High BMP4 expression in low/intermediate risk BCP-ALL identifies children with poor outcomes.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) outcome has improved in the last decades, but leukemic relapses are still one of the main problems of this disease. Bone morphogenetic protein 4 (BMP4) was investigated as a new candidate biomarker with potential prognostic relevance, and its pathogenic role was assessed in the development of disease. A retrospective study was performed with 115 pediatric patients with BCP-ALL, and BMP4 expression was analyzed by quantitative reverse transcription polymerase chain reaction in leukemic blasts at the time of diagnosis. BMP4 mRNA expression levels in the third (upper) quartile were associated with a higher cumulative incidence of relapse as well as a worse 5-year event-free survival and central nervous system (CNS) involvement. Importantly, this association was also evident among children classified as having a nonhigh risk of relapse. A validation cohort of 236 patients with BCP-ALL supported these data. Furthermore, high BMP4 expression promoted engraftment and rapid disease progression in an NSG mouse xenograft model with CNS involvement. Pharmacological blockade of the canonical BMP signaling pathway significantly decreased CNS infiltration and consistently resulted in amelioration of clinical parameters, including neurological score. Mechanistically, BMP4 favored chemoresistance, enhanced adhesion and migration through brain vascular endothelial cells, and promoted a proinflammatory microenvironment and CNS angiogenesis. These data provide evidence that BMP4 expression levels in leukemic cells could be a useful biomarker to identify children with poor outcomes in the low-/intermediate-risk groups of BCP-ALL and that BMP4 could be a new therapeutic target to blockade leukemic CNS disease.

Role of the small GTPase Rab27a during herpes simplex virus infection of oligodendrocytic cells.

BACKGROUND: The morphogenesis of herpes simplex virus type 1 (HSV-1) comprises several events, of which some are not completely understood. It has been shown that HSV-1 glycoproteins accumulate in the trans-Golgi network (TGN) and in TGN-derived vesicles. It is also accepted that HSV-1 acquires its final morphology through a secondary envelopment by budding into TGN-derived vesicles coated with viral glycoproteins and tegument proteins. Nevertheless, several aspects of this process remain elusive. The small GTPase Rab27a has been implicated in regulated exocytosis, and it seems to play a key role in certain membrane trafficking events. Rab27a also seems to be required for human cytomegalovirus assembly. However, despite the involvement of various Rab GTPases in HSV-1 envelopment, there is, to date, no data reported on the role of Rab27a in HSV-1 infection. RESULTS: Herein, we show that Rab27a colocalized with GHSV-UL46, a tegument-tagged green fluorescent protein-HSV-1, in the TGN. In fact, this small GTPase colocalized with viral glycoproteins gH and gD in that compartment. Functional analysis through Rab27a depletion showed a significant decrease in the number of infected cells and viral production in Rab27a-silenced cells. CONCLUSIONS: Altogether, our results indicate that Rab27a plays an important role in HSV-1 infection of oligodendrocytic cells.

A role for the SNARE protein syntaxin 3 in human cytomegalovirus morphogenesis.

As an enveloped virus, replication of human cytomegalovirus (HCMV) is dependent on interaction with cellular membrane systems. Its final envelopment occurs into intracellular membranes prior to its secretion. However the mechanisms underlying these processes are poorly understood. Here, we show that HCMV infection induces expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 3 (STX3), a component of the cellular machinery for membrane fusion. STX3 was located at the plasma membrane and at the assembly site where it was found associated with virus wrapping membranes by immunogold labelling. Depletion of STX3 using RNA interference reduced HCMV production, while expression of a STX3 construct resistant to RNAi inhibition enhanced virus production. Ultrastructural examination of the assembly site in HCMV-infected STX3-depleted cells showed fewer mature virions and more viruses undergoing final envelopment. In contrast, silencing of STX3 did not affect herpes simplex virus type 1 production. The mechanism through which STX3 affected HCMV morphogenesis likely involved late endosomes/lysosomes since STX3 depletion reduced the expression of lysosomal membrane glycoproteins. Our results demonstrate a function for STX3 in HCMV morphogenesis, and unravel a new role for this SNARE protein in late endosomes/lysosomes compartments.

Human cytomegalovirus final envelopment on membranes containing both trans-Golgi network and endosomal markers.

The human cytomegalovirus (HCMV) has been shown to complete its final envelopment on cytoplasmic membranes prior to its secretion to the extracellular medium. However, the nature of these membranes has not been characterized. It is thought that HCMV acquires its final envelope from the trans-Golgi network (TGN), though we and others have previously reported a role for endocytic membranes. Here we studied the localization of cellular markers in HCMV-infected cells and in isolated viruses. Immunofluorescence staining indicated that HCMV induces the recruitment of TGN and endosomal markers to the virus factory. Immuno-gold labelling of isolated viral particles and electron microscopy demonstrated the incorporation of TGN46, endosomal markers early endosomal antigen 1, annexin I, transferrin receptor and CD63, and the cation-independent mannose 6-phosphate receptor, which traffics between the TGN and endosomes into the viral envelope. Virus immunoprecipitation assays demonstrated that virions containing TGN46 and CD63 were infectious. This study reconciles the apparent controversy regarding the nature of the HCMV assembly site and suggests that HCMV has the ability to generate a novel membrane compartment containing markers for both TGN and endosomes, or that the membranes that HCMV uses for its envelope may be vesicles in transit between the TGN and endosomes.

Membrane remodelling during vaccinia virus morphogenesis.

BACKGROUND INFORMATION: VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components. RESULTS: We have used electron tomography of sections of VACV-infected cells to follow, in three dimensions, the remodelling of the membrane components of the virus during envelope maturation. The tomograms obtained suggest that a number of independent 'crescents' interact with each other to enclose the volume of an incomplete ellipsoid in the viral factory area, attaining the overall shape and size characteristic of the first immature form of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to particles with a nucleoid [IVN (IV with nucleoid)] that results in local disorganization of the envelope in regions near the condensed DNA. These particles suffer the progressive disappearance of the membrane outer spikes with a change in the shape of the membrane, becoming locally curled. The transformation of the IVN into the mature virus involves an extreme rearrangement of the particle envelope, which becomes fragmented and undulated. During this process, we also observed connections between the outer membranes with internal ones, suggesting that the latter originate from internalization of the IV envelope. CONCLUSIONS: The main features observed for VACV membrane maturation during morphogenesis resemble the breakdown and reassembly of cellular endomembranes.

African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication.IMPORTANCE African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies.

Acute Lymphoblastic Leukaemia Cells Impair Dendritic Cell and Macrophage Differentiation: Role of BMP4.

Dendritic cells and macrophages are common components of the tumour immune microenvironment and can contribute to immune suppression in both solid and haematological cancers. The Bone Morphogenetic Protein (BMP) pathway has been reported to be involved in cancer, and more recently in leukaemia development and progression. In the present study, we analyse whether acute lymphoblastic leukaemia (ALL) cells can affect the differentiation of dendritic cells and macrophages and the involvement of BMP pathway in the process. We show that ALL cells produce BMP4 and that conditioned media from ALL cells promote the generation of dendritic cells with immunosuppressive features and skew M1-like macrophage polarization towards a less pro-inflammatory phenotype. Likewise, BMP4 overexpression in ALL cells potentiates their ability to induce immunosuppressive dendritic cells and favours the generation of M2-like macrophages with pro-tumoral features. These results suggest that BMP4 is in part responsible for the alterations in dendritic cell and macrophage differentiation produced by ALL cells.

Protein Kinase C δ Regulates the Depletion of Actin at the Immunological Synapse Required for Polarized Exosome Secretion by T Cells.

Multivesicular bodies (MVB) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, ILVs are secreted as Fas ligand-bearing, pro-apoptotic exosomes following T cell receptor (TCR)-induced fusion of MVB with the plasma membrane at the immune synapse (IS). In this study we show that protein kinase C δ (PKCδ), a novel PKC isotype activated by diacylglycerol (DAG), regulates TCR-controlled MVB polarization toward the IS and exosome secretion. Concomitantly, we demonstrate that PKCδ-interfered T lymphocytes are defective in activation-induced cell death. Using a DAG sensor based on the C1 DAG-binding domain of PKCδ and a GFP-PKCδ chimera, we reveal that T lymphocyte activation enhances DAG levels at the MVB endomembranes which mediates the association of PKCδ to MVB. Spatiotemporal reorganization of F-actin at the IS is inhibited in PKCδ-interfered T lymphocytes. Therefore, we propose PKCδ as a DAG effector that regulates the actin reorganization necessary for MVB traffic and exosome secretion.

Tracking Endocytosis and Intracellular Trafficking of Epitope-tagged Syntaxin 3 by Antibody Feeding in Live, Polarized MDCK Cells.

The uptake and trafficking of cell surface receptors can be monitored by a technique called 'antibody-feeding' which uses an externally applied antibody to label the receptor on the surface of cultured, live cells. Here, we adapt the traditional antibody-feeding experiment to polarized epithelial cells (Madin-Darby Canine Kidney) grown on permeable Transwell supports. By adding two tandem extracellular Myc epitope tags to the C-terminus of the SNARE protein syntaxin 3 (Stx3), we provided a site where an antibody could bind, allowing us to perform antibody-feeding experiments on cells with distinct apical and basolateral membranes. With this procedure, we observed the endocytosis and intracellular trafficking of Stx3. Specifically, we assessed the internalization rate of Stx3 from the basolateral membrane and observed the ensuing endocytic route in both time and space using immunofluorescence microscopy on cells fixed at different time points. For cell lines that form a polarized monolayer containing distinct apical and basolateral membranes when cultured on permeable supports, e.g., MDCK or Caco-2, this protocol can measure the rate of endocytosis and follow the subsequent trafficking of a target protein from either limiting membrane.

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes.

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral-but not the apical-plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion.

Role of Proteolipid Protein in HSV-1 Entry in Oligodendrocytic Cells.

Herpes simplex virus type 1 (HSV-1) has the ability to enter many different hosts and cell types by several strategies. This highly prevalent alphaherpesvirus can enter target cells using different receptors and different pathways: fusion at a neutral pH, low-pH-dependent and low-pH-independent endocytosis. Several cell receptors for viral entry have been described, but several observations suggest that more receptors for HSV-1 might exist. In this work, we propose a novel role for the proteolipid protein (PLP) in HSV-1 entry into the human oligodendrocytic cell line HOG. Cells transfected with PLP-EGFP showed an increase in susceptibility to HSV-1. Furthermore, the infection of HOG and HOG-PLP transfected cells with the R120vGF virus--unable to replicate in ICP4-defficient cells--showed an increase in viral signal in HOG-PLP, suggesting a PLP involvement in viral entry. In addition, a mouse monoclonal antibody against PLP drastically inhibited HSV-1 entry into HOG cells. PLP and virions colocalized in confocal immunofluorescence images, and in electron microscopy images, which suggest that PLP acts at the site of entry into HOG cells. Taken together these results suggest that PLP may be involved in HSV-1 entry in human oligodendrocytic cells.

Human cytomegalovirus encoded chemokine receptor US28 activates the HIF-1α/PKM2 axis in glioblastoma cells.

The human cytomegalovirus (HCMV) encoded chemokine receptor US28 promotes tumorigenesis through activation of various proliferative and angiogenic signaling pathways. Upon infection, US28 displays constitutive activity and signals in a G protein-dependent manner, hijacking the host's cellular machinery. In tumor cells, the hypoxia inducible factor-1α/pyruvate kinase M2 (HIF-1α/PKM2) axis plays an important role by supporting proliferation, angiogenesis and reprogramming of energy metabolism. In this study we show that US28 signaling results in activation of the HIF-1α/PKM2 feedforward loop in fibroblasts and glioblastoma cells. The constitutive activity of US28 increases HIF-1 protein stability through a Gαq-, CaMKII- and Akt/mTOR-dependent mechanism. Furthermore, we found that VEGF and lactate secretion are increased and HIF-1 target genes, glucose transporter type 1 (GLUT1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), involved in glucose metabolism, are upregulated in US28 expressing cells. In addition, PKM2 is phosphorylated and found to be in a tumor-associated dimeric state upon US28 expression. Also in HCMV-infected cells HIF-1 activity is enhanced, which in part is US28-dependent. Finally, increased proliferation of cells expressing US28 is abolished upon inhibition of the HIF-1α/PKM2 cascade. These data highlight the importance of HIF-1α and PKM2 in US28-induced proliferation, angiogenesis and metabolic reprogramming.

Rab27a is required for human cytomegalovirus assembly.

Human cytomegalovirus (HCMV) completes its final envelopment on intracellular membranes before it is released from the cell. The mechanisms underlying these processes are not understood. Here we studied the role of Rab27a, a regulator of lysosome-related organelle transport, in HCMV production. HCMV infection increased Rab27a expression, and recruitment of Rab27a to membranous strutures at the assembly site. Immuno-gold labelling demonstrated association of Rab27a with viral envelopes. CMV production was reduced after knock-down of Rab27a, and in Rab27a-deficient ashen melanocytes. This study shows a requirement for Rab27a in the CMV life cycle and suggests that CMV and LRO biogenesis share common molecular mechanisms.

HCMV-encoded chemokine receptor US28 mediates proliferative signaling through the IL-6-STAT3 axis.

US28 is a viral G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor encoded by the human cytomegalovirus (HCMV). In addition to binding and internalizing chemokines, US28 constitutively activates signaling pathways linked to cell proliferation. Here, we show increased concentrations of vascular endothelial growth factor and interleukin-6 (IL-6) in supernatants of US28-expressing NIH 3T3 cells. Increased IL-6 was associated with increased activation of the signal transducer and activator of transcription 3 (STAT3) through upstream activation of the Janus-activated kinase JAK1. We used conditioned growth medium, IL-6-neutralizing antibodies, an inhibitor of the IL-6 receptor, and short hairpin RNA targeting IL-6 to show that US28 activates the IL-6-JAK1-STAT3 signaling axis through activation of the transcription factor nuclear factor kappaB and the consequent production of IL-6. Treatment of cells with a specific inhibitor of STAT3 inhibited US28-dependent [(3)H]thymidine incorporation and foci formation, suggesting a key role for STAT3 in the US28-mediated proliferative phenotype. US28 also elicited STAT3 activation and IL-6 secretion in HCMV-infected cells. Analyses of tumor specimens from glioblastoma patients demonstrated colocalization of US28 and phosphorylated STAT3 in the vascular niche of these tumors. Moreover, increased phospho-STAT3 abundance correlated with poor patient outcome. We propose that US28 induces proliferation in HCMV-infected tumors by establishing a positive feedback loop through activation of the IL-6-STAT3 signaling axis.

The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice.

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3ß (GSK-3ß) function, promoted accumulation of ß-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.

Novel replication complex architecture in rubella replicon-transfected cells.

Rubella virus (RUB) assembles its replication complexes (RCs) in modified organelles of endo-lysosomal origin, known as cytopathic vacuoles (CPVs). These peculiar structures are key elements of RUB factories, where rough endoplasmic reticulum, mitochondria, and Golgi are recruited. Bicistronic RUB replicons expressing an antibiotic resistance gene either in the presence or the absence of the RUB capsid (C) gene were used to study the structure of RCs in transfected cells. Confocal microscopy showed that the RUB replicase components P90 and P150 localized to CPVs, as did double-stranded RNA (dsRNA), a marker for RNA synthesis. Electron microscopy (EM) showed that replicons generated CPVs containing small vesicles and large vacuoles, similar to CPVs from RUB-infected cells and that the replicase proteins were sufficient for organelle recruitment. Some of these CPVs contained straight membranes. When cross-sectioned, these rigid membranes appeared to be sheets of closely packed proteins. Immuno-EM revealed that these sheets, apparently in contact with the cytosol, contained both P150 and P90, as well as dsRNA, and thus could be two-dimensional arrays of functional viral replicases. Labelling of dsRNA after streptolysin-O permeabilization showed that replication of viral genome takes place on the cytoplasmic side of CPVs. When present, C accumulated around CPVs. Mitochondrial protein P32 was detected within modified CPVs, the first demonstration of involvement of this protein, which interacts with C, with RCs.

The ESCRT machinery is not required for human cytomegalovirus envelopment.

The human cytomegalovirus (HCMV) has been proposed to complete its final envelopment on cytoplasmic membranes prior to its release to the extracellular medium. The nature of these membranes and the mechanisms involved in virus envelopment and release are poorly understood. Here we show by immunogold-labelling and electron microscopy that CD63, a marker of multivesicular bodies (MVBs), is incorporated into the viral envelope, supporting the notion that HCMV uses endocytic membranes for its envelopment. We therefore investigated a possible role for the cellular endosomal sorting complex required for transport (ESCRT) machinery in HCMV envelopment. Depletion of tumour suppressor gene 101 and ALIX/AIP1 with small interfering RNAs (siRNAs) in HCMV-infected cells did not affect virus production. In contrast, siRNAs against the vacuolar protein sorting 4 (VPS4) proteins silenced the expression of VPS4A and VPS4B, inhibited the sorting of epidermal growth factor to lysosomes, the formation of HIV Gag-derived virus-like particles and vesicular stomatitis virus infection, but enhanced the number of HCMV viral particles produced. Treatment of infected cells with protease inhibitors also increased viral production. These studies indicate that, in contrast to some enveloped RNA viruses, HCMV does not require the cellular ESCRT machinery to complete its envelopment.

Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway.

Arrestins bind phosphorylated G-protein coupled-receptors (GPCR) and inhibit agonist-induced signal transduction by uncoupling the receptors from their cognate G-proteins. beta-arrestins also act as adaptors that target GPCR to endocytic clathrin-coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of beta-arrestins in US28 trafficking, we used embryonic fibroblasts derived from beta-arrestin knockout mice. In these cells, the internalization of transfected beta2-adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28-mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin-mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the micro2-adaptin subunit of the AP-2 adaptor complex. In cells transfected with micro2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody-feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin-mediated mechanism but is independent of beta-arrestins.

NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

Localization of HCMV UL33 and US27 in endocytic compartments and viral membranes.

The human cytomegalovirus genome encodes four putative seven transmembrane domain chemokine receptor-like proteins. Although important in viral pathogenesis, little is known about the properties or functions of these proteins. We previously reported that US28 is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Here we studied the cellular distributions and trafficking of two other human cytomegalovirus chemokine receptor-like proteins, UL33 and US27, in transfected and human cytomegalovirus-infected cells. Immunofluorescence staining indicated that UL33 and US27 are located at the cell surface, although the majority of both proteins was seen in intracellular organelles located in the perinuclear region of the cell. The intracellular pools of UL33 and US27 showed overlap with markers for endocytic organelles. Antibody-feeding experiments indicated that cell surface US27 undergoes endocytosis. By immunogold labeling of cryosections and electron microscopy, UL33 was seen to localize to multivesicular bodies (MVBs or multivesicular endosomes). Electron microscopy analysis of human cytomegalovirus-infected cells showed that most virus particles wrapped individually into short membrane cisternae, although virus particles were also occasionally seen within and budding into MVBs. Electron microscopy immunolocalization of viral UL33 and US27 on ultrathin cryosections of human cytomegalovirus-infected cells showed gold particles over the membranes into which virions were wrapping, in small membrane tubules and vesicles and in MVBs. Labeling of the human cytomegalovirus glycoproteins gB and gH indicated that these proteins were also present in the same membrane structures. This first electron microscopy analysis of human cytomegalovirus assembly using immunolabeling suggests that the localization of UL33, US27 and US28 to endosomes may allow these proteins to be incorporated into the viral membrane during the final stages of human cytomegalovirus assembly.