The ACE2 Receptor for Coronavirus Entry Is Localized at Apical Cell-Cell Junctions of Epithelial Cells.

Transmembrane proteins of adherens and tight junctions are known targets for viruses and bacterial toxins. The coronavirus receptor ACE2 has been localized at the apical surface of epithelial cells, but it is not clear whether ACE2 is localized at apical Cell-Cell junctions and whether it associates with junctional proteins. Here we explored the expression and localization of ACE2 and its association with transmembrane and tight junction proteins in epithelial tissues and cultured cells by data mining, immunoblotting, immunofluorescence microscopy, and co-immunoprecipitation experiments. ACE2 mRNA is abundant in epithelial tissues, where its expression correlates with the expression of the tight junction proteins cingulin and occludin. In cultured epithelial cells ACE2 mRNA is upregulated upon differentiation and ACE2 protein is widely expressed and co-immunoprecipitates with the transmembrane proteins ADAM17 and CD9. We show by immunofluorescence microscopy that ACE2 colocalizes with ADAM17 and CD9 and the tight junction protein cingulin at apical junctions of intestinal (Caco-2), mammary (Eph4) and kidney (mCCD) epithelial cells. These observations identify ACE2, ADAM17 and CD9 as new epithelial junctional transmembrane proteins and suggest that the cytokine-enhanced endocytic internalization of junction-associated protein complexes comprising ACE2 may promote coronavirus entry.

M-Sec facilitates intercellular transmission of HIV-1 through multiple mechanisms.

BACKGROUND: HIV-1 promotes the formation of tunneling nanotubes (TNTs) that connect distant cells, aiding cell-to-cell viral transmission between macrophages. Our recent study suggests that the cellular protein M-Sec plays a role in these processes. However, the timing, mechanism, and to what extent M-Sec contributes to HIV-1 transmission is not fully understood, and the lack of a cell line model that mimics macrophages has hindered in-depth analysis. RESULTS: We found that HIV-1 increased the number, length and thickness of TNTs in a manner dependent on its pathogenic protein Nef and M-Sec in U87 cells, as observed in macrophages. In addition, we found that M-Sec was required not only for TNT formation but also motility of U87 cells, both of which are beneficial for viral transmission. In fact, M-Sec knockdown in U87 cells led to a significantly delayed viral production in both cellular and extracellular fractions. This inhibition was observed for wild-type virus, but not for a mutant virus lacking Nef, which is known to promote not only TNT formation but also migration of infected macrophages. CONCLUSIONS: By taking advantage of useful features of U87 cells, we provided evidence that M-Sec mediates a rapid and efficient cell-cell transmission of HIV-1 at an early phase of infection by enhancing both TNT formation and cell motility.

Alteration of cell junctions during viral infection.

Cell junctions serve as a protective barrier for cells and provide an important channel for information transmission between cells and the surrounding environment. Viruses are parasites that invade and commandeer components of host cells in order to survive and replicate, and they have evolved various mechanisms to alter cell junctions to facilitate viral infection. In this review, we examined the current state of knowledge on the action of viruses on host cell junctions. The existing evidence suggests that targeting the molecules involved in the virus-cell junction interaction can prevent the spread of viral diseases.

Cell communication by tunneling nanotubes: Implications in disease and therapeutic applications.

Intercellular communication is essential for the development and maintenance of multicellular organisms. Tunneling nanotubes (TNTs) are a recently recognized means of long and short distance communication between a wide variety of cell types. TNTs are transient filamentous membrane protrusions that connect cytoplasm of neighboring or distant cells. Cytoskeleton fiber-mediated transport of various cargoes occurs through these tubules. These cargoes range from small ions to whole organelles. TNTs have been shown to contribute not only to embryonic development and maintenance of homeostasis, but also to the spread of infectious particles and resistance to therapies. These functions in the development and progression of cancer and infectious disease have sparked increasing scrutiny of TNTs, as their contribution to disease progression lends them a promising therapeutic target. Herein, we summarize the current knowledge of TNT structure and formation as well as the role of TNTs in pathology, focusing on viral, prion, and malignant disease. We then discuss the therapeutic possibilities of TNTs in light of their varied functions. Despite recent progress in the growing field of TNT research, more studies are needed to precisely understand the role of TNTs in pathological conditions and to develop novel therapeutic strategies.

Penton-dodecahedral particles trigger opening of intercellular junctions and facilitate viral spread during adenovirus serotype 3 infection of epithelial cells.

Human adenovirus serotypes Ad3, Ad7, Ad11, and Ad14 use the epithelial junction protein desmoglein 2 (DSG2) as a receptor for infection. During Ad infection, the fiber and penton base capsid proteins are produced in vast excess and form hetero-oligomers, called pentons. It has been shown for Ad3 that pentons self-assemble into penton-dodecahedra (PtDd). Our previous studies with recombinant purified Ad3 PtDd (produced in insect cells) showed that PtDd bind to DSG2 and trigger intracellular signaling resulting in the transient opening of junctions between epithelial cells. So far, a definitive proof for a function of Ad3 PtDd in the viral life cycle is elusive. Based on the recently published 3D structure of recombinant Ad3 PtDd, we generated a penton base mutant Ad3 vector (mu-Ad3GFP). mu-Ad3GFP is identical to its wild-type counterpart (wt-Ad3GFP) in the efficiency of progeny virus production; however, it is disabled in the production of PtDd. For infection studies we used polarized epithelial cancer cells or cell spheroids. We showed that in wt-Ad3GFP infected cultures, PtDd were released from cells before viral cytolysis and triggered the restructuring of epithelial junctions. This in turn facilitated lateral viral spread of de novo produced virions. These events were nearly absent in mu-Ad3GFP infected cultures. Our in vitro findings were consolidated in mice carrying xenograft tumors derived from human epithelial cancer cells. Furthermore, we provide first evidence that PtDd are also formed by another DSG2-interacting Ad serotype, the newly emerged, highly pathogenic Ad14 strain (Ad14p1). The central finding of this study is that a subgroup of Ads has evolved to generate PtDd as a strategy to achieve penetration into and dissemination in epithelial tissues. Our findings are relevant for basic and applied virology, specifically for cancer virotherapy.

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.

Mucosal junctions: open doors to HPV and HIV infections?

Throughout adult life, new developmental commitment of adult stem cells causes reversible epithelial replacements in various mucosal surfaces, including the uterine cervix and the anal canal. Located at the squamocolumnar junctions, these metaplastic conversions are associated with chronic inflammation and deregulated expression of soluble and cell-membrane factors important for antiviral immune response. In this paper, we propose that these histological and immunological features increase the susceptibility of these metaplastic microenvironments to human papillomavirus and human immunodeficiency virus infections. Identification of the anatomical sites and cell populations within the anogenital tract, which is the site primary infected by these viruses, is crucial for the understanding of the pathogenesis of viral disease and development of antiviral strategies.

Junctional gating: the achilles' heel of epithelial cells in pathogen infection.

Mucosal epithelial cells are a major barrier restricting pathogen entry and, paradoxically, an important entry port for respiratory and enteric viruses. Elegant studies in this issue of Cell Host & Microbe describe how coxsackievirus B3 (related to human poliovirus) infects polarized epithelial cells by engaging two transmembrane proteins of the tight junctions, occludin and CAR. A distinctive endocytic mechanism opens the junctions and gates infectious virus entry.

Herpes simplex virus gE/gI must accumulate in the trans-Golgi network at early times and then redistribute to cell junctions to promote cell-cell spread.

Herpes simplex virus (HSV) glycoprotein heterodimer gE/gI is necessary for virus spread in epithelial and neuronal tissues. Deletion of the relatively large gE cytoplasmic (CT) domain abrogates the ability of gE/gI to mediate HSV spread. The gE CT domain is required for the sorting of gE/gI to the trans-Golgi network (TGN) in early stages of virus infection, and there are several recognizable TGN sorting motifs grouped near the center of this domain. Late in HSV infection, gE/gI, other viral glycoproteins, and enveloped virions redistribute from the TGN to epithelial cell junctions, and the gE CT domain is also required for this process. Without the gE CT domain, newly enveloped virions are directed to apical surfaces instead of to cell junctions. We hypothesized that the gE CT domain promotes virus envelopment into TGN subdomains from which nascent enveloped virions are sorted to cell junctions, a process that enhances cell-to-cell spread. To characterize elements of the gE CT domain involved in intracellular trafficking and cell-to-cell spread, we constructed a panel of truncation mutants. Specifically, these mutants were used to address whether sorting to the TGN and redistribution to cell junctions are necessary, and sufficient, for gE/gI to promote cell-to-cell spread. gE-519, lacking 32 C-terminal residues, localized normally to the TGN early in infection and then trafficked to cell junctions at late times and mediated virus spread. By contrast, mutants gE-495 (lacking 56 C-terminal residues) and gE-470 (lacking 81 residues) accumulated in the TGN but did not traffic to cell junctions and did not mediate cell-to-cell spread. A fourth mutant, gE-448 (lacking most of the CT domain), did not localize to cell junctions and did not mediate virus spread. Therefore, the capacity of gE/gI to promote cell-cell spread requires early localization to the TGN, but this is not sufficient for virus spread. Additionally, gE CT sequences between residues 495 and 519, which contain no obvious cell sorting motifs, are required to promote gE/gI traffic to cell junctions and cell-to-cell spread.

Human T-lymphotropic virus, type 1, tax protein triggers microtubule reorientation in the virological synapse.

We showed recently that the human T-lymphotropic virus, type 1 (HTLV-1), spreads directly from cell to cell via a virological synapse. The HTLV-1 virological synapse resembles the immunological synapse; each is a specialized contact between a lymphocyte and another cell that contains organized protein microdomains, and each involves repolarization of the T-cell microtubule cytoskeleton. However, formation of the virological synapse is not triggered by T-cell receptor-mediated antigen recognition. On the basis of our previous data, we postulated that formation of the viral synapse was triggered by a conjunction of two signals, one from HTLV-1 infection of the T-cell and one from cell-cell contact. We have recently identified ICAM-1 engagement as a cell-contact signal that causes the microtubule polarization associated with the virological synapse. Here we used confocal microscopy of T-lymphocytes naturally infected with HTLV-1 or transfected with individual HTLV-1 genes to investigate the role of the viral transcriptional transactivator protein Tax. Polarization of the microtubules was induced by cell-cell contact or by cross-linking T-cell surface molecules with monoclonal antibodies adsorbed to latex beads. We show that Tax, which is mainly found in the nucleus, is also present at two specific extranuclear sites as follows: around the microtubule organizing center in association with the cis-Golgi and in the cell-cell contact region. We show that expression of Tax provides an intracellular signal that synergizes with ICAM-1 engagement to cause the T-cell microtubule polarization observed at the virological synapse.

Dangerous liaisons at the virological synapse.

Cell-to-cell viral transmission facilitates the propagation of HIV-1 and human T cell leukemia virus type 1. Mechanisms of cell-to-cell transmission by retroviruses were not well understood until the recent description of virological synapses (VSs). VSs function as specialized sites of immune cell-to-cell contact that direct virus infection. Deciphering the molecular mechanisms of VS formation provides a fascinating insight into how pathogens subvert immune cell communication programs and achieve viral spread.

Retroviral spread by induction of virological synapses.

Cells of the immune system communicate via the formation of receptor-containing adhesive junctions termed immunological synapses. Recently, retroviruses have been shown to subvert this process in order to pass directly from infected to uninfected immune cells. Such cell-cell viral dissemination appears to function by triggering existing cellular pathways involved in antigen presentation and T-cell communication. This mode of viral spread has important consequences for both the virus and the host cells in terms of viral pathogenesis and viral resistance to immune and therapeutic intervention. This review summarises the current knowledge concerning virological synapses induced by retroviruses.

The immune control and cell-to-cell spread of human T-lymphotropic virus type 1.

Human T-lymphotropic virus type 1 (HTLV-1) varies little in sequence compared with human immunodeficiency virus type 1 (HIV) and it is difficult to detect HTLV-1 mRNA, proteins or virions in fresh blood. But the strong and chronically activated T cell response to the virus indicates that HTLV-1 proteins are expressed persistently. It now appears that the efficiency of an individual's cytotoxic T cell (CTL) response to HTLV-1 is the chief single determinant of that person's provirus load, which can differ between HTLV-1-infected people by more than 10 000-fold. Progress is now being made towards defining this CTL 'efficiency' in terms of host genetics, T cell function, T cell gene expression and mathematical dynamics. Lymphocytes that are naturally infected with HTLV-1 do not produce enveloped extracellular virions in short-term culture and this has reinforced the erroneous conclusion that the virus is latent. But recent evidence shows that HTLV-1 can spread directly between lymphocytes across a specialized, virus-induced cell-cell contact - a 'viral synapse'. Instead of making extracellular virions, HTLV-1 uses the mobility of the host cell to spread within and between hosts. In this review the evidence is summarized on the persistent gene expression of HTLV-1 in vivo, the role of the immune system in protection and pathogenesis in HTLV-1 infection, and the mechanism of cell-to-cell spread of HTLV-1.

Spread of HTLV-I between lymphocytes by virus-induced polarization of the cytoskeleton.

Cell contact is required for efficient transmission of human T cell leukemia virus- type 1 (HTLV-I) between cells and between individuals, because naturally infected lymphocytes produce virtually no cell-free infectious HTLV-I particles. However, the mechanism of cell-to-cell spread of HTLV-I is not understood. We show here that cell contact rapidly induces polarization of the cytoskeleton of the infected cell to the cell-cell junction. HTLV-I core (Gag protein) complexes and the HTLV-I genome accumulate at the cell-cell junction and are then transferred to the uninfected cell. Other lymphotropic viruses, such as HIV-1, may similarly subvert normal T cell physiology to allow efficient propagation between cells.

Herpes simplex virus gE/gI expressed in epithelial cells interferes with cell-to-cell spread.

The herpes simplex virus (HSV) glycoprotein heterodimer gE/gI plays an important role in virus cell-to-cell spread in epithelial and neuronal tissues. In an analogous fashion, gE/gI promotes virus spread between certain cell types in culture, e.g., keratinocytes and epithelial cells, cells that are polarized or that form extensive cell junctions. One mechanism by which gE/gI facilitates cell-to-cell spread involves selective sorting of nascent virions to cell junctions, a process that requires the cytoplasmic domain of gE. However, the large extracellular domains of gE/gI also appear to be involved in cell-to-cell spread. Here, we show that coexpression of a truncated form of gE and gI in a human keratinocyte line, HaCaT cells, decreased the spread of HSV between cells. This truncated gE/gI was found extensively at cell junctions. Expression of wild-type gE/gI that accumulates at intracellular sites, in the trans-Golgi network, did not reduce cell-to-cell spread. There was no obvious reduction in production of infectious HSV in cells expressing gE/gI, and virus particles accumulated at cell junctions, not at intracellular sites. Expression of HSV gD, which is known to bind virus receptors, also blocked cell-to-cell spread. Therefore, like gD, gE/gI appears to be able to interact with cellular components of cell junctions, gE/gI receptors which can promote HSV cell-to-cell spread.

Viral interactions with receptors in cell junctions and effects on junctional stability.

Several viruses can use, as entry receptors, cell adhesion molecules that localize to junctional complexes of epithelial cells and other cell types. A recent publication in Cell describes how adenovirus can disrupt cell junctions, thereby effecting its release from basal surfaces of an infected epithelium to the apical or external environment.

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.

Cytoplasmic domain of herpes simplex virus gE causes accumulation in the trans-Golgi network, a site of virus envelopment and sorting of virions to cell junctions.

Alphaherpesviruses express a heterodimeric glycoprotein, gE/gI, that facilitates cell-to-cell spread between epithelial cells and neurons. Herpes simplex virus (HSV) gE/gI accumulates at junctions formed between polarized epithelial cells at late times of infection. However, at earlier times after HSV infection, or when gE/gI is expressed using virus vectors, the glycoprotein localizes to the trans-Golgi network (TGN). The cytoplasmic (CT) domains of gE and gI contain numerous TGN and endosomal sorting motifs and are essential for epithelial cell-to-cell spread. Here, we swapped the CT domains of HSV gE and gI onto another HSV glycoprotein, gD. When the gD-gI(CT) chimeric protein was expressed using a replication-defective adenovirus (Ad) vector, the protein was found on both the apical and basolateral surfaces of epithelial cells, as was gD. By contrast, the gD-gE(CT) chimeric protein, gE/gI, and gE, when expressed by using Ad vectors, localized exclusively to the TGN. However, gD-gE(CT), gE/gI, and TGN46, a cellular TGN protein, became redistributed largely to lateral surfaces and cell junctions during intermediate to late stages of HSV infection. Strikingly, gE and TGN46 remained sequestered in the TGN when cells were infected with a gI(-)HSV mutant. The redistribution of gE/gI to lateral cell surfaces did not involve widespread HSV inhibition of endocytosis because the transferrin receptor and gE were both internalized from the cell surface. Thus, gE/gI accumulates in the TGN in early phases of HSV infection then moves to lateral surfaces, to cell junctions, at late stages of infection, coincident with the redistribution of a TGN marker. These results are related to recent observations that gE/gI participates in the envelopment of nucleocapsids into cytoplasmic vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004-4016, 2000) and that gE/gI can sort nascent virions from cytoplasmic vesicles specifically to the lateral surfaces of epithelial cells (D. C. Johnson, M. Webb, T. W. Wisner, and C. Brunetti, J. Virol. 75:821-833, 2000). Therefore, gE/gI localizes to the TGN, through interactions between the CT domain of gE and cellular sorting machinery, and then participates in envelopment of cytosolic nucleocapsids there. Nascent virions are then sorted from the TGN to cell junctions.