Nearby Clusters of Hemagglutinin Residues Sustain
http://www.100md.com
病菌学杂志 2005年第9期
Molecular Medicine Program
Virology and Gene Therapy Graduate Track, Mayo Clinic College of Medicine, Rochester, Minnesota
Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas
ABSTRACT
Signaling lymphocytic activation molecule (SLAM, CD150) is the universal morbillivirus receptor. Based on the identification of measles virus (MV) hemagglutinin (H) amino acids supporting human SLAM-dependent cell entry, we mutated canine distemper virus (CDV) H and identified residues necessary for efficient canine SLAM-dependent membrane fusion. These residues are located in two nearby clusters in a new CDV H structural model. To completely abolish SLAM-dependent fusion, combinations of mutations were necessary. We rescued a SLAM-blind recombinant CDV with six mutations that did not infect ferret peripheral blood mononuclear cells while retaining full infectivity in epithelial cells.
TEXT
The severe and long-lasting immunosuppression accompanying wild-type morbillivirus infections enhances an individual's susceptibility to secondary infections, which account for most of the high disease-related morbidity and mortality (3, 7, 41). Specific viral entry in lymphocytes through the universal Morbillivirus receptor SLAM (8, 14, 35, 36) is considered one possible immunosuppression determinant. Indeed SLAM expression on activated T cells, immature thymocytes, memory T cells, a proportion of B cells, activated monocytes, and dendritic cells overlaps largely with the dissemination of viral infection through the lymphoid system (5, 30, 38). Other receptors and postentry determinants also influence morbillivirus tropism (2, 4, 20, 23).
The lack of a small animal model has limited studies of the measles virus (MV) tropism determinants, but recently ferrets have been used to characterize the infection of the other Morbillivirus, canine distemper virus (CDV) (38, 39). To evaluate the importance of cell entry through SLAM for CDV infection of peripheral blood mononuclear cells (PBMC), we aimed at producing a recombinant virus unable to interact with this protein but maintaining entry through other receptors. Towards this we set up experiments to characterize residues on the CDV attachment protein necessary for SLAM-dependent cell fusion. We identified several amino acids and localized them on a novel CDV H protein structural model.
Identification of CDV H protein amino acids important for SLAM-dependent membrane fusion. There is no experimental Morbillivirus H-protein structure, but a three-dimensional model of the MV H protein based on the crystal structure of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein (6) was generated (37). This model (Fig. 1A, top view and 1B, side view) suggests that the MV H protein has a globular ectodomain with six beta-sheets, each composed of four strands. The beta-sheets are arranged cyclically around an axis as the blades of a propeller (Fig. 1A, the four strands in each beta-sheet are shown in the same color). Four "anchor" residues with the strongest effect on human SLAM-dependent membrane fusion (Y529, D530, T533, and R553, shown in red) are located on beta-propeller sheet 5 (color coded blue); three other residues (T531, F552 and P554, shown in gold) are also important.
Since CDV and MV H are 36% identical (11, 40), they may share a similar structure and their SLAM-interacting surfaces may roughly correspond. Therefore we operated with the MV H model and mutagenized in CDV H all 23 residues corresponding to the MV amino acids for which alpha carbon atoms were predicted to be within 10 ? of the four MV H anchor residues and solvent exposed. Twenty-two of these residues are located between positions 483 and 543 in the CDV H protein sequence (Fig. 1E). In addition, residue 173 situated outside this segment was mutagenized. CDV numbering and MV H protein numbering are dephased by 4 (e.g., CDV residue 529 corresponds to MV residue 533; compare CDV with MV numbering in Fig. 1E, top).
The H protein of the large plaque-forming variant of the CDV vaccine strain Onderstepoort expressed from plasmid pCG-HOL (40) was selected for initial analysis: this protein has strong SLAM-independent fusion support activity in Vero cells, in addition to SLAM-dependent fusion support activity. Two small amino acids, alanine to substitute charged and with polar residues and serine to replace apolar residues, were used to limit possible structural interferences (Fig. 1E, lowest line). To reduce the risk of reversion in subsequent phases of experimentation, at least two nucleotides were changed in each codon. We also confirmed that the sequence of the relevant H gene segments and the size of the H proteins estimated on denaturing gels were as expected (data not shown).
The function of all mutants was then tested in a fusion support assay based on the complementation of the homologous Onderstepoort F protein expressed from plasmid pCG-FOL either on Vero or on SLAM-expressing CHO cells (CHOdogSLAMtag) (40). The degree of fusion was measured on a four-grade scale described in the legend to Fig. 1 (grades 0 to 3). At least three independent experiments were graded. The results are summarized graphically in the lower half of Fig. 1E. Since Vero cells have a higher intrinsic fusion propensity than CHOdogSLAMtag cells, the thresholds used to define fusion efficiency levels (high, intermediate, low, and none) in the two cell lines are different, as detailed in the legend to Fig. 1.
Proteins with mutations at positions 526, 547, and 548 lost two levels of fusion efficiency in CHOdogSLAMtag cells while maintaining standard activity in Vero cells (Fig. 1E, labeled positions). Proteins with mutations at positions 489, 527, 528, 529, 530, 533, 543, 549, 550, and 552 also lost their fusion activity preferentially in CHOdogSLAM cells. Four mutants completely lost function on both cell lines. The six remaining mutants maintained the same level of function in both cell lines. The seven mutations resulting in strongly reduced fusion activity in SLAM-expressing cells but with standard or near-standard activity in Vero cells are indicated with their ordinal number in Fig. 1E.
To illustrate the position of these amino acids, a new model of CDV H was produced using the same template structure and software suite previously used to model MV H (37). Like MV H, CDV H is predicted to fold as a propeller with six beta-sheets of four strands each. Top and side views of the CDV H model are shown in Fig. 1C and Fig. 1D, respectively. The seven residues necessary for efficient canine SLAM-dependent fusion are highlighted in red (strongest effect) or gold. Six relevant residues are located in two nearby clusters in beta-sheet 5, a situation similar to that of MV H (Fig. 1A and Fig. 1B). The locations of the two clusters vary slightly between CDV H and MV H, but their relative distance is similar: the distances of the alpha-carbon atoms are 12 to 21 ? between the two CDV H clusters and 13 to 22 ? between the two MV H clusters.
Combined mutations in both H residue clusters completely abolish SLAM-dependent fusion. To obtain an H protein that displays no residual SLAM-dependent fusion support, we generated seven combination mutants starting with individual mutations that strongly interfered with SLAM-dependent fusion. In a first mutagenesis round two neighboring mutations were combined, yielding 526/7, 528/9, and 547/8, all of which displayed low fusion support function (1.0, 0.8, and 0.5, respectively) in CHOdogSLAMtag cells. These fusion activities were lower than those of the single mutants (Fig. 1E), all in the 1.5 to 1.8 range. The further combinations of either 526/7 or 528/9 with 547/8, or of all six mutations (526-9 + 547/8), resulted in complete loss of syncytium formation (0 fusion), whereas the fourfold mutant 526-9 maintained some fusion activity. All proteins displayed near-wild-type function in Vero cells (>2.5 fusion), indicating that inefficient protein processing and cell surface transport were unlikely causes for the observed loss of SLAM-mediated fusion support activity. Thus, two clusters of H residues support the CDV H-(Onderstepoort attenuated strain) SLAM-dependent cell fusion. Mutations in both clusters are necessary to eliminate fusion-support function.
A SLAM-blind CDV replicates efficiently in epithelial but not in lymphoid cells. To assess the role of the CDV H-SLAM interactions in PBMC, the alanine or serine mutations in six residues necessary for efficient SLAM-dependent fusion identified in the H protein of the vaccine strain Onderstepoort were transferred to the H protein of the virulent wild-type strain 5804P (39). The Onderstepoort and 5804P H proteins have seven amino acids at variance between residues 480 and 560 (Fig. 1E), but none of these residues aligns with those influencing SLAM-dependent fusion.
To monitor virus recovery, we could not rely on syncytium formation because wild-type viruses are by far less proficient in fusing Vero cells than vaccine strains: cell rounding or very small syncytia dominate the cytopathic effect. Thus to facilitate the identification of cells supporting the replication of a SLAM-blind virus, we introduced the mutations into a cDNA copy of the genome of 5804P-eGFP/H, a virus that expresses enhanced green fluorescent protein (eGFP) from an additional transcription unit inserted between the H and L genes (38). Cells replicating this virus can be identified even if they do not fuse because they emit green light upon stimulation. The mutations were introduced using the unique restriction sites RsrII and AscI (40). The corresponding fragment, which includes the last third of the H gene and the untranslated region between H and eGFP, was assembled by overlap extension PCR (13), yielding p5804SLAMblind-eGFP/H. We then attempted virus recovery using the wild-type CDV rescue system based on B95a cells (39). B95a cells were infected with a T7 polymerase-expressing, replication-deficient vaccinia virus (MVA/T7) (33) and transfected with the plasmid containing the full-length CDV genome as well as expression plasmids for the CDV nucleo-, phospho-, and polymerase proteins. Indeed we recovered a virus that was named 5804P-eGFP/H-SLAMblind. This virus was propagated on VerodogSLAMtag cells, and its growth was analyzed on epithelial cells and PBMC, as shown in Fig. 2.
5804P-eGFP/H-SLAMblind readily infected MDCK cells, a canine epithelial kidney cell line, as well as primary ferret epithelial (PFE) kidney cells. In these cells, the cytopathic effects caused by the parental virus and the SLAM-blind virus 5 days postinoculation were similar (Fig. 2A, columns MDCK and PFE), with multiple small syncytia typical for wild-type strains. In contrast, infection with the SLAM-blind derivative was not detectable at the same time postinoculation in CHOdogSLAMtag cells and ferret PBMC (Fig. 2A; columns CHOdogSLAMtag and PBMC). Thus, the amino acids identified with the SLAM-dependent fusion support assay in cultured cells are essential also for the entry and propagation of a wild-type CDV strain in PBMC.
We then asked which types of PBMC support CDV replication. Using pan-species monoclonal antibodies, we separated ferret T cells (CD3+), B cells (CD79+), and macrophages (CD14+). Figure 2B shows the percentages of each cell type (white columns) detected in cultivated PBMC five days after mock infection (control, left panel) or infection with the two recombinant viruses 5804P-eGFP and 5804P-eGFP-SLAMblind (center and right panel, respectively). CDV infection resulted in a small but significant change in the distribution of T and B cells, possibly due to the preferential death of CD79-positive B lymphocytes. The wild-type virus but not its SLAM-blind derivative entered PBMC efficiently. In Fig. 2B the percentages of green fluorescent CD3, CD79, and CD14 cells five days after infection with the wild-type virus or its SLAM-blind derivative are represented with gray columns. More than half of the CD3, CD79, and CD14 cells emitted green light after wild-type virus infection, whereas GFP expression was minimal after infection with the SLAM-blind virus (Fig. 2B, right panel). Thus efficient CDV entry in all major PBMC types depends on efficient H protein interaction with SLAM.
Discussion. We have identified seven residues in the CDV H protein that are involved in SLAM-mediated fusion support and virus entry. Six of these residues are grouped in two clusters, both situated in propeller beta-sheet 5, and define areas analogous to those previously defined by the SLAM-interacting residues on MV H (37). A study of CDV adaptation to cell entry through marmoset SLAM resulted in the selection of a virus with mutations in residues 530 and 548, one residue in each cluster defined here (29). On the other hand, a mutagenesis study of charged MV H residues (21) identified arginine 533 and aspartates 505 and 507 as crucial for SLAM downregulation and SLAM-dependent fusion. This study did not cover residues downstream of position 537, and another study showed that aspartate 507 had a negative effect not only on SLAM-dependent but also on CD46-dependent fusion (37). It is possible that other amino acids support interactions with different receptors (25). It is remarkable that in CDV H multiple mutations are necessary to abolish canine SLAM-dependent fusion whereas in MV H single mutations strongly compromise human SLAM-dependent fusion (37).
We have shown here that a recombinant SLAM-blind wild-type CDV cannot infect PBMC but infects efficiently epithelial cells. This result and the recent observation in the ferret system that the parental wild-type CDV infects preferentially, if not exclusively, lymphocytes (38) are consistent with a major role of SLAM-dependent cell entry for morbillivirus-induced immunosuppression (35). The fact that in vivo only minimal macrophage infection by the wild-type virus was monitored (38), whereas macrophages are infected efficiently ex vivo, may be due to mitogen activation.
The SLAM-blind CDV retained efficient entry in primary ferret kidney epithelial cells and in canine MDCK cells. Which protein serves as a CDV receptor in these cells? Antibodies to CD9, a tetraspan transmembrane protein, inhibit CDV-induced fusion of primate cells (27), but the cellular molecule responsible for virus binding and uptake in epithelial cells of dogs and other carnivores has not yet been identified. In the case of the human morbillivirus MV, the ubiquitous regulator of complement activation CD46 acts as a cellular receptor for the attenuated and certain wild-type strains (19, 28); other alternative receptors have been sought but not yet identified (1, 12). CDV H does not efficiently interact with human or marmoset CD46 (10, 18, 32), and therefore it was suggested that CD46 may not be the ubiquitous morbillivirus receptor. However, lack of CDV H interaction is documented only for primate CD46, and studies based on canine CD46 cells are necessary to clarify the issue of the epithelial cell receptor for wild-type CDV.
ACKNOWLEDGMENTS
We thank Becky Sanford for excellent secretarial support and Y. Yanagi for kindly providing the CHOdogSLAMtag cells.
This work was supported by grants of the Mayo and Siebens Foundations, NIH grant CA90636 to R.C., and by an Emmy Noether award of the German Research Foundation (DFG) to V. von Messling.
REFERENCES
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.
Avota, E., A. Avots, S. Niewiesk, L. P. Kane, U. Bommhardt, V. ter Meulen, and S. Schneider-Schaulies. 2001. Disruption of Akt kinase activation is important for immunosuppression induced by measles virus. Nat. Med. 7:725-731.
Borrow, P., and M. B. Oldstone. 1995. Measles virus-mononuclear cell interactions. Curr. Top. Microbiol. Immunol. 191:85-100.
Cattaneo, R. 2004. Four viruses, two bacteria and one receptor: membrane cofactor protein (CD46) as a pathogens' magnet. J. Virol. 78:4385-4388.
Cocks, B. G., C. C. 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.
Crennell, S., T. Takimoto, A. Portner, and G. Taylor. 2000. Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat. Struct. Biol. 7:1068-1074.
de Swart, R. L., T. C. Harder, P. S. Ross, H. W. Vos, and A. D. Osterhaus. 1995. Morbilliviruses and morbillivirus diseases of marine mammals. Infect Agents Dis. 4:125-130.
Erlenhoefer, C., W. J. Wurzer, S. L?ffler, 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.
Ferrari, M. 1985. Susceptibility of various cell culture systems to bovine viral diarrhea virus. Microbiologica 8:17-22.
Galbraith, S. E., A. Tiwari, M. D. Baron, B. T. Lund, T. Barrett, and S. L. Cosby. 1998. Morbillivirus downregulation of CD46. J. Virol. 72:10292-10297.
Haas, L., H. Liermann, T. C. Harder, T. Barrett, M. Lochelt, V. von Messling, W. Baumgartner, and I. Greiser-Wilke. 1999. Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses. Vet. Microbiol. 69:15-18.
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.
Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.
Hsu, E. C., C. Iorio, F. Sarangi, A. A. Khine, and C. D. 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.
Ivanciuc, O., N. Oezguen, V. S. Mathura, C. H. Schein, Y. Xu, and W. Braun. 2004. Using property based sequence motifs and 3D modeling to determine structure and functional regions of proteins. Curr. Med. Chem. 11:583-593.
Kelley, L. A., R. M. MacCallum, and M. J. Sternberg. 2000. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299:499-520.
Koradi, R., M. Billeter, and K. Wuthrich. 1996. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14:51-55.
Loffler, S., F. Lottspeich, F. Lanza, D. O. Azorsa, V. ter Meulen, and J. Schneider-Schaulies. 1997. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus. J. Virol. 71:42-49.
Manchester, M., D. S. Eto, A. Valsamakis, P. B. Liton, R. Fernandez-Munoz, P. A. Rota, W. J. Bellini, D. N. Forthal, and M. B. A. Oldstone. 2000. Clinical isolates of measles virus use CD46 as a cellular receptor. J. Virol. 74:3967-3974.
Marie, J. C., J. Kehren, M. C. Trescol-Biemont, A. Evlashev, H. Valentin, T. Walzer, R. Tedone, B. Loveland, J. F. Nicolas, C. Rabourdin-Combe, and B. Horvat. 2001. Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14:69-79.
Massé, N., M. Ainouze, B. Néel, T. F. Wild, R. Buckland, and J. P. Langedijk. 2004. Measles virus (MV) hemagglutinin: evidence that attachment sites for MV receptors SLAM and CD46 overlap on the globular head. J. Virol. 78:9051-9063.
Mathura, V. S., K. V. Soman, T. K. Varma, and W. Braun. 2003. A multimeric model for murine anti-apoptotic protein Bcl-2 and structural insights for its regulation by post-translational modification. J. Mol. Model. 9:298-303.
Palosaari, H., J.-P. Parisien, J. J. Rodriguez, C. M. Ulane, and C. M. Horvath. 2003. STAT protein interference and suppression of cytokine signal transduction by measles virus V protein. J. Virol. 77:7635-7644.
Sanner, M., A. Widmer, H. Senn, and W. Braun. 1989. GEOM: a new tool for molecular modelling based on distance geometry calculations with NMR data. J. Comput.-Aided Mol. Des. 3:195-210.
Santiago, C., E. Bjorling, T. Stehle, and J. M. Casasnovas. 2002. Distinct kinetics for binding of the CD46 and SLAM receptors to overlapping sites in the measles virus hemagglutinin protein. J. Biol. Chem. 277:32294-32301.
Schaumann, T., W. Braun, and K. Wuthrich. 1990. The program FANTOM for energy refinement of polypeptides and proteins using a Newton-Raphson minimizer in torsion angle space. Biopolymers 29:679-694.
Schmid, E., A. Zurbriggen, U. Gassen, B. Rima, V. ter Meulen, and J. Schneider-Schaulies. 2000. Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion. J. Virol. 74:7554-7561.
Schneider, U., V. von Messling, P. Devaux, and R. Cattaneo. 2002. Efficiency of measles virus entry and dissemination through different receptors. J. Virol. 76:7460-7467.
Seki, F., N. Ono, R. Yamaguchi, and Y. Yanagi. 2003. Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells. J. Virol. 77:9943-9950.
Sidorenko, S. P., and E. A. Clark. 1993. Characterization of a cell surface glycoprotein IPO-3, expressed on activated human B and T lymphocytes. J. Immunol. 151:4614-4624.
Soman, K. V., C. H. Schein, H. Zhu, and W. Braun. 2001. Homology modeling and simulations of nuclease structures. Methods Mol. Biol. 160:263-286.
Stern, L. B.-L., M. Greenberg, J. M. Gershoni, and S. Rozenblatt. 1995. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis. J. Virol. 69:1661-1668.
Sutter, G., M. Ohlmann, and V. Erfle. 1995. Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett. 371:9-12.
Takimoto, T., K. G. Murti, T. Bousse, R. A. Scroggs, and A. Portner. 2001. Role of matrix and fusion proteins in budding of Sendai virus. J. Virol. 75:11384-11391.
Tatsuo, H., N. Ono, K. Tanaka, and Y. Yanagi. 2000. SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893-897.
Tatsuo, H., N. Ono, and Y. Yanagi. 2001. Morbilliviruses use signaling lymphocyte activation molecules (CD150) as cellular receptors. J. Virol. 75:5842-5850.
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.
von Messling, V., D. Milosevic, and R. Cattaneo. 2004. Tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system. Proc. Natl. Acad. Sci. USA 101:14216-14221.
von Messling, V., C. Springfeld, P. Devaux, and R. Cattaneo. 2003. A ferret model of canine distemper virus virulence and immunosuppression. J. Virol. 77:12579-12591.
von Messling, V., G. Zimmer, G. Herrler, L. Haas, and R. Cattaneo. 2001. The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity. J. Virol. 75:6418-6427.
Yamanouchi, K. 1980. Comparative aspects of pathogenicity of measles, canine distemper, and rinderpest viruses. Jpn. J. Med. Sci. Biol. 33:41-66.(Veronika von Messling, Nu)
Virology and Gene Therapy Graduate Track, Mayo Clinic College of Medicine, Rochester, Minnesota
Sealy Center for Structural Biology, University of Texas Medical Branch, Galveston, Texas
ABSTRACT
Signaling lymphocytic activation molecule (SLAM, CD150) is the universal morbillivirus receptor. Based on the identification of measles virus (MV) hemagglutinin (H) amino acids supporting human SLAM-dependent cell entry, we mutated canine distemper virus (CDV) H and identified residues necessary for efficient canine SLAM-dependent membrane fusion. These residues are located in two nearby clusters in a new CDV H structural model. To completely abolish SLAM-dependent fusion, combinations of mutations were necessary. We rescued a SLAM-blind recombinant CDV with six mutations that did not infect ferret peripheral blood mononuclear cells while retaining full infectivity in epithelial cells.
TEXT
The severe and long-lasting immunosuppression accompanying wild-type morbillivirus infections enhances an individual's susceptibility to secondary infections, which account for most of the high disease-related morbidity and mortality (3, 7, 41). Specific viral entry in lymphocytes through the universal Morbillivirus receptor SLAM (8, 14, 35, 36) is considered one possible immunosuppression determinant. Indeed SLAM expression on activated T cells, immature thymocytes, memory T cells, a proportion of B cells, activated monocytes, and dendritic cells overlaps largely with the dissemination of viral infection through the lymphoid system (5, 30, 38). Other receptors and postentry determinants also influence morbillivirus tropism (2, 4, 20, 23).
The lack of a small animal model has limited studies of the measles virus (MV) tropism determinants, but recently ferrets have been used to characterize the infection of the other Morbillivirus, canine distemper virus (CDV) (38, 39). To evaluate the importance of cell entry through SLAM for CDV infection of peripheral blood mononuclear cells (PBMC), we aimed at producing a recombinant virus unable to interact with this protein but maintaining entry through other receptors. Towards this we set up experiments to characterize residues on the CDV attachment protein necessary for SLAM-dependent cell fusion. We identified several amino acids and localized them on a novel CDV H protein structural model.
Identification of CDV H protein amino acids important for SLAM-dependent membrane fusion. There is no experimental Morbillivirus H-protein structure, but a three-dimensional model of the MV H protein based on the crystal structure of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein (6) was generated (37). This model (Fig. 1A, top view and 1B, side view) suggests that the MV H protein has a globular ectodomain with six beta-sheets, each composed of four strands. The beta-sheets are arranged cyclically around an axis as the blades of a propeller (Fig. 1A, the four strands in each beta-sheet are shown in the same color). Four "anchor" residues with the strongest effect on human SLAM-dependent membrane fusion (Y529, D530, T533, and R553, shown in red) are located on beta-propeller sheet 5 (color coded blue); three other residues (T531, F552 and P554, shown in gold) are also important.
Since CDV and MV H are 36% identical (11, 40), they may share a similar structure and their SLAM-interacting surfaces may roughly correspond. Therefore we operated with the MV H model and mutagenized in CDV H all 23 residues corresponding to the MV amino acids for which alpha carbon atoms were predicted to be within 10 ? of the four MV H anchor residues and solvent exposed. Twenty-two of these residues are located between positions 483 and 543 in the CDV H protein sequence (Fig. 1E). In addition, residue 173 situated outside this segment was mutagenized. CDV numbering and MV H protein numbering are dephased by 4 (e.g., CDV residue 529 corresponds to MV residue 533; compare CDV with MV numbering in Fig. 1E, top).
The H protein of the large plaque-forming variant of the CDV vaccine strain Onderstepoort expressed from plasmid pCG-HOL (40) was selected for initial analysis: this protein has strong SLAM-independent fusion support activity in Vero cells, in addition to SLAM-dependent fusion support activity. Two small amino acids, alanine to substitute charged and with polar residues and serine to replace apolar residues, were used to limit possible structural interferences (Fig. 1E, lowest line). To reduce the risk of reversion in subsequent phases of experimentation, at least two nucleotides were changed in each codon. We also confirmed that the sequence of the relevant H gene segments and the size of the H proteins estimated on denaturing gels were as expected (data not shown).
The function of all mutants was then tested in a fusion support assay based on the complementation of the homologous Onderstepoort F protein expressed from plasmid pCG-FOL either on Vero or on SLAM-expressing CHO cells (CHOdogSLAMtag) (40). The degree of fusion was measured on a four-grade scale described in the legend to Fig. 1 (grades 0 to 3). At least three independent experiments were graded. The results are summarized graphically in the lower half of Fig. 1E. Since Vero cells have a higher intrinsic fusion propensity than CHOdogSLAMtag cells, the thresholds used to define fusion efficiency levels (high, intermediate, low, and none) in the two cell lines are different, as detailed in the legend to Fig. 1.
Proteins with mutations at positions 526, 547, and 548 lost two levels of fusion efficiency in CHOdogSLAMtag cells while maintaining standard activity in Vero cells (Fig. 1E, labeled positions). Proteins with mutations at positions 489, 527, 528, 529, 530, 533, 543, 549, 550, and 552 also lost their fusion activity preferentially in CHOdogSLAM cells. Four mutants completely lost function on both cell lines. The six remaining mutants maintained the same level of function in both cell lines. The seven mutations resulting in strongly reduced fusion activity in SLAM-expressing cells but with standard or near-standard activity in Vero cells are indicated with their ordinal number in Fig. 1E.
To illustrate the position of these amino acids, a new model of CDV H was produced using the same template structure and software suite previously used to model MV H (37). Like MV H, CDV H is predicted to fold as a propeller with six beta-sheets of four strands each. Top and side views of the CDV H model are shown in Fig. 1C and Fig. 1D, respectively. The seven residues necessary for efficient canine SLAM-dependent fusion are highlighted in red (strongest effect) or gold. Six relevant residues are located in two nearby clusters in beta-sheet 5, a situation similar to that of MV H (Fig. 1A and Fig. 1B). The locations of the two clusters vary slightly between CDV H and MV H, but their relative distance is similar: the distances of the alpha-carbon atoms are 12 to 21 ? between the two CDV H clusters and 13 to 22 ? between the two MV H clusters.
Combined mutations in both H residue clusters completely abolish SLAM-dependent fusion. To obtain an H protein that displays no residual SLAM-dependent fusion support, we generated seven combination mutants starting with individual mutations that strongly interfered with SLAM-dependent fusion. In a first mutagenesis round two neighboring mutations were combined, yielding 526/7, 528/9, and 547/8, all of which displayed low fusion support function (1.0, 0.8, and 0.5, respectively) in CHOdogSLAMtag cells. These fusion activities were lower than those of the single mutants (Fig. 1E), all in the 1.5 to 1.8 range. The further combinations of either 526/7 or 528/9 with 547/8, or of all six mutations (526-9 + 547/8), resulted in complete loss of syncytium formation (0 fusion), whereas the fourfold mutant 526-9 maintained some fusion activity. All proteins displayed near-wild-type function in Vero cells (>2.5 fusion), indicating that inefficient protein processing and cell surface transport were unlikely causes for the observed loss of SLAM-mediated fusion support activity. Thus, two clusters of H residues support the CDV H-(Onderstepoort attenuated strain) SLAM-dependent cell fusion. Mutations in both clusters are necessary to eliminate fusion-support function.
A SLAM-blind CDV replicates efficiently in epithelial but not in lymphoid cells. To assess the role of the CDV H-SLAM interactions in PBMC, the alanine or serine mutations in six residues necessary for efficient SLAM-dependent fusion identified in the H protein of the vaccine strain Onderstepoort were transferred to the H protein of the virulent wild-type strain 5804P (39). The Onderstepoort and 5804P H proteins have seven amino acids at variance between residues 480 and 560 (Fig. 1E), but none of these residues aligns with those influencing SLAM-dependent fusion.
To monitor virus recovery, we could not rely on syncytium formation because wild-type viruses are by far less proficient in fusing Vero cells than vaccine strains: cell rounding or very small syncytia dominate the cytopathic effect. Thus to facilitate the identification of cells supporting the replication of a SLAM-blind virus, we introduced the mutations into a cDNA copy of the genome of 5804P-eGFP/H, a virus that expresses enhanced green fluorescent protein (eGFP) from an additional transcription unit inserted between the H and L genes (38). Cells replicating this virus can be identified even if they do not fuse because they emit green light upon stimulation. The mutations were introduced using the unique restriction sites RsrII and AscI (40). The corresponding fragment, which includes the last third of the H gene and the untranslated region between H and eGFP, was assembled by overlap extension PCR (13), yielding p5804SLAMblind-eGFP/H. We then attempted virus recovery using the wild-type CDV rescue system based on B95a cells (39). B95a cells were infected with a T7 polymerase-expressing, replication-deficient vaccinia virus (MVA/T7) (33) and transfected with the plasmid containing the full-length CDV genome as well as expression plasmids for the CDV nucleo-, phospho-, and polymerase proteins. Indeed we recovered a virus that was named 5804P-eGFP/H-SLAMblind. This virus was propagated on VerodogSLAMtag cells, and its growth was analyzed on epithelial cells and PBMC, as shown in Fig. 2.
5804P-eGFP/H-SLAMblind readily infected MDCK cells, a canine epithelial kidney cell line, as well as primary ferret epithelial (PFE) kidney cells. In these cells, the cytopathic effects caused by the parental virus and the SLAM-blind virus 5 days postinoculation were similar (Fig. 2A, columns MDCK and PFE), with multiple small syncytia typical for wild-type strains. In contrast, infection with the SLAM-blind derivative was not detectable at the same time postinoculation in CHOdogSLAMtag cells and ferret PBMC (Fig. 2A; columns CHOdogSLAMtag and PBMC). Thus, the amino acids identified with the SLAM-dependent fusion support assay in cultured cells are essential also for the entry and propagation of a wild-type CDV strain in PBMC.
We then asked which types of PBMC support CDV replication. Using pan-species monoclonal antibodies, we separated ferret T cells (CD3+), B cells (CD79+), and macrophages (CD14+). Figure 2B shows the percentages of each cell type (white columns) detected in cultivated PBMC five days after mock infection (control, left panel) or infection with the two recombinant viruses 5804P-eGFP and 5804P-eGFP-SLAMblind (center and right panel, respectively). CDV infection resulted in a small but significant change in the distribution of T and B cells, possibly due to the preferential death of CD79-positive B lymphocytes. The wild-type virus but not its SLAM-blind derivative entered PBMC efficiently. In Fig. 2B the percentages of green fluorescent CD3, CD79, and CD14 cells five days after infection with the wild-type virus or its SLAM-blind derivative are represented with gray columns. More than half of the CD3, CD79, and CD14 cells emitted green light after wild-type virus infection, whereas GFP expression was minimal after infection with the SLAM-blind virus (Fig. 2B, right panel). Thus efficient CDV entry in all major PBMC types depends on efficient H protein interaction with SLAM.
Discussion. We have identified seven residues in the CDV H protein that are involved in SLAM-mediated fusion support and virus entry. Six of these residues are grouped in two clusters, both situated in propeller beta-sheet 5, and define areas analogous to those previously defined by the SLAM-interacting residues on MV H (37). A study of CDV adaptation to cell entry through marmoset SLAM resulted in the selection of a virus with mutations in residues 530 and 548, one residue in each cluster defined here (29). On the other hand, a mutagenesis study of charged MV H residues (21) identified arginine 533 and aspartates 505 and 507 as crucial for SLAM downregulation and SLAM-dependent fusion. This study did not cover residues downstream of position 537, and another study showed that aspartate 507 had a negative effect not only on SLAM-dependent but also on CD46-dependent fusion (37). It is possible that other amino acids support interactions with different receptors (25). It is remarkable that in CDV H multiple mutations are necessary to abolish canine SLAM-dependent fusion whereas in MV H single mutations strongly compromise human SLAM-dependent fusion (37).
We have shown here that a recombinant SLAM-blind wild-type CDV cannot infect PBMC but infects efficiently epithelial cells. This result and the recent observation in the ferret system that the parental wild-type CDV infects preferentially, if not exclusively, lymphocytes (38) are consistent with a major role of SLAM-dependent cell entry for morbillivirus-induced immunosuppression (35). The fact that in vivo only minimal macrophage infection by the wild-type virus was monitored (38), whereas macrophages are infected efficiently ex vivo, may be due to mitogen activation.
The SLAM-blind CDV retained efficient entry in primary ferret kidney epithelial cells and in canine MDCK cells. Which protein serves as a CDV receptor in these cells? Antibodies to CD9, a tetraspan transmembrane protein, inhibit CDV-induced fusion of primate cells (27), but the cellular molecule responsible for virus binding and uptake in epithelial cells of dogs and other carnivores has not yet been identified. In the case of the human morbillivirus MV, the ubiquitous regulator of complement activation CD46 acts as a cellular receptor for the attenuated and certain wild-type strains (19, 28); other alternative receptors have been sought but not yet identified (1, 12). CDV H does not efficiently interact with human or marmoset CD46 (10, 18, 32), and therefore it was suggested that CD46 may not be the ubiquitous morbillivirus receptor. However, lack of CDV H interaction is documented only for primate CD46, and studies based on canine CD46 cells are necessary to clarify the issue of the epithelial cell receptor for wild-type CDV.
ACKNOWLEDGMENTS
We thank Becky Sanford for excellent secretarial support and Y. Yanagi for kindly providing the CHOdogSLAMtag cells.
This work was supported by grants of the Mayo and Siebens Foundations, NIH grant CA90636 to R.C., and by an Emmy Noether award of the German Research Foundation (DFG) to V. von Messling.
REFERENCES
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.
Avota, E., A. Avots, S. Niewiesk, L. P. Kane, U. Bommhardt, V. ter Meulen, and S. Schneider-Schaulies. 2001. Disruption of Akt kinase activation is important for immunosuppression induced by measles virus. Nat. Med. 7:725-731.
Borrow, P., and M. B. Oldstone. 1995. Measles virus-mononuclear cell interactions. Curr. Top. Microbiol. Immunol. 191:85-100.
Cattaneo, R. 2004. Four viruses, two bacteria and one receptor: membrane cofactor protein (CD46) as a pathogens' magnet. J. Virol. 78:4385-4388.
Cocks, B. G., C. C. 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.
Crennell, S., T. Takimoto, A. Portner, and G. Taylor. 2000. Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat. Struct. Biol. 7:1068-1074.
de Swart, R. L., T. C. Harder, P. S. Ross, H. W. Vos, and A. D. Osterhaus. 1995. Morbilliviruses and morbillivirus diseases of marine mammals. Infect Agents Dis. 4:125-130.
Erlenhoefer, C., W. J. Wurzer, S. L?ffler, 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.
Ferrari, M. 1985. Susceptibility of various cell culture systems to bovine viral diarrhea virus. Microbiologica 8:17-22.
Galbraith, S. E., A. Tiwari, M. D. Baron, B. T. Lund, T. Barrett, and S. L. Cosby. 1998. Morbillivirus downregulation of CD46. J. Virol. 72:10292-10297.
Haas, L., H. Liermann, T. C. Harder, T. Barrett, M. Lochelt, V. von Messling, W. Baumgartner, and I. Greiser-Wilke. 1999. Analysis of the H gene, the central untranslated region and the proximal coding part of the F gene of wild-type and vaccine canine distemper viruses. Vet. Microbiol. 69:15-18.
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.
Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.
Hsu, E. C., C. Iorio, F. Sarangi, A. A. Khine, and C. D. 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.
Ivanciuc, O., N. Oezguen, V. S. Mathura, C. H. Schein, Y. Xu, and W. Braun. 2004. Using property based sequence motifs and 3D modeling to determine structure and functional regions of proteins. Curr. Med. Chem. 11:583-593.
Kelley, L. A., R. M. MacCallum, and M. J. Sternberg. 2000. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299:499-520.
Koradi, R., M. Billeter, and K. Wuthrich. 1996. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14:51-55.
Loffler, S., F. Lottspeich, F. Lanza, D. O. Azorsa, V. ter Meulen, and J. Schneider-Schaulies. 1997. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus. J. Virol. 71:42-49.
Manchester, M., D. S. Eto, A. Valsamakis, P. B. Liton, R. Fernandez-Munoz, P. A. Rota, W. J. Bellini, D. N. Forthal, and M. B. A. Oldstone. 2000. Clinical isolates of measles virus use CD46 as a cellular receptor. J. Virol. 74:3967-3974.
Marie, J. C., J. Kehren, M. C. Trescol-Biemont, A. Evlashev, H. Valentin, T. Walzer, R. Tedone, B. Loveland, J. F. Nicolas, C. Rabourdin-Combe, and B. Horvat. 2001. Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14:69-79.
Massé, N., M. Ainouze, B. Néel, T. F. Wild, R. Buckland, and J. P. Langedijk. 2004. Measles virus (MV) hemagglutinin: evidence that attachment sites for MV receptors SLAM and CD46 overlap on the globular head. J. Virol. 78:9051-9063.
Mathura, V. S., K. V. Soman, T. K. Varma, and W. Braun. 2003. A multimeric model for murine anti-apoptotic protein Bcl-2 and structural insights for its regulation by post-translational modification. J. Mol. Model. 9:298-303.
Palosaari, H., J.-P. Parisien, J. J. Rodriguez, C. M. Ulane, and C. M. Horvath. 2003. STAT protein interference and suppression of cytokine signal transduction by measles virus V protein. J. Virol. 77:7635-7644.
Sanner, M., A. Widmer, H. Senn, and W. Braun. 1989. GEOM: a new tool for molecular modelling based on distance geometry calculations with NMR data. J. Comput.-Aided Mol. Des. 3:195-210.
Santiago, C., E. Bjorling, T. Stehle, and J. M. Casasnovas. 2002. Distinct kinetics for binding of the CD46 and SLAM receptors to overlapping sites in the measles virus hemagglutinin protein. J. Biol. Chem. 277:32294-32301.
Schaumann, T., W. Braun, and K. Wuthrich. 1990. The program FANTOM for energy refinement of polypeptides and proteins using a Newton-Raphson minimizer in torsion angle space. Biopolymers 29:679-694.
Schmid, E., A. Zurbriggen, U. Gassen, B. Rima, V. ter Meulen, and J. Schneider-Schaulies. 2000. Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion. J. Virol. 74:7554-7561.
Schneider, U., V. von Messling, P. Devaux, and R. Cattaneo. 2002. Efficiency of measles virus entry and dissemination through different receptors. J. Virol. 76:7460-7467.
Seki, F., N. Ono, R. Yamaguchi, and Y. Yanagi. 2003. Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells. J. Virol. 77:9943-9950.
Sidorenko, S. P., and E. A. Clark. 1993. Characterization of a cell surface glycoprotein IPO-3, expressed on activated human B and T lymphocytes. J. Immunol. 151:4614-4624.
Soman, K. V., C. H. Schein, H. Zhu, and W. Braun. 2001. Homology modeling and simulations of nuclease structures. Methods Mol. Biol. 160:263-286.
Stern, L. B.-L., M. Greenberg, J. M. Gershoni, and S. Rozenblatt. 1995. The hemagglutinin envelope protein of canine distemper virus (CDV) confers cell tropism as illustrated by CDV and measles virus complementation analysis. J. Virol. 69:1661-1668.
Sutter, G., M. Ohlmann, and V. Erfle. 1995. Non-replicating vaccinia vector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett. 371:9-12.
Takimoto, T., K. G. Murti, T. Bousse, R. A. Scroggs, and A. Portner. 2001. Role of matrix and fusion proteins in budding of Sendai virus. J. Virol. 75:11384-11391.
Tatsuo, H., N. Ono, K. Tanaka, and Y. Yanagi. 2000. SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893-897.
Tatsuo, H., N. Ono, and Y. Yanagi. 2001. Morbilliviruses use signaling lymphocyte activation molecules (CD150) as cellular receptors. J. Virol. 75:5842-5850.
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.
von Messling, V., D. Milosevic, and R. Cattaneo. 2004. Tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system. Proc. Natl. Acad. Sci. USA 101:14216-14221.
von Messling, V., C. Springfeld, P. Devaux, and R. Cattaneo. 2003. A ferret model of canine distemper virus virulence and immunosuppression. J. Virol. 77:12579-12591.
von Messling, V., G. Zimmer, G. Herrler, L. Haas, and R. Cattaneo. 2001. The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity. J. Virol. 75:6418-6427.
Yamanouchi, K. 1980. Comparative aspects of pathogenicity of measles, canine distemper, and rinderpest viruses. Jpn. J. Med. Sci. Biol. 33:41-66.(Veronika von Messling, Nu)