Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

Does expression of a human complement-regulatory protein on xenograft cells protect them from systemic complement activation?

BACKGROUND: There are many causes of systemic complement activation, which may have detrimental effects on a pig xenograft. Transgenic expression of one or more human complement-regulatory proteins (hCRPs), e.g., hCD46, provides some protection to the xenograft, but it is not known whether it protects the xenograft from the effects of systemic complement activation. We used wild-type (WT) pig aortic endothelial cells (pAECs) to activate complement, and determined whether the expression of hCD46 on a1,3galactosyltransferase gene-knockout (GTKO) pAECs protected them from injury. METHODS: CFSE-labeled and non-labeled pAECs from a WT, a GTKO, or a GTKO/hCD46 pig were separately incubated with heat-inactivated pooled human serum in vitro. Antibody pre-bonded CFSE-labeled and non-labeled pAECs were mixed, and then incubated with rabbit complement. The complement-dependent cytotoxicity was measured by flow cytometry. RESULTS: There was significantly less lysis of GTKO/CD46 pAECs (6%) by 50% human serum compared to that of WT (91%, p<0.001) or GTKO (32%, p<0.01) pAECs. The lysis of GTKO pAECs was significantly increased when mixed with WT pAECs (p<0.05). In contrast, there was no significant change in cytotoxicity of GTKO/CD46 pAECs when mixed with WT pAECs. CONCLUSIONS: The expression of hCD46 protected pAECs from systemic complement activation.

Human adenovirus binding to host cell receptors: a structural view.

Human Adenoviruses (HAdVs) are a family of clinically and therapeutically relevant viruses. A precise understanding of their host cell attachment and entry mechanisms can be applied in inhibitor design and the construction of targeted gene delivery vectors. In this article, structural data on adenovirus attachment and entry are reviewed. HAdVs engage two types of receptors: first, an attachment receptor that is bound by the fibre knob protein protruding from the icosahedral capsid, and next, an integrin entry receptor bound by the pentameric penton base at the capsid vertices. Adenoviruses use remarkably diverse attachment receptors, five of which have been studied structurally in the context of HAdV binding: Coxsackie and Adenovirus Receptor, CD46, the glycans GD1a and polysialic acid, and desmoglein-2. Together with the integrin entry receptors, they display both symmetrical and asymmetrical modes of binding to the virus as demonstrated by the structural analyses reviewed here. The diversity of HAdV receptors contributes to the broad tropism of these viruses, and structural studies are thus an important source of information on HAdV-host cell interactions. The imbalance in structural data between the more and less extensively studied receptors remains to be addressed by future research.

The Role of Membrane Bound Complement Regulatory Proteins in Tumor Development and Cancer Immunotherapy.

It has long been understood that the control and surveillance of tumors within the body involves an intricate dance between the adaptive and innate immune systems. At the center of the interplay between the adaptive and innate immune response sits the complement system-an evolutionarily ancient response that aids in the destruction of microorganisms and damaged cells, including cancer cells. Membrane-bound complement regulatory proteins (mCRPs), such as CD46, CD55, and CD59, are expressed throughout the body in order to prevent over-activation of the complement system. These mCRPs act as a double-edged sword however, as they can also over-regulate the complement system to the extent that it is no longer effective at eliminating cancerous cells. Recent studies are now indicating that mCRPs may function as a biomarker of a malignant transformation in numerous cancer types, and further, are being shown to interfere with anti-tumor treatments. This highlights the critical roles that therapeutic blockade of mCRPs can play in cancer treatment. Furthermore, with the complement system having the ability to both directly and indirectly control adaptive T-cell responses, the use of a combinatorial approach of complement-related therapy along with other T-cell activating therapies becomes a logical approach to treatment. This review will highlight the biomarker-related role that mCRP expression may have in the classification of tumor phenotype and predicted response to different anti-cancer treatments in the context of an emerging understanding that complement activation within the Tumor Microenvironment (TME) is actually harmful for tumor control. We will discuss what is known about complement activation and mCRPs relating to cancer and immunotherapy, and will examine the potential for combinatorial approaches of anti-mCRP therapy with other anti-tumor therapies, especially checkpoint inhibitors such as anti PD-1 and PD-L1 monoclonal antibodies (mAbs). Overall, mCRPs play an essential role in the immune response to tumors, and understanding their role in the immune response, particularly in modulating currently used cancer therapeutics may lead to better clinical outcomes in patients with diverse cancer types.

Neisseria gonorrhoeae evades autophagic killing by downregulating CD46-cyt1 and remodeling lysosomes.

The Gram-negative human pathogen N. gonorrhoeae (Ngo) quickly attaches to epithelial cells, and large numbers of the bacteria remain on the cell surface for prolonged periods. Ngo invades cells but few viable intracellular bacteria are recovered until later stages of infection, leading to the assumption that Ngo is a weak invader. On the cell surface, Ngo quickly recruits CD46-cyt1 to the epithelial cell cortex directly beneath the bacteria and causes its cleavage by metalloproteinases and Presenilin/γSecretease; how these interactions affect the Ngo lifecycle is unknown. Here, we show Ngo induces an autophagic response in the epithelial cell through CD46-cyt1/GOPC, and this response kills early invaders. Throughout infection, the pathogen slowly downregulates CD46-cyt1 and remodeling of lysosomes, another key autophagy component, and these activities ultimately promote intracellular survival. We present a model on the dynamics of Ngo infection and describe how this dual interference with the autophagic pathway allows late invaders to survive within the cell.

A Measles Virus-Based Vaccine Candidate Mediates Protection against Zika Virus in an Allogeneic Mouse Pregnancy Model.

The impact of the Zika virus (ZIKV) epidemic highlights the need for vaccines that reduce or prevent infection and reliably prevent teratogenic complications. The live-attenuated measles virus (MV) vaccine strains are a promising vaccine platform, since they induce robust humoral and cellular immune responses against additional antigens and have an excellent safety record. To explore its potential to protect against ZIKV, we compared a recombinant Schwarz strain MV that encodes ZIKV prM and soluble E proteins (MV-Zika-sE) with a prototypic alum-adjuvanted whole inactivated ZIKV particle vaccine. Analysis of MV-Zika-sE-infected cells confirmed antigen expression, and the virus replicated with vaccine strain characteristics. Immunized IFNAR-/--CD46Ge mice developed E protein-specific and neutralizing antibodies, and ZIKV E-specific cellular immune responses were observed by gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) and in vitro T cell proliferation assays. To analyze protective efficacy, vaccinated female mice were challenged with ZIKV after allogeneic mating. In MV-Zika-sE-vaccinated mice, weight gain was similar to that in uninfected mice, while no plasma viremia was detectable in the majority of the animals. In contrast, infected control animals gained less weight and experienced about 100-fold higher viremia over at least 3 days. Moreover, vaccination with MV-Zika-sE reduced the ZIKV load in different organs and the placentas and prevented infection of the fetus. Consequently, no fetal growth retardation, anemia, or death due to ZIKV infection was seen in MV-Zika-sE-vaccinated dams. In contrast, the inactivated ZIKV vaccine had little to no effect in our studies. Therefore, the MV-derived ZIKV vaccine is a promising candidate for further preclinical and clinical development.IMPORTANCE Zika virus (ZIKV) is a mosquito-borne flavivirus that causes a variety of neurological complications, including congenital birth defects. Despite the urgent need, no ZIKV vaccine has yet been licensed. Recombinant vaccine strain-derived measles viruses (MV) constitute a promising vector platform to induce immunity against foreign pathogens by expressing antigens from additional transcription units while at the same time possessing a remarkable safety profile. This concept has already been validated against different pathogens, including at least 3 other flaviviruses, and our data show that vaccination with MV expressing soluble ZIKV E protein significantly diminishes infection and prevents fetal loss or damage in an allogeneic mouse pregnancy model. It can thus be regarded as a promising emergency vaccine candidate with the potential for inclusion in routine vaccination settings in areas of endemicity to prevent teratogenic effects of circulating ZIKV during pregnancy, comparable to standard rubella virus vaccination.

Safety Study: Intraventricular Injection of a Modified Oncolytic Measles Virus into Measles-Immune, hCD46-Transgenic, IFNαRko Mice.

The modified Edmonston vaccine strain of measles virus (MV) has shown potent oncolytic efficacy against various tumor types and is being investigated in clinical trials. Our laboratory showed that MV effectively kills medulloblastoma tumor cells in both localized disease and when tumor cells are disseminated through cerebrospinal fluid (CSF). Although the safety of repeated intracerebral injection of modified MV in rhesus macaques has been established, the safety of administering MV into CSF has not been adequately investigated. In this study, we assessed the safety of MV-NIS (MV modified to express the human sodium iodide symporter protein) injected into the CSF of measles-immunized and measles virus-susceptible transgenic (CD46, IFNαRko) mice. Treated animals were administered a single intraventricular injection of 1 × 105 or 1 × 106 TCID50 (50% tissue culture infective dose) of MV-NIS. Detailed clinical observation was performed over a 90-day period. Clinically, we did not observe any measles-related toxic effects or behavioral abnormality in animals of any treated cohort. The complete blood count and blood chemistry analysis results were found to be within normal range for all the cohorts. Histologic examination of brains and spinal cords revealed inflammatory changes, mostly related to the needle track; these resolved by day 21 postinjection. To assess viral biodistribution, quantitative RT-PCR to detect the measles virus N-protein was performed on blood and brain samples. Viral RNA was not detectable in the blood as soon as 2 days after injection, and virus cleared from the brain by 45 days postadministration in all treatment cohorts. In conclusion, our data suggest that a single injection of modified MV into the CSF is safe and can be used in future therapeutic applications.

The complement receptors CD46, CD55 and CD59 are regulated by the tumour microenvironment of head and neck cancer to facilitate escape of complement attack.

BACKGROUND: Membrane-bound complement restriction proteins (mCRPs) CD46, CD55 and CD59 enable tumour cells to evade complement dependent cytotoxicity and antibody-dependent killing mechanisms. But less is known about the role of these mCRPs in head and neck cancer. METHODS: In this study we determined the expression of the mCRPs on head and neck squamous cell carcinoma (HNSCC) cell lines, on tumour tissue and TDLNs (tumour-draining lymph nodes) as well as on lymphocytes from HNSCC patients. The influence of the HNSCC microenvironment on the mCRP regulation was analysed using Flow Cytometry, Western blotting and small interfering RNAs (siRNA) transfection studies. RESULTS: We examined the effects of the HNSCC tumour milieu on the expression levels of CD46, CD55 and CD59. We investigated the susceptibility of HNSCC cells to CDC (complement-dependent cytotoxicity) while silencing the mCRPs. Our results demonstrate a huge influence of the HNSCC tumour microenvironment on the regulation of mCRP expression and show a reciprocal regulation between the different mCRPs themselves. CONCLUSIONS: In summary, our data indicate that HNSCC has evolved different strategies to evade complement attacks and that the tumour microenvironment leads to the enhancement of complement resistance of the surrounding tissue.

Membrane-bound complement regulatory proteins as biomarkers and potential therapeutic targets for SLE.

For the last two decades, there had been remarkable advancement in understanding the role of complement regulatory proteins in autoimmune disorders and importance of complement inhibitors as therapeutics. Systemic lupus erythematosus is a prototype of systemic autoimmune disorders. The disease, though rare, is potentially fatal and afflicts women at their reproductive age. It is a complex disease with multiorgan involvement, and each patient presents with a different set of symptoms. The diagnosis is often difficult and is based on the diagnostic criteria set by the American Rheumatology Association. Presence of antinuclear antibodies and more specifically antidouble-stranded DNA indicates SLE. Since the disease is multifactorial and its phenotypes are highly heterogeneous, there is a need to identify multiple noninvasive biomarkers for SLE. Lack of validated biomarkers for SLE disease activity or response to treatment is a barrier to the efficient management of the disease, drug discovery, as well as development of new therapeutics. Recent studies with gene knockout mice have suggested that membrane-bound complement regulatory proteins (CRPs) may critically determine the sensitivity of host tissues to complement injury in autoimmune and inflammatory disorders. Case-controlled and followup studies carried out in our laboratory suggest an intimate relation between the level of DAF, MCP, CR1, and CD59 transcripts and the disease activity in SLE. Based on comparative evaluation of our data on these four membrane-bound complement regulatory proteins, we envisaged CR1 and MCP transcripts as putative noninvasive disease activity markers and the respective proteins as therapeutic targets for SLE. Following is a brief appraisal on membrane-bound complement regulatory proteins DAF, MCP, CR1, and CD59 as biomarkers and therapeutic targets for SLE.

Virion-associated complement regulator CD55 is more potent than CD46 in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization.

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

Protective effects of different combinations of human MCP, DAF, and CD59 on complement-dependent cytolysis in NIH 3T3 cells.

OBJECTIVES: To analyze the protective effects against complement-mediated cytolysis of the MCP, DAF, and CD59 human complement regulatory proteins, alone and in combination, on NIH 3T3 mouse fibroblast cells. MATERIALS AND METHODS: We constructed 3 double and 3 single-human complement regulatory protein plasmids (pIRES-hMCP-hDAF, pIRES-hMCP-hCD59, pIRES-hDAF-hCD59, pIRES-A-hMCP, pIRES-B-hDAF, and pIRES-B-hCD59). The plasmids were transfected into NIH 3T3 cells, and stable transfectants were obtained by treatment with 200 kg/m3 G418 for 2 weeks. Normal human serum (50%) as a source of complement was added to the culture medium of stable transfectants. The 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide assay was used to analyze the protective ability of different human complement regulatory protein plasmids on complement-dependent cytolysis. RESULTS: The viability of double-human complement regulatory protein stable transfectants was significantly higher than that of single-human complement regulatory protein stable transfectants (P < .05). Among the double-transfectants, cells expressing pIRES-hMCP-hDAF and pIRES-hMCPhCD59 survived better than cells expressing pIREShDAF- hCD59 (91.75% ± 3.30% and 84.88% ± 2.36% vs 66.19% ± 6.52%; P < .05). Among the single transfectants, cells expressing pIRES-A-hMCP or pIRES-B-hDAF survived better than cells expressing pIRES-B-hCD59 or pIRES empty vector (53.76% ± 3.84% and 56.32% ± 2.83% vs 43.28% ± 0.96% and 40.27% ± 1.11%; P < .05). CONCLUSIONS: These results suggest that the MCP+DAF and MCP+CD59 combinations could be more effective than DAF+CD59 in protecting the NIH 3T3 cells from injury caused by complement-dependent cytolysis, whereas MCP or DAF alone is stronger than CD59 alone in inhibiting membrane attack complex formation.

CD46 processing: a means of expression.

CD46 is a ubiquitously expressed type I transmembrane protein, first identified as a regulator of complement activation, and later as an entry receptor for a variety of pathogens. The last decade has also revealed the role of CD46 in regulating the adaptive immune response, acting as an additional costimulatory molecule for human T cells and inducing their differentiation into Tr1 cells, a subset of regulatory T cells. Interestingly, CD46 regulatory pathways are defective in T cells from patients with multiple sclerosis, asthma and rheumatoid arthritis, illustrating its importance in regulating T cell homeostasis. Indeed, CD46 expression at the cell surface is tightly regulated in many different cell types, highlighting its importance in several biological processes. Notably, CD46 is the target of enzymatic processing, being cleaved by metalloproteinases and by the presenilin/gamma secretase complex. This processing is required for its functions, at least in T cells. This review will summarize the latest updates on the regulation of CD46 expression and on its effects on T cell activation.

RNA interference characterization of proteins discovered by proteomic analysis of pancreatic cancer reveals function in cell growth and survival.

OBJECTIVES: There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival. METHODS: Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the "normoid" cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi. RESULTS: Integrin ß6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis. CONCLUSIONS: By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.

Mechanism of neuroinflammation: enhanced cytotoxicity and IL-17 production via CD46 binding.

The membrane co-factor protein CD46 is the cellular receptor for a number of pathogens including the human herpesvirus 6 (HHV-6). In addition to its function as an inhibitory complement receptor, engagement of CD46 in the context of T-cell receptor (TCR) signaling influences T-cell activation. Simultaneous cross-linking of the CD3/CD46 molecules led to differentiation of a unique population of CD4+ T-cell subset characterized by enhanced expressions of IFN-gamma, IL-10, granzyme B, adhesion molecule MAdCAM-1 (alpha-4-beta-7), surface-bound cytokine LIGHT, and chemokine receptor CCR9. Multiple sclerosis is a chronic inflammatory neurodegenerative disorder of the central nervous system (CNS) with unknown etiology. The HHV-6 is a candidate pathogen in MS and uses the CD46 molecule as its receptor. We hypothesize that binding of the HHV-6 glycoprotein to CD46 may trigger a pro-inflammatory response that could contribute to CNS tissue damage. To address this question, we examined immunological parameters such as proliferation, cytokine production and cytotoxic functions in CD4+ T cells of healthy individuals and MS patients following CD3/CD46 co-engagement by using anti-CD3 and anti-CD46 monoclonal antibodies as surrogates to mimic T-cell receptor and CD46 signaling. Our results demonstrated that CD3/CD46 cross-linking induced expression of IL-1beta and IL-17A in multiple sclerosis patient T cells. Additionally, increase in transient surface expression of lysosomal associated protein CD107a suggested enhanced CD4+ T-cell cytotoxic functions following CD3/CD46 co-stimulation. Collectively, this study demonstrated evidence to suggest a potential mechanism of virus-induced neuroinflammation that may be involved in MS disease pathogenesis.

Transcellular passage of Neisseria meningitidis across a polarized respiratory epithelium.

Neisseria meningitidis is a major cause of sepsis and meningitis but is also a common commensal, present in the nasopharynx of between 8 and 20% of healthy individuals. During carriage, the bacterium is found on the surface of the nasopharyngeal epithelium and in deeper tissues, while to develop disease the meningococcus must spread across the respiratory epithelium and enter the systemic circulation. Therefore, investigating the pathways by which N. meningitidis crosses the epithelial barrier is relevant for understanding carriage and disease but has been hindered by the lack of appropriate models. Here, we have established a physiologically relevant model of the upper respiratory epithelial cell barrier to investigate the mechanisms responsible for traversal of N. meningitidis. Calu-3 human respiratory epithelial cells were grown on permeable cell culture membranes to form polarized monolayers of cells joined by tight junctions. We show that the meningococcus crosses the epithelial cell barrier by a transcellular route; traversal of the layer did not disrupt its integrity, and bacteria were detected within the cells of the monolayer. We demonstrate that successful traversal of the epithelial cell barrier by N. meningitidis requires expression of its type 4 pili (Tfp) and capsule and is dependent on the host cell microtubule network. The Calu-3 model should be suitable for dissecting the pathogenesis of infections caused by other respiratory pathogens, as well as the meningococcus.

Epithelial-mesenchymal transition abolishes the susceptibility of polarized epithelial cell lines to measles virus.

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.