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. 1998 Apr;72(4):2905-16.
doi: 10.1128/JVI.72.4.2905-2916.1998.

A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells

E C Hsu  1 , F Sarangi, C Iorio, M S Sidhu, S A Udem, D L Dillehay, W Xu, P A Rota, W J Bellini, C D Richardson
Affiliations

A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells

E C Hsu et al. J Virol. 1998 Apr.

Abstract

This paper provides evidence for a measles virus receptor other than CD46 on transformed marmoset and human B cells. We first showed that most tissues of marmosets are missing the SCR1 domain of CD46, which is essential for the binding of Edmonston measles virus, a laboratory strain that has been propagated in Vero monkey kidney cells. In spite of this deletion, the common marmoset was shown to be susceptible to infections by wild-type isolates of measles virus, although they did not support Edmonston measles virus production. As one would expect from these results, measles virus could not be propagated in owl monkey or marmoset kidney cell lines, but surprisingly, both a wild-type isolate (Montefiore 89) and the Edmonston laboratory strain of measles virus grew efficiently in B95-8 marmoset B cells. In addition, antibodies directed against CD46 had no effect on wild-type infections of marmoset B cells and only partially inhibited the replication of the Edmonston laboratory strain in the same cells. A direct binding assay with insect cells expressing the hemagglutinin (H) proteins of either the Edmonston or Montefiore 89 measles virus strains was used to probe the receptors on these B cells. Insect cells expressing Edmonston H but not the wild-type H bound to rodent cells with CD46 on their surface. On the other hand, both the Montefiore 89 H and Edmonston H proteins adhered to marmoset and human B cells. Most wild-type H proteins have asparagine residues at position 481 and can be converted to a CD46-binding phenotype by replacement of the residue with tyrosine. Similarly, the Edmonston H protein did not bind CD46 when its Tyr481 was converted to asparagine. However, this mutation did not affect the ability of Edmonston H to bind marmoset and human B cells. The preceding results provide evidence, through the use of a direct binding assay, that a second receptor for measles virus is present on primate B cells.

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Figures

FIG. 1
FIG. 1
Southern blot of PCR amplification spanning the SCR1 region of the common marmoset (Callithrix jacchus). The brain, heart, liver, lung, kidney, small intestine, spleen, and stomach of a common marmoset were isolated and homogenized in TRIzol, mRNA was extracted, and cDNA was prepared with RT. PCR was performed across the SCR1 region with oligonucleotide primers derived from the conserved signal peptide and SCR3 domains. A 300-bp product was indicative of a deleted SCR1 domain, while a 522-bp product was produced from a complete copy of the CD46 cDNA. Most organs from the marmoset contained the deleted form of CD46, but the brain and heart may contain small amounts of the undeleted species in addition to the major deleted mRNA. B95-8 marmoset B cells were homogenized, and mRNA was extracted and treated in a similar manner to that from the marmoset organs. Undeleted and deleted forms of CD46 were present in the B95-8 cells. PCR analysis was also performed on cDNA clones which had been prepared from mRNA isolated from B95-8 cells and inserted into the pCR Script AMP (SK+) vector. The PCR products from the deleted clone (CD46ΔSCR1) and the nondeleted clone (CD46) templates were also analyzed. PCR products were resolved by agarose gel electrophoresis, transferred to nitrocellulose, probed with 32P-labelled fragments derived from the SCR2 and SCR3 regions of CD46, and subjected to autoradiography with Royal X-OMAT film for 24 h.
FIG. 2
FIG. 2
Growth of the Edmonston strain of measles virus in New World monkey cell lines is impaired when SCR1 is deleted. (A) Human cervical carcinoma (HeLa), African green monkey kidney (Vero), owl monkey kidney (OMK), marmoset kidney (NZP-60), marmoset B (B95-8), and squirrel monkey lung (SML) cells were infected with the Edmonston strain of measles virus which had previously been adapted for growth in Vero cells. The cells were inoculated with 5 PFU of virus per cell, and infections were allowed to proceed for 72 h, after which the infected cells were subjected to immunoblot analysis with monoclonal antibodies to measles virus H protein. Viral protein synthesis was not observed in the OMK and NZP-60 cell lines, but measles virus H protein was detected in B95-8 and SML cells. (B) FACScan analysis was performed on B95-8, OMK, SML, and NZP-60 cells with an antibody to the SCR1 domain of the moustached tamarin (Saguinus mystax) and detected with goat anti-rabbit antibodies which had been conjugated to fluorescein isothiocyanate (solid line). The cells were also tested with rabbit preimmune antisera (dotted line). Shifts in fluorescence were observed in B95-8 and SML cells but not in OMK and NZP-60 cells. (C) mRNA was extracted from B95-8 and SML cells, cDNA was prepared, and PCR products spanning the signal peptide, SCR1, SCR2, and SCR3 domains were prepared and sequenced. The predicted amino acid sequence is shown and was derived from three independent amplification reactions for each sequence. Both deleted and nondeleted forms of mRNA were present in the two cell lines.
FIG. 3
FIG. 3
Infection of cell lines with Edmonston laboratory and Montefiore 89 wild-type strains of measles virus. HeLa cells, Vero cells, Chinese hamster ovary cells transfected with an empty expression vector (CHO-pDRα2), Chinese hamster ovary cells expressing human CD46 (CHO-CD46), and a marmoset B-cell line (B95-8) were inoculated with either Edmonston or wild-type Montefiore 89 strains of measles virus. The cells were incubated for 72 h with either strain of virus, and infection was monitored by immunoblot analysis with a monoclonal antibody to the measles virus M protein (40 kDa). (A) HeLa, Vero, CHO-CD46, and B95-8 cells supported infection by the Edmonston strain, while CHO cells transfected with the expression vector alone (CHO-pDRα2) were not infected. (B) HeLa, Vero, CHO-pDRα2, and B95-8 cells were inoculated with the wild-type Montefiore 89 strain, and infections were allowed to proceed for 72 h. Only the B95-8 cells supported infection. Protein standards (in kilodaltons) are shown at the left of each panel.
FIG. 4
FIG. 4
Polyclonal antibody to CD46 does not inhibit infections by the Montefiore 89 wild-type strain of measles virus. Antibodies to CD46 and the marmoset SCR1 were combined and tested for their ability to inhibit infections by Montefiore 89 and Edmonston measles virus in B95-8 and Vero cells. Cells were treated with CD46 and SCR1 immune antibodies (A, C, and E) or preimmune serum (B, D, and F). CD46 antibodies at dilutions of 1:10 had no effect upon infections of B95-8 cells by the wild-type Montefiore 89 virus (A) but partially inhibited infections of the same type of cells by the Edmonston strain of virus (C). The same antibodies at dilutions as low as 1:400 completely inhibited the infection of Vero cells by the Edmonston virus (E). Infections were assessed by the formation of syncytia or multinucleated cells. Bar, 3 μm.
FIG. 5
FIG. 5
Assays of binding of H proteins from Montefiore 89 and Edmonston measles virus to mouse OST-7 cells expressing CD46 and marmoset B95-8 cells. H proteins from the Montefiore 89 and Edmonston strains of measles virus were cloned and expressed on the surface of Sf9 insect cells by using the recombinant baculovirus system. Sf9 cells also expressed β-galactosidase and were stained blue by the addition of Bluogal substrate. The blue insect cells were incubated with mouse cells expressing human CD46 (A and B), mouse cells expressing marmoset/human chimeric CD46 (C and D), or B95-8 cells (E and F), and loosely adsorbed Sf9 cells were washed away. Insect cells which expressed the Edmonston H protein remained attached to mouse cells expressing human or chimeric CD46 as well as to the marmoset B95-8 cell line (A, C, and E). The wild-type Montefiore H protein did not bind to mouse cells expressing human CD46 or chimeric CD46, but it did adhere to the marmoset B cell line (B, D, and F). Bar, 2.5 μm.
FIG. 6
FIG. 6
Quantitation of Edmonston H and Montefiore H binding to mouse cells expressing human CD46, marmoset/human chimeric CD46, or CD21 or to B95-8 marmoset cells. Sf9 insect cells expressing Edmonston (A) or Montefiore 89 (B) H proteins were incubated with B95-8 marmoset cells or mouse L cells expressing CD46, chimeric CD46, or CD21 as described in the legend to Fig. 5. Loosely attached cells were washed away, and binding was measured colorimetrically with the ONPG substrate for β-galactosidase. Edmonston H bound to marmoset B95-8 cells and mouse cells expressing human CD46 and chimeric CD46. It did not bind to mouse cells expressing CD21 Montefiore H bound to marmoset B95-8 cells but did not adhere to mouse cells expressing human CD46, chimeric CD46, or CD21. Binding is expressed as a percentage relative to either Edmonston H binding or Montefiore H binding to B95-8 cells.
FIG. 7
FIG. 7
The Tyr481Asn mutation inhibits the binding of Edmonston and Montefiore 89 H proteins to CD46. Edmonston H (ED), Montefiore H (WT), and mutated forms of these proteins were expressed in Sf9 insect cells and incubated with CHO cells containing human CD46 (A) or marmoset B95-8 (B) cells. As expected, insect cells expressing Edmonston H (ED) bound to both CHO-CD46 and B95-8 cells, while wild-type Montefiore 89 H (WT) protein bound only to B95-8 cells. The N416D mutation introduced into Montefiore 89 H [WT(N-D)] had no effect on binding to either cell line. However, an N481Y mutation in the wild-type H [WT(N-Y)] converted the protein to a CD46-binding phenotype. In addition, when the Y481N mutation was placed in Edmonston H [ED(Y-N)], binding to CHO-CD46 cells was abolished. None of the mutations affected the binding of either Edmonston or wild-type H proteins to marmoset B95-8 cells. Binding was measured by quantitating β-galactosidase activity and was expressed as a percentage relative to the binding observed for Edmonston H protein.
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