A clathrin, caveolae, and dynamin-independent endocytic pathway requiring free membrane cholesterol drives HIV-1 internalization and infection in polarized trophoblastic cells

In human trophoblastic cells, a correlation between early endosomal trafficking of HIV-1 and virus infection was previously documented. However, if HIV-1 is massively internalized in these cells, the endocytic pathway(s) responsible for viral uptake is still undefined. Here we address this vital question. Amongst all the putative endocytic pathways present in polarized trophoblastic cells, we demonstrate that HIV-1 infection of these cells is independent of clathrin-mediated endocytosis and macropinocytosis. Importantly, treatment with the cholesterol-sequestering drug filipin severely impairs virus internalization, whereas the cholesterol-depleting compound methyl-beta-cyclodextrin has no impact on this pathway. Moreover, viral internalization is unaffected by overexpression of a mutant dynamin 2 or treatment with a kinase or tyrosine phosphatase inhibitor. Thus, HIV-1 infection in polarized trophoblastic cells occurs primarily via a clathrin, caveolae, and dynamin-independent pathway requiring free cholesterol. Notably, even though HIV-1 did not initially co-localize with transferrin, some virions migrate at later time points to transferrin-enriched endosomes, suggesting an unusual transit from the non-classical pathway to early endosomes. Finally, virus internalization in these cells does not involve the participation of microtubules but relies partly on actin filaments. Collectively these findings provide unprecedented information on the route of HIV-1 internalization in polarized human trophoblasts.     

Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells

Trophoblasts, the structural cells of the placenta, are thought to play a determinant role in in utero HIV type 1 (HIV-1) transmission. We have accumulated evidence suggesting that HIV-1 infection of these cells is associated with uptake by an unusual clathrin/caveolae-independent endocytic pathway and that endocytosis is followed by trafficking through multiple organelles. Furthermore, part of this trafficking involves the transit of HIV-1 from transferrin-negative to EEA1 and transferrin-positive endosomes, suggesting a merger from nonclassical to classical endocytic pathways in these cells. In the present article, the relationship between the presence of HIV-1 within specific endosomes and infection was studied. We demonstrate that viral infection is virtually lost when endosome inhibitors are added shortly after exposure to HIV-1. Thus, contrary to what is seen in CD4+ T lymphocytes, the initial presence of HIV-1 within the endosomes is mandatory for infection to take place. Importantly, this process is independent of the viral envelope proteins gp120 and gp41. The Rab family of small GTPases coordinates the vesicular transport between the different endocytic organelles. Experiments performed with various expression vectors indicated that HIV-1 infection in polarized trophoblasts relies on Rab5 and Rab7 without the contribution of Arf6 or Rab11. Furthermore, we conclude that Rab5 drives movements from raft-rich region to early endosomes, and this transit is required for subsequently reaching late endosomes via Rab7. This complex trafficking is mandatory for HIV-1 infection to proceed in human polarized trophoblasts.     

Macrophage bridging conduit trafficking of HIV-1 through the endoplasmic reticulum and Golgi network

Bridging conduits (BC) are tubular protrusions that facilitate cytoplasm and membrane exchanges between tethered cells. We now report that the human immunodeficiency virus type I (HIV-1) exploits these conduits to accelerate its spread and to shield it from immune surveillance. Endosome transport through BC drives HIV-1 intercellular transfers. How this occurs was studied in human monocyte-derived macrophages using proteomic, biochemical, and imaging techniques. Endosome, endoplasmic reticulum (ER), Golgi markers, and HIV-1 proteins were identified by proteomic assays in isolated conduits. Both the ER and Golgi showed elongated and tubular morphologies that extended into the conduits of polarized macrophages. Env and Gag antigen and fluorescent HIV-1 tracking demonstrated that these viral constituents were sequestered into endocytic and ER-Golgi organelles. Sequestered infectious viral components targeted the Golgi and ER by retrograde transport from early and Rab9 late endosomes. Disruption of the ER-Golgi network impaired HIV-1 trafficking in the conduit endosomes. This study provides, for the first time, mechanisms for how BC Golgi and ER direct cell-cell viral transfer.     

Inhibition of endosomal/lysosomal degradation increases the infectivity of human immunodeficiency virus

Productive entry of human immunodeficiency virus type 1 (HIV-1) into a host cell is believed to proceed via fusion of the viral envelope with the host cell's plasma membrane. Interestingly, the majority of HIV-1 particles that bind to the cell surface are taken up by the host cell via endocytosis; however, this mode of internalization generally does not result in infection. Presumably, virus particles remain trapped in the endocytic pathway and are eventually degraded. Here, we demonstrate that treatment of cells with various pharmacological agents known to elevate the pH of endosomes and lysosomes allows HIV-1 to efficiently enter and infect the host cell. Pretreatment of cells with bafilomycin A1 results in up to a 50-fold increase in the infectivity of HIV-1(SF2). Similarly, pretreatment of target cells with amantadine, concanamycin A, concanamycin B, chloroquine, and ammonium chloride resulted in increases in HIV-1 infectivity ranging between 2- and 15-fold. Analysis of receptor and coreceptor expression, HIV-long terminal repeat (LTR) transactivation, and transduction with amphotropic-pseudotyped murine leukemia virus (MLV)-based vectors suggests that the increase in infectivity is not artifactual. The increased infectivity under these conditions appears to be due to the ability of HIV-1 and MLV particles to enter via the endocytic pathway when spared from degradation in the late endosomes and lysosomes. These results could have significant implications for the administration of current and future lysosmotropic agents to patients with HIV disease.     

HIV-1 buds and accumulates in "nonacidic" endosomes of macrophages

Macrophages represent viral reservoirs in HIV-1-infected patients and accumulate viral particles within an endosomal compartment where they remain infectious for long periods of time. To determine how HIV-1 survives in endocytic compartments that become highly acidic and proteolytic and to study the nature of these virus-containing compartments, we carried out an ultrastructural study on HIV-1-infected primary macrophages. The endosomal compartments contain newly formed virions rather than internalized ones. In contrast to endocytic compartments free of viral proteins within the same infected cells, the virus containing compartments do not acidify. The lack of acidification is associated with an inability to recruit the proton pump vacuolar ATPase into the viral assembly compartment. This may prevent its fusion with lysosomes, since acidification is required for the maturation of endosomes. Thus, HIV-1 has developed a strategy for survival within infected macrophages involving prevention of acidification within a devoted endocytic virus assembly compartment.     

HIV-1 Vpu promotes release and prevents endocytosis of nascent retrovirus particles from the plasma membrane

The human immunodeficiency virus (HIV) type-1 viral protein U (Vpu) protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV) Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP) were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes.     

Definition of distinct compartments in polarized Madin-Darby canine kidney (MDCK) cells for membrane-volume sorting, polarized sorting and apical recycling

Previous studies of fibroblasts have demonstrated that recycling of endocytic receptors occurs through a default mechanism of membrane-volume sorting. Epithelial cells require an additional level of polar membrane sorting, but there are conflicting models of polar sorting, some suggesting that it occurs in early endosomes, others suggesting it occurs in a specialized apical recycling endosome (ARE). The relationship between endocytic sorting to the lysosomal, recycling and transcytotic pathways in polarized cells was addressed by characterizing the endocytic itineraries of LDL, transferrin (Tf) and IgA, respectively, in polarized Madin-Darby canine kidney (MDCK) cells. Quantitative analyses of 3-dimensional images of living and fixed polarized cells demonstrate that endocytic sorting occurs sequentially. Initially internalized into lateral sorting endosomes, Tf and IgA are jointly sorted from LDL into apical and medical recycling endosomes, in a manner consistent with default sorting of membrane from volume. While Tf is recycled to the basolateral membrane from recycling endosomes, IgA is sorted to the ARE prior to apical delivery. Quantifications of the efficiency of sorting of IgA from Tf between the recycling endosomes and the ARE match biochemical measurements of transepithelial protein transport, indicating that all polar sorting occurs in this step. Unlike fibroblasts, rab11 is not associated with Tf recycling compartments in either polarized or glass-grown MDCK cells, rather it is associated with the compartments to which IgA is directed after sorting from Tf. These results complicate a suggested homology between the ARE and the fibroblast perinuclear recycling compartment and provide a framework that justifies previous conflicting models of polarized sorting.     

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.     

Vpu and Tsg101 regulate intracellular targeting of the human immunodeficiency virus type 1 core protein precursor Pr55gag

Assembly of human immunodeficiency virus type 1 (HIV-1) is directed by the viral core protein Pr55gag. Depending on the cell type, Pr55gag accumulates either at the plasma membrane or on late endosomes/multivesicular bodies. Intracellular localization of Pr55gag determines the site of virus assembly, but molecular mechanisms that define cell surface or endosomal targeting of Pr55gag are poorly characterized. We have analyzed targeting of newly synthesized Pr55gag in HeLa H1 cells by pulse-chase studies and subcellular fractionations. Our results indicated that Pr55gag was inserted into the plasma membrane and, when coexpressed with the viral accessory protein Vpu, Pr55gag remained at the plasma membrane and virions assembled at this site. In contrast, Pr55gag expressed in the absence of Vpu was initially inserted into the plasma membrane, but subsequently endocytosed, and virus assembly was partially shifted to internal membranes. This endocytosis of Pr55gag required the host protein Tsg101. These results identified a previously unknown role for Vpu and Tsg101 as regulators for the endocytic uptake of Pr55gag and suggested that the site of HIV-1 assembly is determined by factors that regulate the endocytosis of Pr55gag.     

Human immunodeficiency virus type 1 infection of human placenta: potential route for fetal infection.

To determine the potential role of the placenta in transmission of human immunodeficiency virus (HIV) from mother to fetus, the ability of human placental tissue to support HIV type 1 (HIV-1) infection was examined. HIV-1-seronegative first-trimester placentas were maintained in culture and infected with HIV-1. Virus production, measured by HIV-1 antigen release into the supernatant, and HIV-1 DNA, identified by polymerase chain reaction, were detected for at least 12 days postinfection. Western immunoblot analysis showed Gag proteins, precursor p55, and cleavage products p24 and p17 in HIV-1-infected tissues. Double labeling of placental villi with antibodies to CD4 and placental trophoblast-specific alkaline phosphatase indicated that trophoblasts express CD4 antigen. Additionally, immunostaining of HIV-1-infected tissues with anti-p24 antibodies demonstrated HIV-1 protein expression in placental trophoblasts. Evaluation of human chorionic gonadotropin and progesterone production by the placental cultures indicated that there was a 90% decrease in human chorionic gonadotropin and a 70% decrease in progesterone production in HIV-1-infected cultures in comparison with controls. These data demonstrate that trophoblastic cells of human placenta tissue express CD4 and are susceptible to HIV-1 infection; also, placental endocrine function is decreased by HIV-1 infection. Thus, the placenta may serve as a reservoir of HIV-1 infection during pregnancy contributing to infection of the fetus, and decreased placental hormone production may result in impaired fetal development.     

Infectious HIV-1 assembles in late endosomes in primary macrophages

Although human immunodeficiency virus type 1 (HIV-1) is generally thought to assemble at the plasma membrane of infected cells, virions have been observed in intracellular compartments in macrophages. Here, we investigated virus assembly in HIV-1-infected primary human monocyte-derived macrophages (MDM). Electron microscopy of cryosections showed virus particles, identified by their morphology and positive labeling with antibodies to the viral p17, p24, and envelope proteins, in intracellular vacuoles. Immunolabeling demonstrated that these compartments contained the late endosomal marker CD63, which was enriched on vesicles within these structures and incorporated into the envelope of budding virions. The virus-containing vacuoles were also labeled with antibodies against LAMP-1, CD81, and CD82, which were also incorporated into the viral envelope. To assess the cellular source of infectious viruses derived from MDM, virus-containing media from infected cells were precipitated with specific antibodies. Only antibodies against antigens found in late endosomes precipitated infectious virus, whereas antibodies against proteins located primarily on the cell surface did not. Our data indicate that most of the infectious HIV produced by primary macrophages is assembled on late endocytic membranes and acquires antigens characteristic of this compartment. This notion has significant implications for understanding the biology of HIV and its cell-cell transmission.     

Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA.     

Cellubrevin is present in the basolateral endocytic compartment of hepatocytes and follows the transcytotic pathway after IgA internalization

The endocytic compartment of polarized cells is organized in basolateral and apical endosomes plus those endocytic structures specialized in recycling and transcytosis, which are still poorly characterized. The complexity of the various populations of endosomes has been demonstrated by the exquisite repertoire of endogenous proteins. In this study we examined the distribution of cellubrevin in the endocytic compartment of hepatocytes, since its intracellular location and function in polarized cells are largely unknown. Highly purified rat liver endosomes were isolated from estradiol-treated rats, and the early/sorting endosomal fraction was further subfractionated in a multistep sucrose density gradient, and studied. Analysis of dissected endosomal fractions showed that cellubrevin was located in early/sorting endosomes, with Rab4, annexins II and VI, and transferrin receptor, but in a specific subpopulation of these early endosomes with the same density range as pIgA and Raf-1. Interestingly, only in those isolated endosomal fractions, endosomes enriched in transcytotic structures (of livers loaded with IgA), the polymeric immunoglobulin receptor specifically co-immunoprecipitated with cellubrevin. In addition, confocal and immuno-electron microscopy identification of cellubrevin in tubular structures underneath the sinusoidal plasma membrane together with the re-organization of cellubrevin, in the endocytic compartment, after the IgA loading, strongly suggest the involvement of cellubrevin in the transcytosis of pIgA.     

Impact of the placental cytokine-chemokine balance on regulation of cell-cell contact-induced human immunodeficiency virus type 1 translocation across a trophoblastic barrier in vitro

Cells constituting the placental barrier secrete soluble factors that may participate in controlling human immunodeficiency virus type 1 (HIV-1) transmission from the mother to the fetus. In this study, we asked whether placental soluble factors (PSF) could limit cell-cell contact inducing HIV-1 production that occurs after inoculation of HIV-1-infected peripheral blood mononuclear cells (HIV-1+ PBMCs) onto trophoblast-derived BeWo cells grown as tight and polarized barriers in a two-chamber system. The activity of recombinant chemokines and cytokines expressed by placental tissue and of factors secreted by either early or term placentae of HIV-1-negative women, was analyzed. We identified chemokines (RANTES and MIP-1beta) and cytokines (tumor necrosis factor alpha and interleukin-8) that decreased and increased, respectively, viral production in trophoblast barrier cells inoculated with HIV-1+ PBMCs. Unexpectedly, factors secreted by either early or term placentae of HIV-1-negative women enhanced viral production. Nevertheless, the same PSF did not favor infection of trophoblastic barriers with cell-free HIV-1 and strongly reduced viral production in PBMCs infected with cell-free HIV-1. Moreover, PSF contained chemokines (RANTES and MIP-1beta) and a cytokine, leukemia inhibitory factor, exhibiting a strong anti-HIV-1 activity in our model of cell-to-cell infection. Together these data suggested that at the maternal interface the global activity of PSF is related to the synergistic action of several soluble factors with a balance in favor of an enhancing activity on the passage of viruses across the trophoblast barrier. This could explain the presence of viral sequences in trophoblasts in all placentae of HIV-1-infected women.     

Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection.

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.     

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.     

Human immunodeficiency virus type 1 Nef induces accumulation of CD4 in early endosomes.

We have studied the fate of CD4 in CEM T cells expressing a human immunodeficiency virus type 1 HIV-1 Nef protein. Nef triggered a rapid endocytosis and a degradation of CD4, while most of the p56lck was upheld at the cell membrane. In the presence of Nef, CD4 accumulated in acidic intracellular vesicles that were not stained by antibodies against rab6, a marker of the Golgi apparatus complex. Detection of transferrin in CD4-containing vesicles showed that CD4 was trapped in early endosomes, without significant accumulation of CD4 in late endocytic compartments. Internalization pathways taken by CD4 in Nef+ cells may therefore be different from those observed after treatment with phorbol esters.     

The role of LIP5 and CHMP5 in multivesicular body formation and HIV-1 budding in mammalian cells

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release.     

Apical and basolateral endocytic pathways of MDCK cells meet in acidic common endosomes distinct from a nearly-neutral apical recycling endosome

Quantitative confocal microscopic analyses of living, polarized MDCK cells demonstrate different pH profiles for apical and basolateral endocytic pathways, despite a rapid and extensive intersection between the two. Three-dimensional characterizations of ligand trafficking demonstrate that the apical and basolateral endocytic pathways share early, acidic compartments distributed throughout the medial regions of the cell. Polar sorting for both pathways occurs in these common endosomes as IgA is sorted from transferrin to alkaline transcytotic vesicles. While transferrin is directly recycled from the common endosomes, IgA is transported to a downstream apical compartment that is nearly neutral in pH. By several criteria this compartment appears to be equivalent to the previously described apical recycling endosome. The functional significance of the abrupt increase in lumenal pH that accompanies IgA sorting is not clear, as disrupting endosome acidification has no effect on polar sorting. These studies provide the first detailed characterizations of endosome acidification in intact polarized cells and clarify the relationship between the apical and basolateral endocytic itineraries of polarized MDCK cells. The extensive mixing of apical and basolateral pathways underscores the importance of endocytic sorting in maintaining the polarity of the plasma membrane of MDCK cells.