Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tahara, M. Articles by Yanagi, Y. Search for Related Content PubMed PubMed Citation Articles by Tahara, M. Articles by Yanagi, Y.

 Previous Article  |  Next Article 

Journal of Virology, May 2008, p. 4630-4637, Vol. 82, No. 9
0022-538X/08/$08.00+0     doi:10.1128/JVI.02691-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Measles Virus Infects both Polarized Epithelial and Immune Cells by Using Distinctive Receptor-Binding Sites on Its Hemagglutinin

Maino Tahara, Makoto Takeda,^* Yuta Shirogane, Takao Hashiguchi, Shinji Ohno, and Yusuke Yanagi

Department of Virology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan

Received 19 December 2007/ Accepted 12 February 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Measles is one of the most contagious human infectious diseases and remains a major cause of childhood morbidity and mortality worldwide. The signaling lymphocyte activation molecule (SLAM), also called CD150, is a cellular receptor for measles virus (MV), presumably accounting for its tropism for immune cells and its immunosuppressive properties. On the other hand, pathological studies have shown that MV also infects epithelial cells at a later stage of infection, although its mechanism has so far been unknown. In this study, we show that wild-type MV can infect and produce syncytia in human polarized epithelial cell lines independently of SLAM and CD46 (a receptor for the vaccine strains of MV). Progeny viral particles are released exclusively from the apical surface of these polarized epithelial cell lines. We have also identified amino acid residues on the MV attachment protein that are likely to interact with a putative receptor on epithelial cells. All of these residues have aromatic side chains and may form a receptor-binding pocket located in a different position from the putative SLAM- and CD46-binding sites on the MV attachment protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells by using distinctive receptor-binding sites on the attachment protein corresponding to each of their respective receptors. The ability of MV to infect polarized epithelial cells and its exclusive release from the apical surface may facilitate its efficient transmission via aerosol droplets, resulting in its highly contagious nature.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Measles remains a major cause of childhood morbidity and mortality worldwide despite the availability of efficacious vaccines. Measles virus (MV), an enveloped RNA virus belonging to the genus Morbillivirus in the family Paramyxoviridae, is transmitted via aerosol droplets and considered to be one of the most contagious human pathogens. MV has two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins, which are responsible for receptor binding and membrane fusion, respectively (18). MV enters a cell by membrane fusion at the cell surface. The attachment of the H protein to a cellular receptor is believed to induce the conformational change of the H protein, as well as that of the F protein, which promotes the fusion of the viral envelope with the host cell membrane. MV also causes cell-cell fusion in susceptible cells. The signaling lymphocyte activation molecule (SLAM), also known as CD150, has been identified as a receptor for MV (10, 21, 47). SLAM is expressed on immune cells, such as activated lymphocytes, mature dendritic cells, and macrophages, providing a good explanation for the lymphotropism and immunosuppressive nature of MV (4, 51). Although CD46, a ubiquitously expressed complement regulatory molecule, functions as a receptor for the vaccine strains of MV (8, 30), a great majority of viruses circulating in measles patients use SLAM, but not CD46, as a receptor (35, 51). A recent study of MV infection in macaque monkeys also identified SLAM⁺ lymphocytes and dendritic cells as the predominantly infected cell types (6).

However, pathological data from humans and experimentally infected monkeys have shown that MV antigens and syncytia are also detected in epithelial tissues in various organs, such as the skin, oral mucosa, pharynx, trachea, esophagus, intestines, and urinary bladder (5, 24, 26, 27, 31, 33, 34, 37). Epithelial cells do not express SLAM, and wild-type (WT) strains of MV, unlike vaccine strains, do not infect epithelial cell lines. Thus, the mechanism by which MV infects epithelial tissues remains to be determined. Recently, we have reported that a SLAM^– human lung adenocarcinoma cell line, NCI-H358, supports MV entry, replication, and syncytium formation independently of SLAM and CD46 (43). Furthermore, analyses using several monoclonal antibodies (MAbs) against the H protein indicated that the receptor-binding site on the H protein used to infect NCI-H358 cells is different from those used for SLAM and CD46 (43).

In this study, we show that besides NCI-H358, four human polarized epithelial cell lines can support WT MV growth and syncytium formation via a SLAM- and CD46-independent mechanism.

MV is found to be released exclusively from the apical surface of these polarized cell lines. We have also identified amino acid residues on the H protein that are likely to interact with a putative receptor on epithelial cells mediating SLAM- and CD46-independent infection. These residues have aromatic side chains and may form a receptor-binding pocket strategically located on the H protein. These observations provide new insight into MV infection of epithelial cells as well as into measles pathogenesis.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cells. The characteristics and culture conditions for the following cell lines used were as described previously: Vero/hSLAM (35), B95a (25), NCI-H157 (39), NCI-H1299 (15), A549 (16), NCI-H460 (3), NCI-H358 (3), T84 (28), Calu-3 (12), HT29 (13), Caco-2 (12), PC-3 (23), MDCK (14), LLC-PK1 (22), and Vero C1008 (9).

Viruses. All full-length genome plasmids were derived from the p(+)MV323 plasmid encoding the antigenomic full-length cDNA of the IC-B WT strain of MV (44). The p(+)MV323-EGFP and p(+)MV323-Luci plasmids, which have an additional transcriptional unit of the enhanced green fluorescent protein (EGFP) and the Renilla luciferase genes, respectively, were reported previously (20, 43). Recombinant MV strains were generated from the full-length genome plasmids as reported previously (29, 41).

Plasmid constructions. The eukaryotic expression vector pCA7 is a derivative of pCAGGS (32) and has a multiple-cloning site (MCS) for Acc65I, KpnI, SacI, EcoRI, BsaBI, EcoRV, NotI, XhoI, SphI, and NsiI located downstream from two promoters, the CAG and T7 promoters (41, 42). The pCA7ps vector is generated by replacing the MCS of the pCA7 vector with one containing PacI, BstEII, BsmBI, BmtI, NheI, BglI, PmlI, FseI, NaeI, RsrII, and SpeI (the vector contains another SpeI site outside the MCS). The pCA7ps-ICH plasmid was generated by inserting the PacI-SpeI fragment (nucleotide positions 7238 to 9175) of the full-length genome plasmids derived from p(+)MV323 (44) (nucleotide positions are shown in accordance with the sequence of the IC-B strain genome [45]) into the PacI and SpeI sites of the pCA7ps vector. Amino acid substitutions (N481A, N481R, N481E, N481F, F483A, D521S, L522A, Y524S, Y541S, Y543S, S544A, R547S, S550A, and Y551S) were introduced into p(+)MV323-EGFP, p(+)MV323-Luci, or pCA7ps-ICH by site-directed mutagenesis using the complementary primer pairs. pCA7-ICH-N481Y, pCXN2-KAH-N481F, pCXN2-KAH-N481S, pCXN2-KAH-N481T, and pCXN2-EdF have been described previously (38, 50).

Measurement of Renilla luciferase activity. Cells infected with Renilla luciferase-expressing MVs were lysed in Renilla luciferase assay lysis buffer. The Renilla luciferase activity in the cells was then analyzed by a Renilla luciferase assay system (Promega, Madison, WI), according to the manufacturer's instruction. Chemiluminescence was measured using a Mithras LB940 plate reader (Berthold Technologies, Pforzheim, Germany).

Cell-cell fusion assay. B95a or NCI-H358 cells cultured in six-well-cluster plates were cotransfected with each of the H-protein-expressing plasmids (1 µg) and pCXN2-EdF (1 µg), using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA). At 2 or 3 days posttransfection, the cells were observed under a phase-contrast imaging microscope.

Virus titration. The titers of EGFP-expressing viruses were determined as described previously (20, 42) and expressed in cell infectious units (CIU). The multiplicity of infection (MOI) was calculated in accordance with the number of CIU determined on Vero/hSLAM cells. The titers of luciferase-expressing viruses were determined by plaque titration.

Virus growth. Various cell lines cultured in 96-well-cluster plates were infected with luciferase-expressing MV strains at an MOI of 1. At various time intervals, the cells were harvested, and the Renilla luciferase activities were analyzed. Caco-2, T84, and HT29 cells were seeded at confluence on 24-mm-diameter Transwell filter supports with 0.4-µm pores (Corning Inc., Corning, NY) and cultured for 8 to 12 days to produce polarized monolayers. EGFP-expressing MV strains were inoculated into either the apical or basolateral medium and incubated for 2 h at 37°C. The cells were then washed with phosphate-buffered saline (PBS) from both sides and incubated in culture medium at 37°C. At various days postinfection (p.i.), both apical and basolateral media were collected to determine the numbers of CIU in them.

Immunofluorescence staining and confocal microscopy. The cells were cultured on collagen-coated coverslips. After MV infection, cells were fixed and permeabilized with PBS containing 2.5% formaldehyde and 0.5% Triton X-100. The cells were then washed with PBS and incubated with the mouse MAb against ZO-1 (Invitrogen) for 1 h at 37°C, followed by incubation with Alexa Fluor 594-conjugated secondary antibody (Molecular Probes, Eugene, OR). Thirty fluorescence images of the cells were obtained sequentially from the top to the bottom of the cells by using a confocal microscope (Radiance 2100; Bio-Rad, Hercules, CA) and merged by using Lasersharp software (Bio-Rad).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Infection of epithelial cells with WT MV. In an attempt to understand why WT MV can grow in the NCI-H358 cell line, microarray analysis was performed to compare its gene expression pattern with the patterns of other nonsusceptible human lung carcinoma cell lines. The results showed that the NCI-H358 cells express several genes encoding molecules involved in tight-junction formation or cell adhesion at significantly higher levels than those of the nonsusceptible cell lines (data not shown). This prompted us to examine the susceptibility of several polarized epithelial cell lines to WT MV. Caco-2, HT29, T84, Calu-3, MDCK, LLC-PK1, and Vero C1008 cells are known to be "polarized cells," which form tight junctions between adjacent cells (1, 7, 9, 17, 36, 40, 49). These cell lines showed a circumferential immunofluorescent staining pattern of ZO-1, a major component of the tight junction (40), indicating one of their properties as polarized cells (Fig. 1A). NCI-H358 cells also exhibited an incompletely circumferential staining pattern of ZO-1, suggesting that they may have an ability to form tight junctions. By contrast, scattered dots or short fragments of ZO-1 were observed on the surfaces or at the margins of nonpolarized cell lines (HeLa, HEK293, 293T, NCI-H460, NCI-H1299, NCI-H322, A549, NCI-H157, and PC3) (Fig. 1A and data not shown). No signal for ZO-1 was observed on B-lymphoblastoid B95a cells, which are susceptible to all MV strains. The subconfluent monolayers of these cell lines were infected with IC323-EGFP, a recombinant WT MV strain (based on the IC-B WT strain of MV) expressing EGFP (20, 44), and observed daily. The MV-infected cells were visualized with EGFP autofluorescence. As shown previously (20, 43), IC323-EGFP grew and produced syncytia in B95a (using SLAM as a receptor) and NCI-H358 (using an unknown receptor) cells (Fig. 1A). The virus also grew and produced syncytia in all of the human polarized cell lines examined (Caco-2, HT29, T84, and Calu-3), disrupting tight junctions. By contrast, the polarized nonhuman cell lines (dog MDCK, porcine LLC-PK1, and monkey Vero C1008) and the nonpolarized human cell lines only rarely supported IC323-EGFP infection, and no syncytia were detected in them even when the cells were observed for 2 weeks (Fig. 1A and data not shown).

View larger version (55K): [in this window] [in a new window]

FIG. 1. WT MV spreads in human polarized epithelial cells. (A) Subconfluent monolayers of various cell lines were infected with IC323-EGFP at an MOI of 1. At the indicated days p.i., the distribution of ZO-1 in monolayers was examined using a MAb specific to ZO-1, followed by incubation with Alexa Fluor 549-conjugated anti-mouse secondary antibody. The MV-infected cells were visualized with EGFP autofluorescence. (B) Subconfluent monolayers of various cell lines were infected with IC323-Luci at an MOI of 1. The Renilla luciferase activities in cells were determined at the various time points indicated.

Subconfluent monolayers of these cell lines were also infected with IC323-Luci, a recombinant WT MV strain expressing the Renilla luciferase (43). At various time intervals, the Renilla luciferase activity in cells was quantified (Fig. 1B). In Vero/hSLAM cells (Vero cells constitutively expressing human SLAM), IC323-Luci replicated efficiently, showing the maximum luciferase activity at 45 h p.i. Although slower than that in the Vero/hSLAM cells, the luciferase activities increased in all four of the human polarized cell lines (Caco-2, HT29, T84, and Calu-3) and in the NCI-H358 cells after infection with IC323-Luci. The maximum luciferase activities in the Caco-2 and HT29 cells were as high as that in the Vero/hSLAM cells. By contrast, the luciferase activity did not increase significantly in the nonhuman polarized or human nonpolarized cell lines.

These data indicate that WT MV has an ability to grow and form syncytia in human polarized epithelial cells as well as in NCI-H358 cells.

Exclusive budding of WT MV at the apical surface of polarized cells. The confluent monolayers of the Caco-2 cells forming tight junctions were prepared on the filters of Transwell permeable supports, and 1 x 10⁴ CIU of IC323-EGFP was inoculated into the apical medium. After 2 h of incubation with the virus, the cells were washed with PBS and then cultured in fresh medium at 37°C. At various days p.i., the amounts of the virus in the apical and basolateral media were determined (Fig. 2A). At 4 days p.i., more than 1 x 10² CIU/ml of infectious virus was detected in the apical medium. At 18 days p.i., the virus titer in the apical medium reached 6 x 10⁴ CIU/ml. The virus titer in the basolateral medium increased only after 24 days p.i., when tight junctions were disrupted by an MV-induced cytopathic effect. Similar data were obtained with the HT29 (Fig. 2B) and T84 (data not shown) cells. The monolayers of the Caco-2 cells were also infected with IC323-EGFP from the basolateral side. The virus entered the cells much less efficiently than it did from the apical side, and infectious virus particles were again released mostly from the apical side (data not shown). Analyses by confocal microscopy showed that H, F, and matrix proteins were predominantly transported to the apical surface of the WT-MV-infected cells (data not shown). All these data indicate that WT MV buds almost exclusively from the apical surface of polarized epithelial cells.

View larger version (14K): [in this window] [in a new window]

FIG. 2. WT MV buds from the apical surface of polarized epithelial cells. Caco-2 (A) and HT29 (B) cells were cultured at confluence on 24-mm-diameter Transwell filter supports with 0.4-µm pores. At 8 to 12 days after plating, polarized monolayers of cells were infected with IC323-EGFP from the apical side. At various time intervals, the numbers of CIU/ml of both the apical and basolateral media were measured (filled and open circles, respectively). Average numbers of CIU/ml in duplicate experiments are shown.

Identification of residues on the H protein critical for fusion of NCI-H358 cells induced by MV H and F proteins. Amino acid residues on the H protein that are important in supporting cell-cell fusion in epithelial cells induced by MV glycoproteins were examined. Our previous study suggested that WT MV uses a novel receptor-binding site on the H protein which is different from those for SLAM and CD46 to infect epithelial cells (43). That receptor-binding site is, however, likely to overlap that for CD46, as the N481Y substitution, which enables WT MV to use CD46 as a receptor, also enhances the ability of WT MV to grow and induce syncytia in the NCI-H358 epithelial cells via a CD46-independent mechanism (43). Asn at position 481 of the H protein (of the IC-B WT strain) was replaced with Phe, Ser, Thr, Ala, Arg, Glu, or Tyr, and the mutated H proteins were transiently expressed, together with the F protein, in B95a and NCI-H358 cells. None of the substitutions affected the cell-cell fusion of B95a cells (Fig. 3 and data not shown), consistent with the observation that the residue at position 481 is not involved in the H protein interaction with SLAM (48). Similarly, none of the substitutions inhibited the cell-cell fusion of NCI-H358 cells, indicating that the residue at position 481 is not critical for the H protein to support cell-cell fusion of the cells (Fig. 3 and data not shown). However, the H protein with the N481F substitution, which cannot support CD46-dependent cell-cell fusion (50), enhanced the cell-cell fusion of the NCI-H358 cells as efficiently as that with the N481Y substitution (Fig. 3). These results suggest that aromatic residues (Tyr and Phe) at position 481 positively modulate the interaction of the H protein with the putative receptor on NCI-H358 cells.

View larger version (118K): [in this window] [in a new window]

FIG. 3. Role of residues at position 481 of the H protein in cell-cell fusion of NCI-H358 cells. NCI-H358 or B95a cells cultured in six-well-cluster plates were transfected with the WT or mutant-H-protein-expressing plasmid (1 µg) together with the F-protein-expressing plasmid (1 µg). -, no H protein plasmid. At 2 (B95a) or 3 (NCI-H358) days posttransfection, the cells were observed under a phase-contrast imaging microscope.

Recently, we have determined the crystal structure of the MV H protein, which allowed us to precisely locate the amino acid residues presumed to be interacting with SLAM and CD46 (19). The structure also revealed that there are many residues on the surface of the molecule which are well conserved among different morbilliviruses and reside outside the putative SLAM- and CD46-binding sites (Fig. 4A). We speculated that these conserved residues may be involved in SLAM- and CD46-independent infection of epithelial cells. To test this possibility, the H proteins with substitutions at these residues (Phe483, Asp521, Leu522, Tyr524, Tyr541, Tyr543, Ser544, Arg547, Ser550, and Tyr551) were examined for their ability to support the cell-cell fusion of NCI-H358 cells induced by H and F proteins. The L522A and Y524S substitutions caused the H protein to lose the ability to support cell-cell fusion in both B95a and NCI-H358 cells, whereas the D521S, S544A, R547S, and S550A substitutions exhibited little effect on the cell-cell fusion of either cell line (data not shown). The Y551S substitution inhibited the cell-cell fusion of B95a cells but not that of NCI-H358 cells (data not shown). Importantly, the F483A, Y541S, and Y543S substitutions caused the H protein to lose the ability to support syncytium formation in NCI-H358 cells but not in B95a cells (Fig. 4B).

View larger version (95K): [in this window] [in a new window]

FIG. 4. Identification of critical amino acid residues on the H protein to support cell-cell fusion of NCI-H358 cells. (A) The conserved residues on H proteins of seven morbilliviruses (measles, rinderpest, peste-des-petits-ruminants, canine distemper, dolphin distemper, porpoise distemper, and phocine distemper viruses). The H protein monomer is illustrated as observed almost from the top. Red, identical; salmon pink, strong similarity; wheat yellow, weak similarity; gray, little similarity. The residues at the putative SLAM-binding sites are shown with asterisks, and those at the putative CD46-binding sites are denoted in italics. The underlined residues, which are conserved among morbilliviruses and reside outside the putative SLAM- and CD46-binding sites, were studied by mutagenesis. (B) NCI-H358 and B95a cells cultured in six-well-cluster plates were transfected with the WT or mutant-H-protein-expressing plasmid (1 µg) together with the F-protein-expressing plasmid (1 µg). At 2 (B95a) or 3 (NCI-H358) days posttransfection, cells were observed under a phase-contrast imaging microscope.

Infection of human epithelial cells with recombinant MV strains bearing the H protein with specific substitutions. Substitutions in the H protein that enhanced (N481Y and N481F) or suppressed (F483A and Y543S) syncytium formation in NCI-H358 cells were introduced into the genomes of infectious MVs by reverse genetics techniques (29, 41, 44). The recombinant MV with the N481Y substitution in the H protein was reported previously (38). All of the recombinant viruses with the mutated H proteins efficiently replicated and produced syncytia in B95a cells (Fig. 5A). The viruses with the N481Y or N481F substitution induced much larger syncytia in NCI-H358 cells than the parental virus did (Fig. 5A), consistent with the findings obtained with plasmid-mediated fusion analysis (Fig. 3). These mutant viruses also induced larger syncytia in the four human polarized epithelial cell lines, HT29, Calu-3, Caco-2, and T84, than the parental virus did (Fig. 5A). On the other hand, the mutant viruses with the F483A or Y543S substitution neither grew well nor induced syncytia in NCI-H358 cells (Fig. 5A). The strains with the F483A or Y543S substitution did not induce syncytia in HT29 and Calu-3 cells, but they did produce syncytia in Caco-2 and T84 cells (Fig. 5A).

View larger version (57K): [in this window] [in a new window]

FIG. 5. Infection of human epithelial cells with recombinant MV strains bearing the H protein with specific substitutions. (A) EGFP autofluorescence in MV-infected cells. B95a, H358, HT29, Calu-3, Caco-2, and T84 cells were infected with recombinant MVs at an MOI of 0.5. The panels show representative images with a fluorescence microscope at 2 days (B95a and H358 cells), 6 days (HT29, Calu-3, and Caco-2 cells), or 14 days (T84 cells) p.i. (B) The monolayers of B95a, H358, HT29, Calu-3, Caco-2, and T84 cells were infected with recombinant MVs at an MOI of 1.0. At 2 (for B95a) or 3 (for other cell lines) days p.i., the Renilla luciferase activities in the cells were determined. The bar graph shows the percent Renilla luciferase activities (average and standard deviation) of the cells infected with recombinant MVs bearing the WT H protein (dark gray) or mutant H protein with the Y543S (white) or F483A (light gray) substitution. The Renilla luciferase activities of the respective cell lines infected with IC323-Luci bearing the WT H protein were set to 100%.

The growth of the mutant viruses possessing the F483A or Y543S substitution in the H protein was also examined by using recombinant MVs expressing Renilla luciferase (Fig. 5B). These mutant viruses grew in B95a cells as efficiently as the virus carrying the WT H protein. The virus possessing the Y543S substitution grew very poorly in NCI-H358, HT29, and Calu-3 cells but replicated well in Caco-2 and T84 cells. The replication of the virus with the F483A substitution was also greatly reduced in NCI-H358, HT29, Calu-3, and Caco-2 cells but less so in T84 cells. These data suggest that although the same residues on the H protein may play an important role in WT MV infection of epithelial cells, the virus-receptor interactions are somehow different among different epithelial cell lines.

Taken together, our results indicate that Phe483, Tyr541, and Tyr543 on the H protein are important for WT MV to infect NCI-H358 and polarized epithelial cell lines. The residue at position 481 is not critical, but the presence of aromatic residues at that position greatly enhances WT MV infection of epithelial cells. These four positions are indicated on the three-dimensional structure of the H protein, together with putative SLAM- and CD46-binding sites (19) (Fig. 6A and B). (The structure shown in Fig. 6 is based on the H protein of the Edmonston strain, but all indicated residues except that at position 481 are conserved between the Edmonston and WT IC-B strains.) The aromatic side chains of these residues may form a receptor-binding pocket interacting with a putative receptor allowing MV infection of epithelial cells (Fig. 6C).

View larger version (43K): [in this window] [in a new window]

FIG. 6. Cluster of aromatic residues at the putative receptor-binding site of the H protein. (A) Surface presentation of the H protein seen from the top. The amino acid residues assumed to interact with SLAM and CD46 are shown in magenta and cyan, respectively. Residues important for infection of epithelial cells (F483, Y541 and Y543) are shown in orange. (B) Ribbon and stick model at the same angle as that used in panel A. The head of the H protein exhibits a six-bladed β-propeller fold. The colors blue, green, light green, yellow, orange, and red represent each propeller from β-sheets 1, 2, 3, 4, 5, and 6, respectively. (C) Magnification of the putative receptor-binding site on the H protein used to infect epithelial cells.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CD46 is the first identified MV receptor (8, 30). The ubiquitous expression of CD46 had been thought to explain the findings that MV can infect various types of cells in vivo and in vitro. Then, SLAM was identified as a receptor for both the WT and vaccine strains of MV, and the use of CD46 was shown to be limited mostly to the vaccine strains of MV (47), making us rethink measles pathogenesis. From the known distribution of SLAM in the body, the first targets for MV are likely to be dendritic cells, lymphocytes, and/or macrophages in the lymphoid tissues of the respiratory tract (51). After the initial infection of those cells, the virus enters the blood and replicates in lymphoid tissues and organs throughout the body, causing profound immunosuppression. At a later stage of infection, infected dendritic cells may transmit MV to epithelial cells (6). Previous studies using a panel of cell lines showed that only SLAM⁺ cells support efficient WT MV infection and syncytium formation (11). Epithelial cells do not express SLAM, and WT MV uses SLAM but not CD46 as a receptor. How then does MV infect epithelial cells in vivo? Although previous studies have shown that a low level of SLAM-independent WT MV infection occurs in various cell lines (the efficiency is 100 to 1,000 times lower than that using SLAM), this type of infection does not produce syncytia in infected cells (20), except in primary cultures of human small-airway epithelial cells and endothelial cells (2, 46).

Here and in our previous paper (43), we have demonstrated that WT MV has the ability to infect and produce syncytia in certain epithelial cell lines. The infection is independent of SLAM and CD46. Furthermore, we have identified residues on the attachment H protein that presumably interact with the putative epithelial cell receptor. Those residues are in a location different from putative SLAM- and CD46-binding sites on the H protein. Thus, our results indicate that MV has an intrinsic ability to infect both polarized epithelial and immune cells using distinctive receptor-binding sites on the H protein corresponding to respective receptors.

All of the identified amino acid residues relevant for infection of epithelial cells are those with aromatic side chains (Phe483, Try541, and Try543), suggesting that hydrophobic interactions play an important role in the binding of the H protein to the putative receptor on epithelial cells. This is also supported by the finding that the presence of aromatic residues at position 481, which is located close to the aforementioned three positions, enhances cell-cell fusion in epithelial cells after expression of the H and F proteins or recombinant virus infection (Fig. 3 and 5). These results suggest that there exists a cluster of hydrophobic residues on the putative epithelial cell receptor, which interacts with the aforementioned aromatic residues on the H protein. We have previously shown that residues at the SLAM-binding site on the H protein are highly conserved among different morbilliviruses (19). Tyr541 and Tyr543 are also highly conserved among morbilliviruses. Phe483 is found only in MV, but all other morbilliviruses have a hydrophobic Leu residue at that position. Thus, it is likely that morbilliviruses other than MV also use the same molecule as a receptor to infect epithelial cells. These relevant aromatic residues are located upward from the viral envelope (as portrayed in Fig. 6), because of the tilted orientation of the molecules forming the H protein dimer (19). Thus, they should be able to interact readily with the putative receptor on epithelial cells.

Why does MV infect epithelial cells? Polarized epithelial cells form tight junctions covering the external epithelial surface. The tight junctions prevent the passage of most dissolved molecules from one side of the epithelium to the other. Thus, it is possible that MV cannot efficiently release progeny virions into the external surface through its ability to infect immune cells alone. At present, the identity of the receptor which MV uses to infect epithelial cells is unknown, but our results showed that progeny virions are selectively released into the apical side of polarized epithelial cells (the luminal side). This suggests that the ability to infect epithelial cells is necessary for MV to spread efficiently from person to person, rather than to spread only within an individual host. This may explain why human immunodeficiency virus, which shares a similar tropism for immune cells with MV, is transmitted exclusively via sexual contact or blood, whereas MV is transmitted efficiently via aerosol droplets.

 
We thank M. Sato, T. Yano, Y. Fujinaga, Y. Ito, S. Naito, and T. Harada for providing cell lines; T. Seya and T. A. Sato for antibodies; and K. Maenaka for helpful discussions. We also thank C. Tsuda (Tomy Digital Biology Co., Ltd.) for the analysis of microarray data.

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labor and Welfare of Japan. M. Tahara is supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists.

 
* Corresponding author. Mailing address: Department of Virology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6138. Fax: 81-92-642-6140. E-mail: mtakeda{at}virology.med.kyushu-u.ac.jp

Published ahead of print on 20 February 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Anderson, J. M., C. M. Van Itallie, M. D. Peterson, B. R. Stevenson, E. A. Carew, and M. S. Mooseker. 1989. ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells. J. Cell Biol. 109:1047-1056.[Abstract/Free Full Text]
2
2. Andres, O., K. Obojes, K. S. Kim, V. ter Meulen, and J. Schneider-Schaulies. 2003. CD46- and CD150-independent endothelial cell infection with wild-type measles viruses. J. Gen. Virol. 84:1189-1197.[Abstract/Free Full Text]
3
3. Brower, M., D. N. Carney, H. K. Oie, A. F. Gazdar, and J. D. Minna. 1986. Growth of cell lines and clinical specimens of human non-small cell lung cancer in a serum-free defined medium. Cancer Res. 46:798-806.[Abstract/Free Full Text]
4
4. Cocks, B. G., C.-C. J. Chang, J. M. Carballido, H. Yssel, J. E. de Vries, and G. Aversa. 1995. A novel receptor involved in T-cell activation. Nature 376:260-263.[CrossRef][Medline]
5
5. Craighead, J. E. 2000. Rubeola (measles), p. 397-410. In J. E. Craighead (ed.), Pathology and pathogenesis of human viral disease. Elsevier, London, United Kingdom.
6
6. de Swart, R. L., M. Ludlow, L. de Witte, Y. Yanagi, G. van Amerongen, S. McQuaid, S. Yuksel, T. B. Geijtenbeek, W. P. Duprex, and A. D. Osterhaus. 2007. Predominant infection of CD150(+) lymphocytes and dendritic cells during measles virus infection of macaques. PLoS Pathog. 3:e178.[CrossRef][Medline]
7
7. Dharmsathaphorn, K., J. A. McRoberts, K. G. Mandel, L. D. Tisdale, and H. Masui. 1984. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am. J. Physiol. 246:G204-G208.[Medline]
8
8. Dörig, R. E., A. Marcil, A. Chopra, and C. D. Richardson. 1993. The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 75:295-305.[CrossRef][Medline]
9
9. Earley, E., and K. Johnson. 1988. The lineage of the Vero, Vero 76 and its clone C1008 in the United States, p. 26-29. In B. Simizu, and T. Terasima. (ed.), Vero cells: origin, properties and biomedical applications. Chiba University, Tokyo, Japan.
10
10. Erlenhoefer, C., W. J. Wurzer, S. Löffler, S. Schneider-Schaulies, V. ter Meulen, and J. Schneider-Schaulies. 2001. CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition. J. Virol. 75:4499-4505.[Abstract/Free Full Text]
11
11. Erlenhöfer, C., W. Duprex, B. Rima, V. ter Meulen, and J. Schneider-Schaulies. 2002. Analysis of receptor (CD46, CD150) usage by measles virus. J. Gen. Virol. 83:1431-1436.[Abstract/Free Full Text]
12
12. Fogh, J., J. M. Fogh, and T. Orfeo. 1977. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59:221-226.[Medline]
13
13. Fogh, J., and G. Trempe. 1975. New human tumor cell lines, p. 115-160. In J. Fogh (ed.), Human tumor cells in vitro. Plenum Publishing Corp., New York, NY.
14
14. Gaush, C. R., W. L. Hard, and T. F. Smith. 1966. Characterization of an established line of canine kidney cells (MDCK). Proc. Soc. Exp. Biol. Med. 122:931-935.[CrossRef][Medline]
15
15. Giaccone, G., J. Battey, A. F. Gazdar, H. Oie, M. Draoui, and T. W. Moody. 1992. Neuromedin B is present in lung cancer cell lines. Cancer Res. 52:S2732-S2736.
16
16. Giard, D. J., S. A. Aaronson, G. J. Todaro, P. Arnstein, J. H. Kersey, H. Dosik, and W. P. Parks. 1973. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51:1417-1423.[Medline]
17
17. Gopalakrishnan, S., M. A. Hallett, S. J. Atkinson, and J. A. Marrs. 2003. Differential regulation of junctional complex assembly in renal epithelial cell lines. Am. J. Physiol. Cell Physiol. 285:C102-C111.[Abstract/Free Full Text]
18
18. Griffin, D. E. 2007. Measles virus, p. 1551-1585. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
19
19. Hashiguchi, T., M. Kajikawa, N. Maita, M. Takeda, K. Kuroki, K. Sasaki, D. Kohda, Y. Yanagi, and K. Maenaka. 2007. Crystal structure of measles virus hemagglutinin provides insight into effective vaccines. Proc. Natl. Acad. Sci. USA 104:19535-19540.[Abstract/Free Full Text]
20
20. Hashimoto, K., N. Ono, H. Tatsuo, H. Minagawa, M. Takeda, K. Takeuchi, and Y. Yanagi. 2002. SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein. J. Virol. 76:6743-6749.[Abstract/Free Full Text]
21
21. Hsu, E., C. Iorio, F. Sarangi, A. Khine, and C. Richardson. 2001. CDw150(SLAM) is a receptor for a lymphotropic strain of measles virus and may account for the immunosuppressive properties of this virus. Virology 279:9-21.[CrossRef][Medline]
22
22. Hull, R. N., W. R. Cherry, and G. W. Weaver. 1976. The origin and characteristics of a pig kidney cell strain, LLC-PK. In Vitro 12:670-677.[Medline]
23
23. Kaighn, M. E., K. S. Narayan, Y. Ohnuki, J. F. Lechner, and L. W. Jones. 1979. Establishment and characterization of a human prostatic carcinoma cell line (PC-3). Invest. Urol. 17:16-23.[Medline]
24
24. Kimura, A., K. Tosaka, and T. Nakao. 1975. Measles rash. I. Light and electron microscopic study of skin eruptions. Arch. Virol. 47:295-307.[CrossRef][Medline]
25
25. Kobune, F., H. Sakata, and A. Sugiura. 1990. Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus. J. Virol. 64:700-705.[Abstract/Free Full Text]
26
26. Lightwood, R., and R. Nolan. 1970. Epithelial giant cells in measles as an acid in diagnosis. J. Pediatr. 77:59-64.[CrossRef][Medline]
27
27. Moench, T. R., D. E. Griffin, C. R. Obriecht, A. J. Vaisberg, and R. T. Johnson. 1988. Acute measles in patients with and without neurological involvement: distribution of measles virus antigen and RNA. J. Infect. Dis. 158:433-442.[Medline]
28
28. Murakami, H., and H. Masui. 1980. Hormonal control of human colon carcinoma cell growth in serum-free medium. Proc. Natl. Acad. Sci. USA 77:3464-3468.[Abstract/Free Full Text]
29
29. Nakatsu, Y., M. Takeda, M. Kidokoro, M. Kohara, and Y. Yanagi. 2006. Rescue system for measles virus from cloned cDNA driven by vaccinia virus Lister vaccine strain. J. Virol. Methods 137:152-155.[CrossRef][Medline]
30
30. Naniche, D., G. Varior-Krishnan, F. Cervoni, T. F. Wild, B. Rossi, C. Rabourdin-Combe, and D. Gerlier. 1993. Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. J. Virol. 67:6025-6032.[Abstract/Free Full Text]
31
31. Nii, S., J. Kamahora, Y. Mori, M. Takahashi, S. Nishimura, and Y. Okuno. 1964. Experimental pathology of measles in monkeys. Biken J. 6:271-297.[Medline]
32
32. Niwa, H., K. Yamamura, and J. Miyazaki. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193-199.[CrossRef][Medline]
33
33. Nommensen, F. E., and N. W. Dekkers. 1981. Detection of measles antigen in conjunctival epithelial lesions staining by lissamine green during measles virus infection. J. Med. Virol. 7:157-162.[CrossRef][Medline]
34
34. Olding-Stenkvist, E., and B. Bjorvatn. 1976. Rapid detection of measles virus in skin rashes by immunofluorescence. J. Infect. Dis. 134:463-469.[Medline]
35
35. Ono, N., H. Tatsuo, Y. Hidaka, T. Aoki, H. Minagawa, and Y. Yanagi. 2001. Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. J. Virol. 75:4399-4401.[Abstract/Free Full Text]
36
36. Polak-Charcon, S., and Y. Ben-Shaul. 1979. Degradation of tight junctions in HT29, a human colon adenocarcinoma cell line. J. Cell Sci. 35:393-402.[Abstract]
37
37. Sakaguchi, M., Y. Yoshikawa, K. Yamanouchi, T. Sata, K. Nagashima, and K. Takeda. 1986. Growth of measles virus in epithelial and lymphoid tissues of cynomolgus monkeys. Microbiol. Immunol. 30:1067-1073.[Medline]
38
38. Seki, F., M. Takeda, H. Minagawa, and Y. Yanagi. 2006. Recombinant wild-type measles virus containing a single N481Y substitution in its haemagglutinin cannot use receptor CD46 as efficiently as that having the haemagglutinin of the Edmonston laboratory strain. J. Gen. Virol. 87:1643-1648.[Abstract/Free Full Text]
39
39. Sherwin, S. A., J. D. Minna, A. F. Gazdar, and G. J. Todaro. 1981. Expression of epidermal and nerve growth factor receptors and soft agar growth factor production by human lung cancer cells. Cancer Res. 41:3538-3542.[Abstract/Free Full Text]
40
40. Stevenson, B. R., J. M. Anderson, D. A. Goodenough, and M. S. Mooseker. 1988. Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance. J. Cell Biol. 107:2401-2408.[Abstract/Free Full Text]
41
41. Takeda, M., S. Ohno, F. Seki, K. Hashimoto, N. Miyajima, K. Takeuchi, and Y. Yanagi. 2005. Efficient rescue of measles virus from cloned cDNA using SLAM-expressing Chinese hamster ovary cells. Virus Res. 108:161-165.[CrossRef][Medline]
42
42. Takeda, M., S. Ohno, F. Seki, Y. Nakatsu, M. Tahara, and Y. Yanagi. 2005. Long untranslated regions of the measles virus M and F genes control virus replication and cytopathogenicity. J. Virol. 79:14346-14354.[Abstract/Free Full Text]
43
43. Takeda, M., M. Tahara, T. Hashiguchi, T. A. Sato, F. Jinnouchi, S. Ueki, S. Ohno, and Y. Yanagi. 2007. A human lung carcinoma cell line supports efficient measles virus growth and syncytium formation via a SLAM- and CD46-independent mechanism. J. Virol. 81:12091-12096.[Abstract/Free Full Text]
44
44. Takeda, M., K. Takeuchi, N. Miyajima, F. Kobune, Y. Ami, N. Nagata, Y. Suzaki, Y. Nagai, and M. Tashiro. 2000. Recovery of pathogenic measles virus from cloned cDNA. J. Virol. 74:6643-6647.[Abstract/Free Full Text]
45
45. Takeuchi, K., N. Miyajima, F. Kobune, and M. Tashiro. 2000. Comparative nucleotide sequence analysis of the entire genomes of B95a cell-isolated and Vero cell-isolated measles viruses from the same patient. Virus Genes 20:253-257.[CrossRef][Medline]
46
46. Takeuchi, K., N. Miyajima, N. Nagata, M. Takeda, and M. Tashiro. 2003. Wild-type measles virus induces large syncytium formation in primary human small airway epithelial cells by a SLAM(CD150)-independent mechanism. Virus Res. 94:11-16.[CrossRef][Medline]
47
47. Tatsuo, H., N. Ono, K. Tanaka, and Y. Yanagi. 2000. SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893-897.[CrossRef][Medline]
48
48. Vongpunsawad, S., N. Oezgun, W. Braun, and R. Cattaneo. 2004. Selectively receptor-blind measles viruses: identification of residues necessary for SLAM- or CD46-induced fusion and their localization on a new hemagglutinin structural model. J. Virol. 78:302-313.[Abstract/Free Full Text]
49
49. Wan, H., H. L. Winton, C. Soeller, G. A. Stewart, P. J. Thompson, D. C. Gruenert, M. B. Cannell, D. R. Garrod, and C. Robinson. 2000. Tight junction properties of the immortalized human bronchial epithelial cell lines Calu-3 and 16HBE14o. Eur. Respir. J. 15:1058-1068.[Abstract]
50
50. Xie, M.-F., K. Tanaka, N. Ono, H. Minagawa, and Y. Yanagi. 1999. Amino acid substitutions at position 481 differently affect the ability of the measles virus hemagglutinin to induce cell fusion in monkey and marmoset cells co-expressing the fusion protein. Arch. Virol. 144:1689-1699.[CrossRef][Medline]
51
51. Yanagi, Y., M. Takeda, and S. Ohno. 2006. Measles virus: cellular receptors, tropism and pathogenesis. J. Gen. Virol. 87:2767-2779.[Abstract/Free Full Text]

---

Journal of Virology, May 2008, p. 4630-4637, Vol. 82, No. 9
0022-538X/08/$08.00+0     doi:10.1128/JVI.02691-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Sharma, L. B., Ohgimoto, S., Kato, S., Kurazono, S., Ayata, M., Takeuchi, K., Ihara, T., Ogura, H. (2009). Contribution of Matrix, Fusion, Hemagglutinin, and Large Protein Genes of the CAM-70 Measles Virus Vaccine Strain to Efficient Growth in Chicken Embryonic Fibroblasts. J. Virol. 83: 11645-11654 [Abstract] [Full Text]  
• Runkler, N., Dietzel, E., Carsillo, M., Niewiesk, S., Maisner, A. (2009). Sorting signals in the measles virus wild-type glycoproteins differently influence virus spread in polarized epithelia and lymphocytes. J. Gen. Virol. 90: 2474-2482 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tahara, M. Articles by Yanagi, Y. Search for Related Content PubMed PubMed Citation Articles by Tahara, M. Articles by Yanagi, Y.