3 x 10-27), interferon signaling (2.9 x 10-7), antigen processing (8.8 x 10-7) and down-modulation of cell surface receptors (4.7 x 10-5). Most interestingly, we also found that 16 proteins overexpressed in the ABC group could be potential drug targets for an FDA approved drug, e.g. high affinity immunoglobulin gamma Fc receptor I, CD47, HDAC2, ELANE and carbonic anhydrase 1. Conclusions: In this large proteomic study we found a number of overexpressed proteins in the ABC subtype, previously not described in DLBCL. Even though functional studies aimed at individual proteins and protein interactions to evaluate potential clinical effect are needed, our findings reveal novel proteins that could be potential druggable targets in ABC DLBCL patients. Figure 1 Disclosures Enblad: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees. Mellqvist:Amgen, Janssen, Oncopeptides, Sanofi, Sandoz, Takeda: Honoraria. Andersson:Abbvie and Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead, Janssen and Roche: Consultancy; Gilead: Research Funding.

Abstract Non-Hodgkin Lymphoma (NHL) is the most common hematological malignancy, with B-cell lymphoma (NHL-B) accounting for 85% of all lymphomas. In the United States, there are ~500,000 lymphoma patients currently living with this disease and ~20,000 lymphoma-related deaths occur annually. The current overall cure rate for B-cell lymphoma is estimated at ~30%, indicating that new innovative therapeutic approaches are needed to significantly reduce the high mortality rate, particularly of relapsed/refractory (r/r) NHL-B. The poor quality of life in patients suffering from chronic diseases like cancer has forced many patients to pursue alternative treatment options, including medicinal cannabinoids (CB), in order to improve their clinical prospect/outcomes. Medicinal cannabinoids have been legalized in 23 states and DC for several medical conditions such as cachexia, chronic pain, epilepsy and other similar disorders characterized by seizures, glaucoma, HIV- AIDS, Multiple Sclerosis, muscle spasticity and GI enteritis. Lately however, cannabis has been shown to have a broader biologic activity spectrum with various cannabis compounds functioning as ligands binding the two principle cannabinoid-specific G protein-coupled receptors (GPCR) CB1 (in neural cells), and CB2, in immune lymphoid, particularly B cells, but have also been identified, showing aberrant expression in a wide variety of important human cancers. This suggests not only a wider spectrum of cellular usage of cannabinoids and their cognate receptors, but also their potential utility as novel therapeutic targets. Gene expression profiling data has demonstrated, however, that B-cell lymphoma is one of the top three cancers (glioma and gastric are the other two) showing high expression of CB1 and CB2 receptors. Our studies showed that CB1 receptor is highly expressed in aggressive NHL-B, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) cells in comparison to normal unstimulated (G0) B cells, and that targeting CB1 using an siRNA approach leads to cell growth inhibition. Furthermore, pharmacological approaches targeting CB1 with small molecule antagonists (Rimonabant and Otenabant) inhibited lymphoma cell viability, leading to the induction of apoptosis and G2M cell cycle arrest. Using proteomic approach via reverse-phase protein array (RPPA), we have demonstrated that lymphoma cells treated with the CB1 antagonist Rimonabant showed a robust effect on apoptosis (increases in caspase 3 and 7, Bad, and bak), cell cycle (increases in p27 and cyclin D1), DNA damage (increases in gH2AX), and autophagy (increases in LC3A) associated proteins. In addition, Rimonabant treatment also inhibited several growth and survival pathways, including STAT3, SRC, and b-catenin, while enhancing the PI3K/ATK pathway. Of note, Rimonabant treatment also activated the DNA damage response (DDR) pathway through stimulating two checkpoint kinases (Chk1 and Chk2). Blocking Rimonabant-induced Chk1 and Chk2 with a selective ATP-competitive inhibitor of Chk1 and Chk2 leads to a robust synergistic effect on cell growth inhibition and apoptotic induction, suggesting that blocking the DDR pathway with Chk kinase inhibitors prevents cells recovering from rimonabant-induced DNA damage. These findings suggest that targeting the cannabinoid receptors and the DDR pathway represents a new therapeutic strategy against resistant r/r NHL-B cells. Disclosures Pham: Vyripharm Biopharmaceuticals: Research Funding. Bryant:Vyripharm Biopharmaceuticals: Equity Ownership. Yang:Vyripharm Biopharmaceuticals: Employment.

Treatment of HIV-1-infected patients results in improved clinical and immunological conditions, but severe non-AIDS-related conditions still persist. Novel proteomic platforms have identified inflammatory proteins where abundance is dysregulated in adult treated patients, whereas limited data are available in treated HIV-1 infection of children. Using a proteomic plasma profiling approach comprising 92 inflammation-related molecules, we analyzed specimens from 43 vertically HIV-1-infected children receiving antiretroviral treatment (ART) and matched controls in Ethiopia. The infected children were analyzed as a group and separately, according to age of treatment initiation. Proteins displaying a significantly different abundance between groups were hierarchically clustered and presented in heat maps. Random forest analysis was performed to pin-point proteins discriminating between groups; five proteins (STAMBP, CD5, TFG-α, TRANCE, AXIN1) were the strongest prediction factors for treated HIV-1 infection. TRANCE was previously linked to reduced bone mass levels in HIV-1-infected children. CCL4 chemokine, ligand to HIV-1 co-receptor CCR5, was the most critical protein for successful classification between children who initiated ART at different time points. Our data provide evidence that a dysregulated expression of proteins linked to immunological abnormalities and bone metabolism can be found in HIV-1-infected children with prolonged exposure to ART.

The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.

ABSTRACT The primary roles attributed to the human immunodeficiency virus type 1 (HIV-1) Vpu protein are the degradation of the viral receptor CD4 and the enhancement of virion release. With regard to CD4 downregulation, Vpu has been shown to act as an adapter linking CD4 with the ubiquitin-proteasome machinery via interaction with the F-box protein βTrCP. To identify additional cellular βTrCP-dependent Vpu targets, we performed quantitative proteomics analyses using the plasma membrane fraction of HeLa cells expressing either wild-type Vpu or a Vpu mutant (S52N/S56N) that does not bind βTrCP. One cellular protein, BST-2 (CD317), was consistently underrepresented in the membrane proteome of cells expressing wild-type Vpu compared to the proteome of cells expressing the Vpu mutant. To verify the biological relevance of this phenotype for HIV pathogenesis, we showed that in T cells infected with HIV-1, BST-2 downregulation occurred in a Vpu-dependent manner. Recently, BST-2 has been identified as the interferon-inducible cellular factor Tetherin, which restricts HIV virion release in the absence of Vpu. We address here the unresolved mechanism of Vpu-mediated BST-2 downregulation. Our data show that the presence of wild-type Vpu reduced cell surface and total steady-state BST-2 levels, whereas that of the mutant Vpu had no effect. In addition, treatment of cells with the lysosome acidification inhibitor concanamycin A, but not treatment with the proteasome inhibitor MG132, reduced BST-2 downregulation by wild-type Vpu, thereby suggesting that the presence of Vpu leads to the degradation of BST-2 via an endosome-lysosome degradation pathway. The importance of βTrCP in this process was confirmed by demonstrating that in the absence of βTrCP, BST-2 levels were restored despite the presence of Vpu. Taken together, these data support the hypothesis that, in similarity to its role in CD4 degradation, Vpu acts as an adapter molecule linking BST-2 to the cellular ubiquitination machinery via βTrCP. However, in contrast to the proteasome-dependent degradation of CD4, which occurs in the endoplasmic reticulum, Vpu appears to interact with BST-2 in the trans-Golgi network or in early endosomes, leading to lysosomal degradation of BST-2. Via this action, Vpu could counter the tethering function of BST-2, resulting in enhanced HIV-1 virion release. Interestingly, although HIV-2 does not express Vpu, an isolate known to exhibit enhanced viral egress can downregulate surface BST-2 by an as-yet-unknown mechanism that does not appear to involve degradation. Understanding the molecular mechanisms of both Vpu-dependent and -independent mediated antagonism of BST-2 will be critical for therapeutic strategies that exploit this novel viral function.

The tumor microenvironment is best characterized as a fluctuation of hypoxia and nutrient deprivation, which leads to epigenetic and genetic adaptation of clones and increased invasiveness and metastasis. In turn, these hypoxic adaptations make the tumors more difficult to treat and confer increased resistance to current therapies. Part of this adaptation is the regulation of gene products in response to hypoxia. Many of these hypoxia-regulated genes are mediated by the hypoxia-inducible factor 1 (HIF-1) complex, which is composed of a heterodimer pair of HIF-1α and HIF-1β. This heterodimer binds to the promoter of hypoxia-responsive genes, while interacting with other transcription factors, such as p300, signal and transducer of transcription 3, and Redox effector factor 1/apurinic/apyrimidinic endonuclease. HIF-1α levels itself can be regulated by hypoxia transcriptionally and post-translationally through ubiquitination; but the magnitude of the response is modulated by several other pathways, including free radicals that affect crosstalk with HIF-1α/HIF-1β transcriptional activities. HIF-1α has emerged as an important transcription factor in breast cancer and prostate cancer biology, and is expressed in the early stages of mammary and prostate carcinogenesis. Its expression is correlated with diagnostic and prognostic indicators for early relapse and metastatic disease, thus making HIF-1α a potential prognostic biomarker in proteomic assessments of breast and prostate cancers. The importance of HIF-1α in tumor progression makes it a logical target for chemoprevention strategies in patients at higher genetic risk of breast and prostate cancer with Cox 2 inhibitors or 2-methoxyestradiol, as well as a target for new approaches to inhibiting angiogenesis. The crosstalk between estrogen signaling pathways and HIF-1α is still not fully defined in breast cancer, but downstream estrogen receptor signaling may be a candidate for estrogen modulation of HIF-1α levels. In prostate cancer, androgens upregulate HIF-1α through androgen-regulated autocrine receptor tyrosine kinase receptor signaling. This review will put into perspective the role of HIF-1α in endocrine oncology and present new data on HIF-1α signaling and the potential for targeted therapies, including combinatory hormonal therapies.

CD161 expression on CD4+ T cells is associated with a Th17 functional phenotype, as well as with an innate capacity to respond to interleukin (IL)-12 and IL-18 without T cell receptor (TCR) stimulation. Chronic HIV-1 infection is associated with loss of the CD161+ CD4 T cell population, and non-human primate studies suggest that their depletion is associated with disease progression. However, the dynamics of the CD161+ CD4+ T cell population during acute HIV-1 infection remains unknown. In this study, we characterize peripheral blood CD161+ CD4+ T cells in detail, and examine how they are affected during the earliest stages of HIV-1 infection. Unbiased surface proteome screening and principal component analysis indicated that CD161+ CD4+ T cells are relatively phenotypically homogeneous between donors, and are intermediates between conventional CD4 T cells and innate-like T cells. In acute untreated HIV-1 infection, the circulating CD161+ CD4+ T cell population decreased in frequency, as did absolute cell counts starting from peak viral load, with elevated levels of activation and exhaustion markers expressed throughout acute HIV-1 infection. The capacity of these cells to respond to stimulation with IL-12 and IL-18 was also reduced. Early initiation of anti-retroviral treatment (ART) during acute HIV-1 infection restored the functionality of peripheral blood CD161+ CD4+ T cells, but not their frequency. In contrast, early ART initiation prevented the decline of colonic CD161+ CD4+ T cells that otherwise started during acute infection. Furthermore, loss of peripheral and colonic CD161+ CD4+ T cells in untreated infection was associated with levels of viral load. These results suggest that acute HIV-1 infection has profound effects on the CD161+ CD4+ T cell population that could not be completely prevented by the initiation of ART.

HIV infection and disease is a multistage process that involves a variety of cell types as the virus spreads through the body. Initially, dendritic cells (DCs) present at the mucosal site of infection bind and internalise HIV for degradation and presentation to T cells. As the DCs migrate to lymph nodes and mature, part of the internalised virions remains infective inside endosomal compartments. During formation of the immunological synapse between CD4 T cells and DCs, infective virions from dendritic cells are transferred to CD4 T cells leading to a strong infection of those cells allowing rapid virus dissemination throughout the body and establishment of the typical HIV infection. Various membrane receptors are involved in this process. Initial HIV binding to DCs is mediated by C-type lectin receptors such as the mannose receptor or DC-SIGN (DC specific intracellular adhesion molecule 3 grabbing non integrin) which is followed by virus internalisation and lysis albeit virus induced changes in endocytic routing prevents a proportion from degradation. Productive infection of DCs has also been observed allowing trans infection of CD4 T cells through a different mechanism. HIV infection of CD4 T cells, DCs and other cells is a multistep process initiated by binding of HIV envelope gp120 to the CD4 receptor, a 55 kDa transmembrane glycoprotein. Subsequent conformational changes in gp120 allow binding to a chemokine receptor, either CCR5 or CXCR4, followed by membrane fusion and infection. The aim of this thesis was to investigate protein associations with the HIV receptors DC-SIGN and CD4 in order to elucidate the mechanism of complex formation, virus entry and/or defining target sites for antiretroviral drugs. This thesis used a proteomic approach for studying the receptors with mass spectrometry-based protein identification as its core technology. A range of different approaches were developed and compared for identification of protein interactions and characterisation of the identified protein associations. An affinity purification of the CD4 receptor complex from lymphoid cells was used as the basis for detecting novel CD4-binding proteins. For this approach a strategy based on mass spectrometry identification of CD4 associating proteins using affinity chromatography and affinity-tag mediated purification of tryptic peptides was developed. This method proved successful for the identification of CD4 interacting proteins such as the strongly associated kinase p56lck, however a limited number of non-specifically bound proteins were also identified along the receptor complex. Using one-dimensional SDS-polyacrylamide gel electrophoresis followed by in-gel digests and mass spectrometry analysis, a large number of non-specifically binding proteins were identified along the CD4/lck complex. Evaluation of different lysis buffers in several independent experiments demonstrated that there was a large and inconsistent array of proteins that were obviously non-specifically bound to the receptor. No further specific binding partners were detected. These data suggested that protein interactions of CD4 on this cell type are of weak and/or transient nature. It also demonstrated a need for careful interpretation of proteomic data in the light of the propensity of non-specific binding under these conditions. To overcome dissociation of weak protein interactions, a method was developed using chemical cross-linking to preserve weak protein interactions on lymphoid cells. Affinity purification was used to purify CD4 along with cross-linked associated proteins and mass spectrometry analysis identified an interaction with the transferrin receptor CD71 and the tyrosine phosphatase CD45. The CD45-CD4 interaction is well known. The CD4-CD71 interaction was demonstrated to be a result from colocalization of the two molecules during formation of endocytic vesicles. Flow cytometry-based fluorescence resonance energy transfer (FRET) measurements were applied to confirm colocalization. A similar interaction was suspected for CD4 and DC-SIGN on the plasma membrane of DCs as cis infection of DCs has been demonstrated i.e. initial binding to DC-SIGN then to CD4/CCR5 on the same cell. Therefore, protein associations of DC-SIGN were investigated using the developed techniques. Using cross-linking, DC-SIGN was shown to assemble in large complexes on the surface of immature monocyte-derived DCs. Mass spectrometry analysis of the purified complexes identified them as homo-oligomers of DC-SIGN. The absence of CD4 suggested that the fraction interacting with CD4 at any one time must be small. The complexes of DC-SIGN were further characterised to be tetramers and successfully co-immunoprecipitated with HIV gp120 and mannan. DC-SIGN monomers were not evident demonstrating that the assembly of DC-SIGN into tetramers is required for high affinity binding of its natural and viral ligands. Thus potential antiviral agents aimed at blocking the early stage of HIV binding to DCs must simulate tetramers in order to neutralise the virus efficiently. Overall the thesis provides new information on protein interactions of CD4 and DC-SIGN, a careful investigation of "proteomics" techniques for identifying the proteins in affinity-purified samples and demonstrates the need for multifaceted analytical approaches to probe complex cellular systems.