Receptor use by vesicular stomatitis virus pseudotypes with glycoproteins of defective variants of measles virus isolated from brains of patients with subacute sclerosing panencephalitis

Masashi Shingai1, Minoru Ayata1, Hiroshi Ishida1,2, Isamu Matsunaga1, Yuko Katayama1, Tsukasa Seya3, Hironobu Tatsuo4, Yusuke Yanagi4 and Hisashi Ogura1

1 Department of Virology, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka 545-8585, Japan
2 Department of Pediatrics, Osaka City University Medical School, Asahimachi, Abeno-ku, Osaka 545-8585, Japan
3 Department of Immunology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537-8511, Japan
4 Department of Virology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

Correspondence
Hisashi Ogura
ogurah{at}med.osaka-cu.ac.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The vaccine or Vero cell-adapted strains of measles virus (MV) have been reported to use CD46 as a cell entry receptor, while lymphotropic MVs preferentially use the signalling lymphocyte activation molecule (SLAM or CD150). In contrast to the virus obtained from patients with acute measles, little is known about the receptor that is used by defective variants of MV isolated from patients with subacute sclerosing panencephalitis (SSPE). The receptor-binding properties of SSPE strains of MV were analysed using vesicular stomatitis virus pseudotypes expressing the envelope glycoproteins of SSPE strains of MV. Such pseudotype viruses could use SLAM but not CD46 for entry. The pseudotype viruses with SSPE envelope glycoproteins could enter Vero cells, which do not express SLAM. In addition, their entry was not blocked by the monoclonal antibody to CD46, pointing to another entry receptor for SSPE strains on Vero cells. Furthermore, the unknown receptor(s), distinct from SLAM and CD46, may be present on cell lines derived from lymphoid and neural cells. Biochemical characterization of the receptor present on Vero cells and SK-N-SH neuroblastoma cells was consistent with a glycoprotein. Identification of additional entry receptors for MV will provide new insights into the mechanism of spread of MV in the central nervous system and possible reasons for differences between MVs isolated from patients with acute measles and SSPE.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Measles virus (MV) has been reported to use CD46 (membrane cofactor protein) as an entry receptor (Dörig et al., 1993; Naniche et al., 1993). In addition, SLAM (signalling lymphocyte activation molecule), also designated CD150, has been proposed to be a primary entry receptor for MV (Tatsuo et al., 2000b; Erlenhoefer et al., 2001; Hsu et al., 2001). MV vaccine and laboratory strains, including the Edmonston strain, can use both CD46 and SLAM, while wild-type MV uses SLAM preferentially (Tatsuo et al., 2000b; Ono et al., 2001). CD46 is present on most human cell types, with the exception of erythrocytes (Dörig et al., 1993; Naniche et al., 1993), while SLAM expression is restricted to lymphocytes, monocytes and dendritic cells (Sidorenko & Clark, 1993; Cocks et al., 1995; Minagawa et al., 2001; Ohgimoto et al., 2001; Murabayashi et al., 2002). Some cell lines, such as B95a, an Epstein–Barr virus (EBV)-transformed marmoset B cell line of lymphoblastoid cells, also express a simian homologue of human SLAM (Tatsuo et al., 2000b). Such differences in distribution of suitable receptors for MV are likely to explain the difficulty of isolating wild-type MV in Vero cells and ease of isolation in B95a cells. Receptor use in vivo for MV is controversial (Manchester et al., 2000; Ono et al., 2001; Yanagi, 2001). The interaction of the SLAM and CD46 receptors with MV glycoproteins plays a key role in triggering profound immunosuppression (Schlender et al., 1996; Karp, 1999; Oldstone et al., 1999; Klagge et al., 2000; Hsu et al., 2001; Schneider-Schaulies et al., 2001; Hahm et al., 2003) but the molecular mechanisms are unknown.

In contrast to the MV strains obtained from patients with acute measles, little is known about the cellular receptors used by defective variants of MV isolated from the brains of patients with subacute sclerosing panencephalitis (SSPE). These viruses have been isolated by cocultivation of brain cells with nonlymphatic cells, such as Vero (Doi et al., 1972; Kratzsch et al., 1977; Makino et al., 1977; Ogura et al., 1997; Homma et al., 1982), BSC-1 (Burnstein et al., 1974) and human embryonic lung cells (Ueda et al., 1975). Isolation of virus from brain tissue is usually problematic because of the limited quantity of biopsy specimens, poor quality of postmortem tissue and restricted expression of viral mRNAs and proteins in infected brains (Baczko et al., 1986; Liebert et al., 1986; Cattaneo et al., 1987; Sidhu et al., 1994). However, despite these difficulties, viruses have been isolated successfully from three cases of SSPE. Recovery of virus was more efficient in Vero cells than in B95a cells (Ogura et al., 1997), suggesting that Vero cells express a host molecule(s) that makes them suitable for growth of SSPE strains of MV. Although the receptor-independent spread of MV in the central nervous system is proposed (Lawrence et al., 2000), the entry receptor for MV is potentially one of the most important factors in the neurotropism and subsequent pathogenesis of SSPE. Recent studies have presented evidence supporting a role for CD46 as the receptor for MV in the brain (Buchholz et al., 1996; Ogata et al., 1997). Since neither Vero nor neural cells display SLAM, CD46 is a logical candidate for the SSPE strain entry receptor. However, the haemagglutinin (H) protein derived from SSPE strains does not adsorb to African green monkey erythrocytes, indicating the absence of an interaction between the SSPE H protein and CD46 (Furukawa et al., 2001). Adaptation of the MV H protein to CD46 receptor use in cell culture could be achieved by substitution of tyrosine for asparagine at position 481 (Nielsen et al., 2001) or substitution of glycine for serine at position 546 (Shibahara et al., 1994; Furukawa et al., 2001; Li & Qi, 2002; K. Furukawa, unpublished observation). Our sequence analysis of the H genes of the Osaka-1, Osaka-2, Osaka-3 SSPE strains, including sibling viruses isolated in either B95a or Vero cells, revealed that there were no substitutions at positions 481 and 546, even after serial passage in Vero cells (Furukawa et al., 2001). In addition, H protein sequences from Vero cell isolate Osaka-2/Fr/V and B95a cell isolate Osaka-2/Fr/B of the Osaka-2 strain, as well as the Vero cell isolate Osaka-3/Bs/V and B95a cell isolate Osaka-3/Bs/B of the Osaka-3 strain, were identical between the two sibling viruses (Furukawa et al., 2001). The H genes from the Osaka-2/Fr/V, Osaka-2/Fr/B, Osaka-3/Bs/V and Osaka-3/Bs/B strains of MV were cloned into plasmids. After transfection of Vero cells, the H proteins were expressed on the cell surface and, in combination with the fusion (F) protein, syncytia were formed (Furukawa et al., 2001; K. Furukawa, unpublished observation; M. Ayata, unpublished observation). Therefore, adaptation by changes in nucleotide and amino acid sequence of envelope glycoproteins cannot be the reason that the SSPE strains of MV replicate in Vero cells.

SSPE strains of MV are highly cell-associated and cell-free virions are not usually produced. These viruses replicate and spread from cell to cell by forming syncytia in Vero cells. The cell tropism of SSPE strains has not been studied because of the problems inherent in culturing SSPE strains in vitro. We have used cell-free virus-like particles, prepared by treating cells infected with MVs from patients with SSPE with cytochalasin D, to study the tropism of particles that resulted in syncytia formation on either Vero or B95a cells (Ito et al., 2002; H. Ishida, unpublished observation). Large syncytia developed after infection of both Vero and B95a cells, indicating the existence of host molecules on both cells suitable for infection and growth of SSPE strains of MV.

In this study, we have analysed the receptor use by SSPE strains of MV using the vesicular stomatitis virus (VSV) pseudotype system. The VSV pseudotype system allowed us to eliminate the internal host factors involved in virus replication that might affect cell tropism and to focus on the interaction between SSPE glycoproteins and candidate receptor molecules.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells.
Cell lines used in this study and their derivations are listed in Table 1. OCUPSN, OCUPGK and OCUPON cells were established and provided by T. Kawamura (Department of Pediatrics, Osaka City University Medical School, Japan). These cell lines, as well as CHO, CHO/CD46 (a gift from Y. Murakami, Department of Pharmaceutical Sciences, Hokkaido University, Japan) and CHO/SLAM cells (Tatsuo et al., 2000b) were cultured in RPMI-1640 medium supplemented with 10 % heat-inactivated FCS. B95a cells (Kobune et al., 1990) were cultured in RPMI-1640 supplemented with 5 % FCS. Vero cells were cultured in DMEM supplemented with 1 % FCS and 4 % newborn calf serum. HeLa, NIH-3T3 and L929 cells were cultured in DMEM supplemented with 10 % FCS. SK-N-MC, SK-N-SH, U-343, U-87 and U-251 cells were provided by H. Mori (Department of Neuroscience, Osaka City University Medical School, Japan). These cell lines, as well as A172 and T98G cells, were cultured in DMEM supplemented with 10 % FCS, 1 % non-essential amino acids and 1 mM sodium pyruvate.


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Table 1. Derivations and expression of SLAM and CD46 on cells used in this study

 
Viruses.
The year of virus isolation, the characteristics of the MV and SSPE strains used in this study and the amino acid sequence of the H protein for each virus at positions 481 and 546 are shown in Table 2. All strains were isolated in Osaka, Japan. The Toyoshima laboratory strain was isolated originally in FL cells (Toyoshima et al., 1959) and passaged in Vero cells. The Nagahata strain was isolated first in primary human embryonic kidney cell cultures and passaged subsequently in human embryonic cells and then Vero or CV-1 cells (Wong et al., 1991). The Masusako strain was isolated and passaged in Vero cells. The Nagahata and Masusako strains of MV are genetically closely related to three SSPE strains and are clustered in the same genotype (Ayata et al., 1998; Furukawa et al., 2001). The method for the haemadsorption assay has been described previously (Furukawa et al., 2001).


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Table 2. Summary of MV strains used in this study

 
Construction of pseudotype viruses and quantification in various cell lines.
VSV pseudotypes complemented with MV envelope glycoproteins were prepared as described previously (Tatsuo et al., 2000a). Briefly, a recombinant VSV (VSV{Delta}G*), in which the G protein gene of the parental VSV was replaced by the green fluorescent protein (GFP) gene, was complemented with the F and H glycoproteins from one of the six strains of MV (Table 2) (Furukawa et al., 2001; Ning et al., 2002). The fusion inhibition peptide (FIP, z-D-Phe-Phe-Gly) (Peptide Institute) (Richardson et al., 1980) was used for preparing all pseudotype viruses (Tatsuo et al., 2000a). The pseudotypes complemented with the F and H proteins homologous to the Toyoshima, Nagahata and Masusako strains were designated VSV{Delta}G*-Toy-F/H, VSV{Delta}G*-Nag-F/H and VSV{Delta}G*-Mas-F/H, respectively. The pseudotypes complemented with the F and H proteins homologous to the Osaka-1, Osaka-2 and Osaka-3 strains were designated VSV{Delta}G*-Osa1-F/H, VSV{Delta}G*-Osa2-F/H and VSV{Delta}G*-Osa3-F/H, respectively. For a negative control, we used uncomplemented VSV{Delta}G*. The positive control was VSV{Delta}G*-G complemented with the VSV G protein. The quality of these pseudotype viruses was assessed by quantifying the RNA obtained from the identical quantities of virus stock. The GFP genes were amplified by RT-PCR and the amounts of each product were compared prior to saturating amplification cycles. The difference in expression levels between the pseudotype viruses was minimal, with quantities within a threefold range, indicating that the genome content of the pseudotype virus stocks was similar. In our assessment of the infectivity of these stocks on different cell types and their potential to interact with various receptors, we used a single stock of each pseudotype in all our experiments to ensure comparability of results.

Ninety-six-well microtitre plates seeded with 2x104 adherent cells or 4x104 lymphoid cells were used for titrations, as described previously (Tatsuo et al., 2000a). At 24 h after infection, the number of GFP-expressing cells was counted using a fluorescence microscope and results were expressed as infectious units ml-1 of pseudotype virus stock. When suspension cultures were used for the assay, cells were dispersed by gentle pipetting before positive cells were counted.

Antibodies, FIP and entry blocking experiments.
IPO-3 to human SLAM (Sidorenko & Clark, 1993) was from Kamiya Biomedical. The manufacturer specifies that IPO-3 cross-reacts with the homologous molecule on lymphocytes from African green monkeys. M177 or M160 recognizes the short consensus repeat (SCR) 2 or SCR3 domain of human CD46, respectively (Seya et al., 1995). The L77 neutralizing mAb against the MV H protein was a gift from V. ter Meulen (Institute of Virology and Immunology, University of Wurzburg, Germany). Cells were dispensed in 100 µl volumes to 96-well microplates and cultured overnight. Either antibody or FIP was added to culture wells to a final concentration of 40 or 50 µg ml-1, respectively, and, after 1 h of incubation, 50 µl of serially diluted virus stock was added to the culture. GFP-positive cells were counted 24 h later.

Flow cytometry analysis.
Cells were incubated with primary antibodies IPO-3, M177, M160 or L77, followed by staining with FITC-labelled goat anti-mouse IgG. Stained cells were analysed on a FACScan machine (Becton Dickinson). Since the CD46 homologue on B95a cells is structurally different from human CD46, it was detected by M160 antibody but not M177 (Murakami et al., 1998).

Chemical modification of cells.
To treat cell surface proteins, Vero or SK-N-SH cells were washed twice with serum-free medium and incubated with pronase (Sigma), trypsin (Sigma) or chymotrypsin (Sigma) in serum-free medium at 37 °C for 20 min. To treat carbohydrates, cells were incubated with NaIO4 (Sigma) or neuraminidase from Vibrio cholerae (Sigma) in serum-free medium at 37 °C for 1 h. For tunicamycin (Sigma) treatment, cells were incubated with the drug in complete medium at 37 °C for 8 h. Treated cells were washed twice with serum-free medium, incubated with pseudotype viruses for 1 h, washed twice again and then replenished with fresh complete medium. After 24 h of incubation, GFP-positive virus-infected cells were counted.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
SSPE strains can use SLAM but not CD46 as the entry receptor
We examined the entry of the VSV pseudotypes into B95a and Vero cells. Pseudotype viruses were titrated in both cell types by quantification of fluorescence-positive cells at 24 h. Because we did not evaluate or adjust the quantity of the envelope glycoproteins incorporated into VSV envelope, we simply compared the entry efficiency among different cell types by the individual pseudotype viruses. All pseudotype viruses, with the exception of VSV{Delta}G*, entered B95a cells efficiently (Fig. 1A). Both the pseudotype viruses with either the Toyoshima or the Nagahata envelope glycoproteins and the positive control virus entered Vero cells efficiently (Fig. 1A) but there was no evidence for entry of either the pseudotype viruses with the Masusako glycoprotein or the negative control virus (Fig. 1A). Pseudotype viruses with SSPE glycoproteins also entered Vero cells (Fig. 1A). These results of pseudotype viruses with the SSPE glycoproteins essentially paralleled the observations in Vero cells infected by each virus.



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Fig. 1. Entry of the pseudotype viruses into B95a and Vero cells (A) or CHO cells expressing SLAM (CHO/SLAM), CHO cells expressing CD46 (CHO/CD46) and the CHO parental line (CHO) (B). Each cell line was infected with similar quantities of virus stock for each of the eight pseudotype viruses (VSV{Delta}G*-G, VSV{Delta}G*, VSV{Delta}G*-Toy-F/H, VSV{Delta}G*-Nag-F/H, VSV{Delta}G*-Mas-F/H, VSV{Delta}G*-Osa1-F/H, VSV{Delta}G*-Osa2-F/H and VSV{Delta}G*-Osa3-F/H). Each bar represents the infectivity titres of three independent experiments (mean±SD) measured by counting the number of GFP-expressing cells 24 h after infection.

 
The entry of VSV pseudotypes into CHO cells stably expressing either CD46 or SLAM (CHO/CD46 or CHO/SLAM, respectively) was studied to explore the use by glycoproteins of the SSPE strains of known MV receptors. The pseudotype viruses, with the exception of VSV{Delta}G*, entered CHO/SLAM cells efficiently (Fig. 1B). The pseudotype viruses with either the Toyoshima or the Nagahata envelope glycoproteins, as well as the positive control virus, entered CHO/CD46 cells efficiently (Fig. 1B). However, no cell entry was observed by either the pseudotype viruses with the Masusako or SSPE glycoproteins or by the negative control virus (Fig. 1B). As expected, only the VSV{Delta}G*-G pseudotype virus entered the parental CHO cell line (Fig. 1B).

To confirm the specificity of the entry through the receptor further, we used mAbs against both SLAM (IPO-3) and CD46 (M177 and M160). M177, but not M160, will block MV receptor activity (Seya et al., 1995). Optimal conditions for antibody blocking of virus entry were determined by quantifying virus entry into either CHO/SLAM or CHO/CD46 cells with the pseudotype viruses VSV{Delta}G*-Toy-F/H or VSV{Delta}G*-Nag-F/H. The entry of these pseudotype viruses through SLAM was blocked by IPO-3 but was not affected by M177 (Fig. 2A). The entry of the same pseudotype viruses through CD46 was blocked by M177 but was not affected by M160 (Fig. 2A). These three antibodies were used to examine blocking of the entry of SSPE pseudotype viruses into Vero cells. The pseudotype viruses with either the Toyoshima or the Nagahata envelope glycoproteins were blocked completely by M177 (Fig. 2B). The entry of pseudotype viruses with SSPE glycoproteins was not affected by M177 or by a mixture of M177 and IPO-3 (Fig. 2B). Both the L77 neutralizing antibody to H glycoprotein and FIP could block all pseudotype virus infection (Fig. 2B). Neither the M160 nor the IPO-3 antibodies inhibited the entry of any pseudotype viruses into Vero cells (Fig. 2B).



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Fig. 2. Effect of mouse mAbs against SLAM and CD46 on the entry of pseudotype viruses into CHO/SLAM or CHO/CD46 cells (A), Vero cells (B), SK-N-SH cells (C), OCUPSN cells (D) and B95a cells (E). Cells were pretreated for 1 h either with media only or with 40 µg SLAM antibody ml-1 (IPO-3), CD46 antibodies (M160 and M177), a cocktail of M177 and IPO-3 (M177+IPO-3), neutralizing monoclonal anti-H MV (L77) or 50 µg FIP ml-1. Infection was with the pseudotype viruses VSV{Delta}G*-Toy-F/H, VSV{Delta}G*-Nag-F/H, VSV{Delta}G*-Osa1-F/H, VSV{Delta}G*-Osa2-F/H or VSV{Delta}G*-Osa3-F/H. Infectivity titres were measured by counting the number of GFP-expressing cells 24 h after infection.

 
A putative third entry receptor for MV distributes on various cell lines
Since results described in the previous section showed that entry of SSPE strains of MV into cells could not be explained entirely by interaction with either SLAM or CD46, we explored the possible existence of a third receptor. We examined the infectivity of VSV pseudotypes in various cell lines, including lymphoid and neural cells of human origin (Table 1). Cell surface expression of SLAM and CD46 molecules was evaluated by flow cytometry (data not shown). B95a cells are currently widely used because of their efficiency for MV isolation, probably because they express large amounts of SLAM homologue. All of the cell lines tested, except for the CHO cells, were positive for CD46. The only SLAM-positive cells, in addition to CHO/SLAM cells, were the B95a cells and three EBV-transformed human B cell lines (Table 1). When the pseudotype virus with the glycoproteins of the Osaka-2 strain (VSV{Delta}G*-Osa2-F/H) was tested for infection (Fig. 3), 293T cells were comparable to Vero cells for permissive infection. HeLa cells were also infected by VSV{Delta}G*-Osa2-F/H but not as efficiently as 293T cells. All cell lines of neural origin (SK-N-MC, SK-N-SH, U-343, U-87, A172 and U-251) could be infected by VSV{Delta}G*-Osa2-F/H. The single exception in this study was the T98G cell line. The murine cell lines NIH-3T3 and L929 were not permissive. Lymphoid cell lines, including three EBV-transformed B cell lines, all expressed SLAM and all were infected by the pseudotype virus with the Osaka-2 glycoproteins. Similar results were obtained using B95a cells. The pseudotype virus with the glycoproteins of the Masusako strain could infect only limited types of cell lines but could readily infect lymphoid cells.



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Fig. 3. Cell lines permissive for the entry of the pseudotype viruses. Each of the cell lines was infected individually with the four pseudotype viruses (VSV{Delta}G*-G, VSV{Delta}G*-Mas-F/H, VSV{Delta}G*-Osa2-F/H and VSV{Delta}G*). Infectivity titres were measured by counting the number of GFP-expressing cells 24 h after infection.

 
To confirm the specificity of receptor-mediated entry into selected cell lines further, cells were treated with our panel of mAbs to SLAM and CD46. The entry of the pseudotype viruses with either the Toyoshima or the Nagahata envelope glycoproteins into a human neuroblastoma cell line SK-N-SH was blocked completely by M177 antibody to CD46, L77 antibody to the H glycoprotein and FIP, but was not affected by M160 antibody to CD46 or IPO-3 antibody to SLAM (Fig. 2C). The entry into SK-N-SH cells by pseudotype viruses with SSPE glycoproteins was blocked by L77 or FIP but was not blocked by M160, M177 or IPO-3 or by the combination of M177 and IPO-3 receptor antibodies (Fig. 2C). These results were comparable to those obtained in Vero cells shown in Fig. 2(B).

The effect of receptor antibodies on virus entry was tested on cell lines of lymphoid origin. Results from the OCUPSN human B cell line and B95a cells are shown in Fig. 2(D, E). The entry into OCUPSN cells of the pseudotype viruses with either the Toyoshima or the Nagahata envelope glycoproteins was blocked partially by IPO-3 or a polyclonal antibody to SLAM but was not affected by M160 or M177 antibodies (Fig. 2D). The reason why the M177 antibody could not block the entry into OCUPSN cells is unknown but the high affinity of the H protein for SLAM may account for the negative effect of the antibody to CD46 in such SLAM and CD46 double-positive cells. The L77 antibody, FIP and the cocktail of IPO-3 and M177 antibodies could block the entry into OCUPSN cells completely by the Toyoshima or Nagahata pseudotype viruses. The entry into OCUPSN cells of the pseudotype viruses with the Masusako envelope glycoproteins was blocked completely by IPO-3 or a polyclonal antibody to SLAM without addition of M177 antibody. The entry into OCUPSN cells of pseudotype viruses with SSPE glycoproteins was also blocked partially by IPO-3 or a polyclonal antibody to SLAM (Fig. 2D). The combination of M177 antibody and IPO-3, however, did not block the entry into OCUPSN cells by the SSPE pseudotype viruses (Fig. 2D). The results of the blocking pseudotype virus entry with either antibodies or FIP were similar in both B95a and OCUPSN cells (Fig. 2E). The CD46 homologue on B95a cells is defective and cannot be used for entry by most MV strains (Hsu et al., 1997; Murakami et al., 1998). As expected, the entry into B95a cells of the pseudotype viruses expressing either the Toyoshima or the Nagahata envelope glycoproteins was blocked completely by IPO-3 or a polyclonal antibody to SLAM without adding M177 antibody to CD46 (Fig. 2E). The entry of pseudotype viruses with SSPE glycoproteins into B95a cells was blocked partially by IPO-3 or a polyclonal antibody to SLAM (Fig. 2E).

These results show that a third unknown receptor was expressed on the cell surface of various cell lines, including neural and lymphoid cells. A molecule on Vero cells used by SSPE strains for entry may be different from a molecule(s) found on other cell lines but it can be used only by pseudotype viruses with the SSPE glycoproteins.

Biochemical characterization of a putative third entry receptor suggests a glycoprotein
To characterize the biochemical nature of a molecule involved in the entry of SSPE strains, we examined the infectivity of pseudotype virus with the glycoproteins of the Osaka-2 strain (VSV{Delta}G*-Osa2-F/H) on Vero or SK-N-SH cells that were chemically modified by various reagents. Pseudotype virus with the G protein of VSV (VSV{Delta}G*-G), which has a known phospholipid receptor, was used as a control (Schlegel et al., 1983).

Vero cells were preincubated with pronase, trypsin or chymotrypsin at various concentrations and then infected with VSV{Delta}G*-G or VSV{Delta}G*-Osa2-F/H (Fig. 4A). Pronase treatment of cells markedly reduced the infectivity of VSV{Delta}G*-Osa2-F/H in a dose-dependent manner, while VSV{Delta}G*-G infectivity was not affected even at the highest concentration used (Fig. 4A). Trypsin treatment had no inhibitory effect but rather increased the infectivity of VSV{Delta}G*-Osa2-F/H (Fig. 4A). The dose-responsiveness of VSV{Delta}G*-Osa2-F/H was different from that of pseudotype virus with the Toyoshima glycoprotein. Infectivity of VSV{Delta}G*-Toy-F/H for Vero cells was reduced sharply by trypsin treatment at a concentration of 500 µg ml-1. Chymotrypsin treatment had no effect, even at concentration of 500 µg ml-1, on the infectivity of VSV{Delta}G*-Osa2-F/H (data not shown). These results suggest that the third, putative cellular receptor for SSPE strain on Vero cells is sensitive to pronase and resistant to trypsin and chymotrypsin.



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Fig. 4. Infectivity of pseudotype viruses on cells whose surface proteins were chemically modified. Vero (A) and SK-N-SH (B) cells were preincubated with the indicated concentrations of pronase, neuraminidase, NaIO4, tunicamycin or trypsin (Vero cells only) and virus infectivity was quantified. Three wells per treatment modality were used. Each bar represents the per cent infectivity (% IU, mean±SD) calculated by comparing the ratio of GFP-expressing cells in treated to untreated cells.

 
Vero cells were treated with NaIO4 or tunicamycin before infecting with pseudotype viruses to determine the role of carbohydrates in virus entry (Fig. 4A). The treatment of cells with these reagents reduced the infectivity of VSV{Delta}G*-Osa2-F/H in a dose-dependent manner but only affected VSV{Delta}G*-G infectivity slightly. In addition, we treated Vero cells with neuraminidase but this treatment had no effect on the infectivity of either pseudotype virus, indicating the absence of the role of sialic acids in virus entry (Fig. 4A).

SK-N-SH cells were also pretreated by pronase, tunicamycin, NaIO4 or neuraminidase and then VSV{Delta}G*-G and VSV{Delta}G*-Osa2-F/H were tested for infectivity (Fig. 4B). Neuraminidase treatment had no effect, even at a concentration that was 10 times higher than that sufficient to abolish the susceptibility of cells to influenza virus (Takada et al., 1997). However, the infectivity of VSV{Delta}G*-Osa2-F/H was reduced in a dose-dependent manner by the treatment of cells with pronase, tunicamycin or NaIO4. The pattern observed in SK-N-SH cells was essentially the same as that of Vero cells, indicating the similarity of the biochemical nature of the molecule in both cell lines.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we demonstrate that the Toyoshima and Nagahata strains of MV can use both SLAM and CD46 as an entry receptor in a manner similar to the Edmonston laboratory strain of MV (Ono et al., 2001). In contrast, the Osaka-1, Osaka-2 and Osaka-3 SSPE strains of MV use SLAM but not CD46 as an entry receptor, as has been described for recent lymphotropic field isolates such as the KA strain of MV (Tatsuo et al., 2000b). With the current VSV pseudotype system, we could analyse the differential receptor use of SSPE strains in a manner that was independent of possible internal host factors that could affect cell tropism (Takeda et al., 1998; Kouomou & Wild, 2002). Some reports propose that CD46 is the receptor for MV infection in the brain (Buchholz et al., 1996; Ogata et al., 1997). CD46 isoforms with cytoplasmic tail 2 were isolated from a brain of SSPE, expressed and found to interact with H protein (Buchholz et al., 1996). The disappearance of CD46 in SSPE lesions in the brain, which has also been reported by Ogata et al. (1997), can be explained by the H protein-mediated downregulation of the CD46 molecule. However, CD46 was not used as an entry receptor by the three strains of SSPE used in our study. Thus, the absence of CD46 in SSPE lesions may not be related directly to lack of receptor presence but rather a consequence of SSPE-induced dysfunction of infected brain cells. Furthermore, the two key amino acid changes on the H glycoprotein of asparagine to tyrosine at position 481 and/or serine to glycine at position 546, which are closely related to its interaction with CD46 (Furukawa et al., 2001), did not occur in our three SSPE strains. By site-directed mutagenesis, we introduced the tyrosine at position 481 or glycine at position 546 substitutions into our three SSPE H genes and tested for the interaction with CD46 (unpublished data). The H protein of the Osaka-3 strain gains the ability to interact with CD46 after substitution of tyrosine for asparagine at position 481 or substitution of glycine for serine at position 546. However, the H protein of the Osaka-1 strain requires an additional two substitutions of phenylalanine for leucine at position 549 and of phenylalanine for serine at position 552. Similarly, the H protein of the Osaka-2 strain requires a substitution of leucine for serine at position 526 in addition to either of the key substitutions. These data indicate the requirements of specific amino acid changes for the interaction with the H protein and CD46 (Massé et al., 2002) and supports the negative interaction of our SSPE H protein with CD46. Unlike CD46, SLAM is not usually expressed on neural cells (McQuaid & Cosby, 2002) and there are no reports of its involvement in MV spread in the brain, effectively eliminating receptors described previously as means of MV spread in neural tissue.

There are reports of successful MV infection of rat brain in vivo or rat hippocampal tissue in vitro in the absence of detectable CD46 (Duprex et al., 2000; Ehrengruber et al., 2002). Immature rat neurons may possess alternative receptors that can be used by MV. Interferon {alpha}/{beta} receptor-knocked out mice are also susceptible to the Edmonston strain of MV, although the transgenic addition of CD46 facilitated the lethal outcome (Mrkic et al., 1998). These reports support the hypothesis that there are entry receptors for MV other than CD46 and SLAM. The data in our study also support the possibility that there is a third unidentified receptor for SSPE strains of MV on Vero cells. It is possible that our SSPE strains could have attained the ability to bind to the third receptor during passages on Vero cells in vitro. We have analysed recently a brain tissue obtained at autopsy from a patient with SSPE. The Osaka-2 strain was isolated from the patient 8 years before autopsy. The nucleotide sequence of MV was determined directly without culture and found that it was similar to the sequence determined from the Osaka-2 strain. Although the nucleotide sequence of the envelope glycoprotein was variable in some positions, the key amino acid sequences of the F and H proteins were not changed during 8 years in the brain (unpublished data). Therefore, it is likely that the virus used in this study, at least the Osaka-2 strain, represents the virus replicating in a brain of a patient with SSPE. An alternative explanation for the entry of pseudotype virus of SSPE into Vero cells is that, despite the lack of detection of SLAM on Vero cells (Ono et al., 2001), there may be small amounts of SLAM that can be used very efficiently as a receptor.

Recently, Lawrence et al. (2000) proposed a mechanism by the receptor-independent spread of MV in neurons. We do not exclude such a possibility that MV may spread in differentiated cell types, such as neurons, with different mechanisms. MV glycoproteins may be sorted to the specific portion of the neuron and could serve for transmission, as demonstrated by Ehrengruber et al. (2002). However, these papers are not presuming a third receptor other than CD46 and SLAM and the unidentified molecule may be involved in virus spread without significant fusion. Firsching et al. (1999) proposed such a microfusion event at sites of cell-to-cell contact. It should be noted that we have observed massive fusion in the hippocampal neurons in a hamster brain that was infected with the Osaka-2 strain of MV (Ito et al., 2002).

We found that the yet to be identified receptor(s) may also exist on other cell lines, including human neural and lymphoid cells, since blocking experiments using mAbs against known receptors did not inhibit infection by pseudotype viruses with the SSPE glycoproteins. SLAM-independent MV entry was reported by Hashimoto et al. (2002), who used recombinant MV expressing GFP based on the wild-type IC-B strain of MV to demonstrate infection of SLAM-negative cells such as Vero cells. The infection of the original IC-B strain of MV to Vero cells was not obvious when the infection criterion was morphological change of cells (Hashimoto et al., 2002). Pseudotype virus with such wild-type MV glycoproteins does not enter Vero cells (Tatsuo et al., 2000a). The infectivity of the virus for SLAM-negative cells was two to three logs lower than that for SLAM-positive cells (Hashimoto et al., 2002). The discrepancy between the two assay systems using the recombinant IC-B and the pseudotype virus with the IC-B glycoprotein is similar to the results obtained in this study using our Masusako strain. The Masusako strain was isolated originally in, and replicated in, Vero cells, while the pseudotype virus with the Masusako glycoproteins failed to enter Vero cells. The molecular basis of the ability of the parental Masusako strain of MV to grow in Vero cells after entry is not known. However, this strain is considered, along with the IC-B strain, to be a wild-type, based on the sequences of the F and H glycoproteins (Furukawa et al., 2001; Ning et al., 2002). Whether the receptor used by the Masusako and IC-B strains is different from that used by the SSPE strains is unknown.

Identification of all possible receptor(s) used by SSPE strains is necessary to understand how MV enters into and spreads throughout the central nervous system. Such information may provide insight into the conditions that lead to SSPE instead of the typical measles disease.


   ACKNOWLEDGEMENTS
 
We thank M. A. Whitt for allowing us to use the VSV{Delta}G*-GFP system. We are grateful to all of our scientific colleagues for their generous gifts of reagents used in this study. We also thank K. Tatsuta, E. Uenaka, M. Egami and Y. Nishio for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (No. 11670779) from the Ministry of Education, Science, Sports and Culture of Japan, a grant from the Osaka City University Medical Research Foundation Fund and a grant from the Osaka Medical Research Foundation for Incurable Diseases.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 January 2003; accepted 28 April 2003.