Differential downregulation of CD46 by measles virus strains

JOURNAL OF VIROLOGY, Nov. 1995, p. 7257–7259 0022-538X/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 69, No. 11

Differential Downregulation of CD46 by Measles Virus Strains ¨ RGEN SCHNEIDER-SCHAULIES,1* LEE M. DUNSTER,1 FUMIO KOBUNE,2 JU BERT RIMA,3 AND VOLKER TER MEULEN1 Institut fu ¨r Virologie und Immunbiologie, D-97078 Wu ¨rzburg, Germany1; Department of Viral Disease and Vaccine Control, National Institute of Health, Tokyo 208, Japan2; and Division of Molecular Biology, School of Biology and Biochemistry, The Queen’s University of Belfast, Belfast BT9 7BL, Northern Ireland3 Received 27 March 1995/Accepted 28 July 1995

Recently, we found that several lymphotropic wild-type isolates of measles virus (MV) did not lead to the downregulation of CD46 following infection. We hypothesized that either the site of virus isolation, e.g., throat swab versus peripheral blood mononuclear cells, or the cell type used for the isolation may exert selective pressure on a mixed population of viruses, resulting in isolates with the differential properties observed. This hypothesis has been tested by simultaneously isolating MV from a throat swab and peripheral blood mononuclear cells from a single patient by cultivation on B95 and Vero cells. We report that neither the source of MV nor the cell type used for isolation directly influenced the capacity for CD46 modulation of these MV isolates.

material obtained from patients (4, 5). Therefore, strains of MV isolated by passage in lymphocytes are thought more likely to resemble the circulating wild-type strains than those obtained by Vero cell passage. In addition to the 19 MV strains described (16), we investigated 15 further wild-type isolates: BIL (18); CL, SE, TT, Y22, and R118 (12); JM (1); AK-4 (13); EB-L and EB-T (14); Kohno (6); and YL-B3, YT-B3, YL-V4, and YT-V4. The cells used for isolation, the country of origin, the year of isolation, and the results concerning CD46 modulation after infection of human target cells with these strains are presented in Table 1. Of the 15 new strains, 4 (BIL, CL, TT, and Y22) led to the downregulation of CD46 and 11 did not. Figure 1 shows the surface expression of CD46 on B95a, Raji, and HeLa cells, which were infected with seven representative MV strains before having CD46 expression tested by flow cytometry as described elsewhere (16). The MV strains TT and Y22 modulated CD46 expression by approximately 40 and 50% and strain CL modulated CD46 expression by only approximately 30%, whereas strains YL-B3, YT-B3, SE, and R118 did not induce downregulation of CD46 (Fig. 1A, C, and E). The level of infection for all strains was standardized on the basis of the expression of MV-H (Fig. 1B, D, and F). Since the procedure for virus isolation and passages in certain cell types might have led to the selection of MV strains with differences in lymphotropism, we tested this hypothesis with isolates derived from a throat swab (T) and peripheral blood mononuclear cells (L) from a single MV patient using B95a cells (Epstein-Barr virus-transformed marmoset leukocytes) and Vero cells (African green monkey kidney cells). The resulting strains were designated YT-V4, YT-B3, YL-V4, and YL-B3 (Fig. 2). In addition to these four strains, we tested the MV strains EB-T and EB-L, which were derived from a different MV patient from a throat swab (T) and peripheral blood mononuclear cells (L) by cultivation on Vero cells. None of these isolates led to the downregulation of CD46 after infection of B95a cells (results for YL-B3 and YT-B3 are shown in Fig. 1A) and BJAB cells (transformed human B lymphocytes [data not shown]). These results suggest that neither the cell type nor the source of infectious material directly deter-

Recently, two cell surface molecules, CD46 (2, 10) and moesin (3), have been identified to be functionally associated with measles virus (MV) susceptibility of cells. CD46, the major MV receptor, can be found in close proximity with moesin at the cell surface, forming a receptor complex (15). CD46, or MCP (membrane cofactor protein), is a classical transmembrane protein binding the complement proteins C3b and C4b and inhibiting lysis of host cells by complement (7, 8). The binding of MV to CD46 is dependent on its N glycosylation, which probably is necessary for the proper conformation of this molecule (9). Following infection with MV strain Halle, CD46 is strongly downregulated from the surface of the host cell (11). All MV vaccine strains tested to date, including Edmonston and CAM, modulated the cell surface expression of CD46, whereas lymphotropic wild-type isolates did not downregulate CD46 following infection (16). Vaccinia virus-encoded recombinants expressing MV hemagglutinin (MV-H) from different strains exert these differential effects on CD46 downregulation (16). Since the normal cellular function of CD46 in vivo is to inhibit the deposition of the complement proteins C3b and C4b on host cells, a consequence of the modulation of CD46 following MV infection is the increased susceptibility of cells to complement lysis (17). The recently observed differences in nucleotide sequences and biological properties of MV strains suggest that the heterogeneity of MV strains may be greater than had originally been thought. For the investigation of these differences, it is important to take into account that the method chosen for the isolation of individual MV strains will exert selective pressure upon the original virus population and will promote the growth of variants better able to adapt to growth in cell culture. Classically, MV strains were established by passage of infected blood or biopsy material of patients in Vero cells. However, it has been noted that MV-induced fusion occurs more rapidly after infection of a marmoset B-lymphocytic line (B95a) and that these cells were much more sensitive to infection with

* Corresponding author. Mailing address: Institut fu ¨r Virologie und Immunbiologie, Versbacher Str. 7, D-97078 Wu ¨rzburg, Germany. Phone: 931-2015954. Fax: 931-2013934. 7257

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J. VIROL.

FIG. 1. Quantification of surface CD46 and MV-H expression by flow cytometry. B95a cells were infected with MV isolates YL-B3 and YT-B3 (multiplicity of infection 5 1) for 3 days (A and B); Raji cells were infected with MV strains TT, CL, and SE for 3 days (C and D); and HeLa cells were infected with MV strains Y22 and R118 for 2 days (E and F). The negative control (CTRL) was done with a nonbinding antibody, CD46 expression was detected with the CD46-specific monoclonal antibody (MAb) 13/42 (15) on uninfected (UNINF) and MV-infected cells (A, C, and E), and MV-H expression was detected with the hemagglutinin-specific MAb K83 (16) on MV-infected cells (B, D, and F). The strains YL-B3 and YT-B3 (mean fluorescence intensities [mfi] 5 67.80 and 66.78, respectively, in comparison with the value for uninfected cells [mfi 5 53.79]) and SE (mfi 5 71.98 in comparison with 78.77 for uninfected cells) and R118 (mfi 5 111.49 in comparison with 123.29) did not lead to a decrease in the mfi of CD46, whereas the strains TT, CL, and Y22 did (mfi 5 48.56, 56.61, and 57.11, respectively).

mines the phenotype of a particular virus isolate from a single source. We further investigated whether the phenotype of an MV strain, with respect to CD46 modulation, is altered by adaptation of a lymphotropic CD46 nondownregulating strain to Vero or HeLa cells. The lymphotropic MV strains DL and WTF were adapted to nonlymphocytes by repeated (at least 30) passages over Vero cells. We then tested their capacity to modulate CD46 expression after infection of HeLa cells. At no time was the phenotype of the MV strains altered with respect to CD46 downregulation, although the potential for infecting fibroblasts was clearly improved by the adaptation process (data not shown). Since neither the isolation procedure nor the adaptation in cell culture changed the potential of these strains to modulate CD46, we suggest that this is an intrinsic property of a particular MV strain dependent on the sequence of its H protein and that selection from a mixed virus population is unlikely. The degree of downregulation observed after infection of human cells with MV strains BIL and CL was only 25 to 30%, whereas the degree of downregulation of CD46 observed with other modulating MV strains was between 40 and 80%. With 10% reduction of CD46 expression, the MV strain SE was classified as nondownregulating under the stringencies we set for the interpretation of the results. These data suggest that certain

strains might represent intermediate variants of MVs between extremely downregulating and nondownregulating forms. It would be interesting to know what role the receptor downregulation has in vivo and whether nondownregulating variants might occur by antigenic drift directed by evolutionary pressure.

FIG. 2. Flow diagrammatic representation of isolation of MV from a throat swab (T) and peripheral blood mononuclear cells (L) from a single patient by using the lymphocytic cell line B95a (B) and the African green monkey kidney cell line Vero (V). The four resulting isolates were designated YT-V4, YT-B3, YL-V4, and YL-B3.

VOL. 69, 1995

NOTES

TABLE 1. Downregulation of CD46 after infection with MV strains Group

Vaccine group I

MV strain

Cell type used for isolation

CAM Edmonston Edm Zagreb EDW ELV EVA Moraten

Vero HK, Vero Vero Vero Vero Vero Vero

Japan United United United United United United

BCLL Raji Vero Vero Vero Vero Raji Vero Vero HeLa, Vero HeLa, CV-1 HeLa, Vero BJAB B95 BJAB BJAB Vero Vero Vero B95 Vero Raji BJAB B95 B95 Vero Vero

Wild-type BIL group II CL CM JOY MY Woodfolk TT Y22 Braxator Halle LEC Mantooth Wild-type AB group III AK-4 DF DL EB-L EB-T JM Kohno R118 SE WTF YL-B3 YT-B3 YL-V4 YT-V4

Country of isolation

DownYr of regulation isolation of CD46a

1968 1954 1966 Late 50’s Late 50’s Late 50’s 1964

1 1 1 1 1 1 1

The Netherlands England United States United States United States United States England Cameroon Germany United States

1992 1988 Late 70’s Late 70’s Late 70’s 1970 1991 1983 1971 1971

25%b 1 1 1 1 1 1 1 1 77%

United States

1970

1

United States

1969

1

Germany Japan Germany Germany Japan Japan United States Japan Gabon England Germany Japan Japan Japan Japan

1992 1988 1992 1992 1985 1985 1977 1985 1984 1988 1990 1994 1994 1994 1994

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

States States States States States States

a CD46 expression on the cell surface was determined by flow cytometry with monoclonal antibody 13/42. A reduction of the mean fluorescence intensity by $25% was found for downregulating strains and is indicated as 1; lack of downregulation (,10%) is indicated as 2. A summary of data obtained exclusively with human cell lines is shown. As target cells of MV strains isolated on Vero cells, we used human HeLa cells. As target cells of the lymphotropic strains isolated on various B-cell lines, we used the human transformed B-cell lines BJAB and Raji (Burkitt lymphoma). Cells were infected with a multiplicity of infection of 1 and incubated for 2 to 4 days, depending on the rate of replication of each MV isolate, until high levels of MV-H were detected. b The MV strain BIL was the least potent downregulating MV strain, with 25% downregulation after infection, whereas Halle was the most potent downregulating virus, with 77% downregulation, as indicated.

For providing us with MV strains we thank A. D. M. E. Osterhaus, Utrecht, The Netherlands (BIL, F. Wild, Lyon, France (R118 and Y22); T. Schulz and R. Weiss, London, United Kingdom (CL, SE, and TT); and M. Morita, Akita, Japan (AK-4). We thank S. Lo ¨ffler and K. Pech for excellent technical assistance.

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This work was supported by the Deutsche Forschungsgemeinschaft and Bundesministerium fu ¨r Forschung und Technologie.

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