Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling–liquid chromatography–tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.Subject terms: Breast cancer, Predictive markers     

ExoCarta: A compendium of exosomal proteins and RNA

Exosomes, membrane microvesicles (40–100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary‐conserved set of protein molecules have been identified in most exosomes studied to date. However, with the complexity of tissue/cell type‐specific proteins being incorporated in the exosomes, some of these so‐called exosomal markers are not always present in all the exosomes. The presence of tissue/cell type‐specific proteins in exosomes allows researchers to isolate them using immunoaffinity capture methods. A compendium for exosomal proteomes will aid researchers in identifying proteins that were more commonly found in various exosomes (exosome markers) and those that are specific to certain tissue/cell type‐derived exosomes. Here, we describe ExoCarta, a compendium for proteins and RNA molecules identified in exosomes. ExoCarta is first of its kind and the resource is freely available to the scientific community through the web ( http://exocarta.ludwig.edu.au ). We believe that this community resource will be of great biological importance for any future exosome analyses.     

Exosomal ANXA2 derived from ovarian cancer cells regulates epithelial-mesenchymal plasticity of human peritoneal mesothelial cells

Ovarian cancer, one of the malignant gynaecological tumours with the highest mortality rate among female reproductive system, is prone to metastasis, recurrence and chemotherapy resistance, causing a poor prognosis. Exosomes can regulate the epithelial-mesenchymal plasticity of tumour cells, remodel surrounding tumour microenvironment, and affect tumour cell proliferation, invasion and metastasis. However, the function and mechanism of exosomes in the intraperitoneal implantation of ovarian cancer remain unclear. In this study, exosomal annexin A2 (ANXA2) derived from ovarian cancer cells was co-cultured with human peritoneal mesothelial (HMrSV5) cells; functional experiments were conducted to explore the effects of exosomal ANXA2 on the biological behaviour of HMrSV5 and the related mechanisms. This study showed that ANXA2 in ovarian cancer cells can be transferred to HMrSV5 cells through exosomes, exosomal ANXA2 can not only promote the migration, invasion and apoptosis of HMrSV5 cells, but also regulates morphological changes and fibrosis of HMrSV5 cells. Furthermore, ANXA2 promotes the mesothelial-mesenchymal transition (MMT) and degradation of the extracellular matrix of HMrSV5 cells through PI3K/AKT/mTOR pathway, finally affects pre-metastasis microenvironment of ovarian cancer, which provides a new theoretical basis for the mechanism of intraperitoneal implantation and metastasis of ovarian cancer.     

Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles

Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.     

Secretion of active membrane type 1 matrix metalloproteinase (MMP-14) into extracellular space in microvesicular exosomes

Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP14) is an efficient extracellular matrix (ECM) degrading enzyme that plays important roles in tissue homeostasis and cell invasion. Like a number of type I membrane proteins, MT1-MMP can be internalized from the cell surface through early and recycling endosomes to late endosomes, and recycled to the plasma membrane. Late endosomes participate in the biogenesis of small (30-100 nm) vesicles, exosomes, which redirect plasma membrane proteins for extracellular secretion. We hypothesized that some of the endosomal MT1-MMP could be directed to exosomes for extracellular release. Using cultured human fibrosarcoma (HT-1080) and melanoma (G361) cells we provide evidence that both the full-length 60 kDa and the proteolytically processed 43 kDa forms of MT1-MMP are secreted in exosomes. The isolated exosomes were identified by their vesicular structure in electron microscopy and by exosomal marker proteins CD9 and tumor susceptibility gene (TSG101). Furthermore, exosomes contained beta1-integrin (CD29). The exosomes were able to activate pro-MMP-2 and degrade type 1 collagen and gelatin, suggesting that the exosomal MT1-MMP was functionally active. The targeting of MT1-MMP in exosomes represents a novel mechanism for cancer cells to secrete membrane type metalloproteolytic activity into the extracellular space.     

Oncogenic and Non‐Malignant Pancreatic Exosome Cargo Reveal Distinct Expression of Oncogenic and Prognostic Factors Involved in Tumor Invasion and Metastasis

Exosomes are small extracellular membrane vesicles important in intercellular communication, with their oncogenic cargo attributed to tumor progression and pre‐metastatic niche formation. To gain an insight into key differences in oncogenic composition of exosomes, human non‐malignant epithelial and pancreatic cancer cell models and purified and characterized resultant exosome populations are utilized. Proteomic analysis reveals the selective enrichment of known exosome markers and signaling proteins in comparison to parental cells. Importantly, valuable insights into oncogenic exosomes (362 unique proteins in comparison to non‐malignant exosomes) of key metastatic regulatory factors and signaling molecules fundamental to pancreatic cancer progression (KRAS, CD44, EGFR) are provided. It is reported that oncogenic exosomes contain factors known to regulate the pre‐metastatic niche (S100A4, F3, ITGβ5, ANXA1), clinically‐relevant proteins which correlate with poor prognosis (CLDN1, MUC1) as well as protein networks involved in various cancer hallmarks including proliferation (CLU, CAV1), invasion (PODXL, ITGA3), metastasis (LAMP1, ST14) and immune surveillance escape (B2M). The presence of these factors in oncogenic exosomes offers an understanding of select differences in exosome composition during tumorigenesis, potential components as prognostic and diagnostic biomarkers in pancreatic cancer, and highlights the role of exosomes in mediating crosstalk between tumor and stromal cells.     

OX40 ligation of CD4+ T cells enhances virus-specific CD8+ T cell memory responses independently of IL-2 and CD4+ T regulatory cell inhibition

We have previously shown that CD4(+) T cells are required to optimally expand viral-specific memory CD8(+) CTL responses using a human dendritic cell-T cell-based coculture system. OX40 (CD134), a 50-kDa transmembrane protein of the TNFR family, is expressed primarily on activated CD4(+) T cells. In murine models, the OX40/OX40L pathway has been shown to play a critical costimulatory role in dendritic cell/T cell interactions that may be important in promoting long-lived CD4(+) T cells, which subsequently can help CD8(+) T cell responses. The current study examined whether OX40 ligation on ex vivo CD4(+) T cells can enhance their ability to "help" virus-specific CTL responses in HIV-1-infected and -uninfected individuals. OX40 ligation of CD4(+) T cells by human OX40L-IgG1 enhanced the ex vivo expansion of HIV-1-specific and EBV-specific CTL from HIV-1-infected and -uninfected individuals, respectively. The mechanism whereby OX40 ligation enhanced help of CTL was independent of the induction of cytokines such as IL-2 or any inhibitory effect on CD4(+) T regulatory cells, but was associated with a direct effect on proliferation of CD4(+) T cells. Thus, OX40 ligation on CD4(+) T cells represents a potentially novel immunotherapeutic strategy that should be investigated to treat and prevent persistent virus infections, such as HIV-1 infection.     

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin‐1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non–small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin‐1 and flotillin‐2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin‐1 as well as its EV‐to‐cellular ratio vary drastically depending on cell type.     

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom‐up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.     

HIV-1 infection abrogates CD8+ T cell mitogen-activated protein kinase signaling responses

Mitogen-activated protein kinase (MAPK) signaling pathways are dynamic and sensitive regulators of T cell function and differentiation. Altered MAPK signaling has been associated with the inflammatory and autoimmune diseases lupus and arthritis and with some pathogenic viral infections. HIV-1 infection is characterized by chronic immune inflammation, aberrantly heightened CD8⁺ T cell activation levels, and altered T cell function. The relationship between MAPK pathway function, HIV-1-induced activation (CD38 and HLA-DR), and exhaustion (Tim-3) markers in circulating CD8⁺ T cells remains unknown. Phosphorylation of the MAPK effector proteins ERK and p38 was examined by “phosflow” flow cytometry in 79 recently HIV-1-infected, antiretroviral-treatment-naïve adults and 21 risk-matched HIV-1-negative controls. We identified a subset of CD8⁺ T cells refractory to phorbol 12-myristate 13-acetate plus ionomycin-induced ERK1/2 phosphorylation (referred to as p-ERK1/2-refractory cells) that was greatly expanded in HIV-1-infected adults. The CD8⁺ p-ERK1/2-refractory cells were highly activated (CD38⁺ HLA-DR⁺) but not exhausted (Tim-3 negative), tended to have low CD8 expression, and were enriched in intermediate and late transitional memory states of differentiation (CD45RA⁻ CD28⁻ CD27^+/−). Targeting MAPK pathways to restore ERK1/2 signaling may normalize immune inflammation levels and restore CD8⁺ T cell function during HIV-1 infection.     

Evaluation of human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T-lymphocyte responses utilizing B-lymphoblastoid cell lines transduced with the CD4 gene and infected with HIV-1.

Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.     

The CD85j+ NK cell subset potently controls HIV-1 replication in autologous dendritic cells

Natural killer (NK) cells and dendritic cells (DC) are thought to play critical roles in the first phases of HIV infection. In this study, we examined changes in the NK cell repertoire and functions occurring in response to early interaction with HIV-infected DC, using an autologous in vitro NK/DC coculture system. We show that NK cell interaction with HIV-1-infected autologous monocyte-derived DC (MDDC) modulates NK receptor expression. In particular, expression of the CD85j receptor on NK cells was strongly down-regulated upon coculture with HIV-1-infected MDDC. We demonstrate that CD85j(+) NK cells exert potent control of HIV-1 replication in single-round and productively HIV-1-infected MDDC, whereas CD85j(-) NK cells induce a modest and transient decrease of HIV-1 replication. HIV-1 suppression in MDCC by CD85j(+) NK cells required cell-to-cell contact and did not appear mediated by cytotoxicity or by soluble factors. HIV-1 inhibition was abolished when NK-MDDC interaction through the CD85j receptor was blocked with a recombinant CD85j molecule, whereas inhibition was only slightly counteracted by blocking HLA class I molecules, which are known CD85j ligands. After masking HLA class I molecules with specific antibodies, a fraction of HIV-1 infected MDDC was still strongly stained by a recombinant CD85j protein. These results suggest that CD85j(+) NK cell inhibition of HIV-1 replication in MDDC is mainly mediated by CD85j interaction with an unknown ligand (distinct from HLA class I molecules) preferentially expressed on HIV-1-infected MDDC.     

Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End‐Products (RAGE)

Exosomes are nano‐sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end‐products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599–606, 2018. © 2017 Wiley Periodicals, Inc.     

Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines

Exosomes are important mediators in cell‐to‐cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma‐specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro‐migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells’ aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.     

Cocaine‐Specific Effects on Exosome Biogenesis in Microglial Cells

Cocaine is a highly addictive stimulant and a well-known drug, with multiple effects on physiology. Cocaine can have direct effects on all cell types in the brain, including microglia. Microglia can be activated by other conditions, such as infection, inflammation, or injury. However, how cocaine regulates microglia and the influence of cocaine on microglial-derived exosomes remains unknown. Exosomes are nanovesicles that are responsible for intercellular communications, signaling, and trafficking necessary cargo for cell homeostasis. In this study, we hypothesized that cocaine affects exosome biogenesis and composition in BV2 microglial cells. BV2 microglial cells were cultured in exosome-depleted RPMI-1640 media and were treated according to the experimental designs. We observed that cell viability decreased by 11% at 100 µM cocaine treatment but was unaffected at other concentrations. After treatments, the exosomes were isolated from the condition media. Purified exosomes were characterized and quantified using transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). By NTA, there was a significant decrease in particles/mL after cocaine treatment. There was a 39.5%, 58.1%, 32.3% and 28.1% decrease in particles/mL at 100 nM, 1 μM, 10 μM and 100 μM cocaine, respectively. The characterization of exosomes and exosomal protein was performed by western/dot blot analyses. Tetraspanins CD11b, CD18 and CD63 were relatively unchanged after cocaine treatment. The heat shock proteins (Hsps), Hsp70 and Hsp90, were both significantly increased at 10 μM and 100 μM, but only hsp70 was significantly increased at 10 nM. The Rab proteins were assessed to investigate their role in cocaine-mediated exosomal decrease. Rab11 was significantly decreased at 10 nM, 100 nM, 1 μM, 10 μM and 100 μM by 15%, 28%, 25%, 38% and 22%, respectively. Rab27 was decreased at all concentrations but only significantly decreased at 100 nM, 1 μM and 100 μM cocaine by 21%, 24% and 23%, respectively. Rab35 had no significant changes noted when compared to control. Rab7 increased at all cocaine concentrations but only a significant increase in expression at 100 nM and 10 μM by 1.32-fold and 1.4-fold increase. Cocaine was found to alter exosome biogenesis and composition in BV2 microglial cells. Western and dot blot analyses verified the identities of purified exosomes, and the specific protein compositions of exosomes were found to change in the presence of cocaine. Furthermore, cocaine exposure modulated the expression of exosomal proteins, such as Hsps and Rab GTPases, suggesting the protein composition and formation of microglial-derived exosomes were regulated by cocaine.

     

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.     

Vpu augments the initial burst phase of HIV-1 propagation and downregulates BST2 and CD4 in humanized mice

While human cells express potent antiviral proteins as part of the host defense repertoire, viruses have evolved their own arsenal of proteins to antagonize them. BST2 was identified as an inhibitory cellular protein of HIV-1 replication, which tethers virions to the cell surface to prevent their release. On the other hand, the HIV-1 accessory protein, Vpu, has the ability to downregulate and counteract BST2. Vpu also possesses the ability to downmodulate cellular CD4 and SLAMF6 molecules expressed on infected cells. However, the role of Vpu in HIV-1 infection in vivo remains unclear. Here, using a human hematopoietic stem cell-transplanted humanized mouse model, we demonstrate that Vpu contributes to the efficient spread of HIV-1 in vivo during the acute phase of infection. Although Vpu did not affect viral cytopathicity, target cell preference, and the level of viral protein expression, the amount of cell-free virions in vpu-deficient HIV-1-infected mice was profoundly lower than that in wild-type HIV-1-infected mice. We provide a novel insight suggesting that Vpu concomitantly downregulates BST2 and CD4, but not SLAMF6, from the surface of infected cells. Furthermore, we show evidence suggesting that BST2 and CD4 impair the production of cell-free infectious virions but do not associate with the efficiency of cell-to-cell HIV-1 transmission. Taken together, our findings suggest that Vpu downmodulates BST2 and CD4 in infected cells and augments the initial burst of HIV-1 replication in vivo. This is the first report demonstrating the role of Vpu in HIV-1 infection in an in vivo model.     

Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production

A previous study using a Nef-defective human immunodeficiency virus type 1 (HIV-1) mutant suggested that Nef-mediated down-regulation of HLA class I on the infected cell surface affects the cytolytic activity of HIV-1-specific cytotoxic T-lymphocyte (CTL) clones for HIV-1-infected primary CD4(+) T cells. We confirmed this effect by using a nef-mutant HIV-1 strain (NL-M20A) that expresses a Nef protein which does not induce down-regulation of HLA class I molecules but is otherwise functional. HIV-1-specific CTL clones were not able to kill primary CD4(+) T cells infected with a Nef-positive HIV-1 strain (NL-432) but efficiently lysed CD4(+) T cells infected with NL-M20A. Interestingly, CTL clones stimulated with NL-432-infected CD4(+) T cells were able to produce cytokines, albeit at a lower level than when stimulated with NL-M20A-infected CD4(+) T cells. This indicates that Nef-mediated HLA class I down-regulation affects CTL cytokine production to a lesser extent than cytolytic activity. Replication of NL-432 was partially suppressed in a coculture of HIV-1-infected CD4(+) T cells and HIV-1-specific CTL clones, while replication of NL-M20A was completely suppressed. These results suggest that HIV-1-specific CD8(+) T cells are able to partially suppress the replication of HIV-1 through production of soluble HIV-1-suppressive factors such as chemokines and gamma interferon. These findings may account for the mechanism whereby HIV-1-specific CD8(+) T cells are able to partially but not completely control HIV-1 replication in vivo.     

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.