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Journal of Virology, September 2004, p. 9666-9674, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9666-9674.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Mechanism of CD150 (SLAM) Down Regulation from the Host Cell Surface by Measles Virus Hemagglutinin Protein

G. Grant Welstead, Eric C. Hsu, Caterina Iorio, Shelly Bolotin, and Christopher D. Richardson^*

Department of Medical Biophysics, University of Toronto, and Ontario Cancer Institute, Toronto, Canada

Received 25 March 2004/ Accepted 10 May 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Measles virus has been reported to enter host cells via either of two cellular receptors, CD46 and CD150 (SLAM). CD46 is found on most cells of higher primates, while SLAM is expressed on activated B, T, and dendritic cells and is an important regulatory molecule of the immune system. Previous reports have shown that measles virus can down regulate expression of its two cellular receptors on the host cell surface during infection. In this study, the process of down regulation of SLAM by measles virus was investigated. We demonstrated that expression of the hemagglutinin (H) protein of measles virus was sufficient for down regulation. Our studies provided evidence that interactions between H and SLAM in the endoplasmic reticulum (ER) can promote the down regulation of SLAM but not CD46. In addition, we demonstrated that interactions between H and SLAM at the host cell surface can also contribute to SLAM down regulation. These results indicate that two mechanisms involving either intracellular interactions between H and SLAM in the ER or receptor-mediated binding to H at the surfaces of host cells can lead to the down regulation of SLAM during measles virus infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Measles virus is a major killer of children in the developing countries of Africa and South America. It is currently estimated that 44 million children are infected each year and that about 1 million of these individuals succumb to the disease or to secondary infections (2, 46). Inhibitory effects of measles virus on the immune system have been documented for decades, but a clear mechanism for this phenomenon has eluded investigators (5, 14, 44, 47).

Several laboratories, including ours, have reported that CD150 (SLAM) is a receptor for measles virus (11, 18, 41). SLAM is a 70-kDa type I transmembrane glycoprotein found on activated T, B, and dendritic cells. SLAM has structural features that place it within the CD2 family, which includes CD2, CD48, CD58, 2B4, and Ly-9. Like other members of the CD2 family, SLAM has two extracellular domains, an N-terminal V-set domain and a membrane-proximal C2-set domain. Following the C2 domain is the transmembrane segment and a cytoplasmic tail, which contains four potential phosphorylation sites, three of which are located in consensus SH2 docking sites (3, 32). SLAM is a homophilic molecule that self-associates with very low affinity, and, to date, it is believed that SLAM is its own ligand (10, 28, 42). Several functions have been attributed to SLAM. In general, it seems that SLAM is a modifier of T- or B-cell signals, with the final outcome of this modification dependent on the cell type, i.e., T cell or B cell, and the character of the initial signal (4, 6, 7, 16, 29, 35, 39). SLAM is expressed solely on activated lymphocytes and seems to play a role in maintaining a balance between a Th1 and Th2 immune responses. Measles virus infection may affect SLAM function and potentially cause the Th1-to-Th2 shift observed over the course of measles virus infection. It has been shown that an effective immune response to viral infections requires a repertoire of Th1 cells (24). To determine whether SLAM plays a role in the process of measles virus-induced immunosuppression, the effect of infection on SLAM expression and signaling is currently being examined in our laboratory.

In the present study, the down regulation of SLAM surface expression during measles virus infection was characterized. Using a vaccinia virus expression system, we showed that the presence of the H protein of measles virus was sufficient to reduce SLAM expression. It was also determined that two mechanisms are involved in H-induced down regulation of SLAM. First, biochemical analysis of SLAM in measles virus-infected cells indicated that SLAM transport to the cell surface could be inhibited by a specific interaction between H and SLAM in the endoplasmic reticulum (ER). Studies involving a mutant H protein that was expressed only in the ER confirmed that an ER retention mechanism was involved in H-induced down regulation of SLAM surface expression. We also showed that interactions between H and SLAM at the surface of infected cells could lead to SLAM down regulation and that the two mechanisms worked together to promote SLAM down regulation during infection.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell lines and viruses. Marmoset B95-8 cells were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen cultured-cell collection (Braunschweig, Germany), and human 1A2 B lymphoma cells were obtained from the Ontario Cancer Institute cell repository (Toronto, Canada). B95-8 cells were grown in RPMI 1640 (GIBCO/BRL) supplemented with 10% fetal calf serum. 1A2 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum and sodium pyruvate. Sf9 insect cells were grown in Grace's insect medium supplemented with 10% fetal calf serum. The Edmonston strain of measles virus was originally obtained from Erling Norrby (Karolinska Institute, Stockholm, Sweden) and was cultivated in Vero monkey kidney cells as previously described (13). The Montefiore 89 strain of measles virus (lymphotropic or wild-type strain) was obtained from Ilya Spigland and Amy Fox (Montefiore Medical Center, Bronx, N.Y.) and was amplified in B95-8 cells as previously described (21). The baculovirus expressing the H protein was previously made in our laboratory (17, 43).

Antibodies and glycosidases. Monoclonal antibodies specific for CD150 were purchased from Advanced ImmunoChemical (clone IPO-3; Long Beach, Calif.) and Pharmingen (clone A12). The monoclonal antibody specific for CD46 (clone M75) was purchased from Seikagaku Corporation (Tokyo, Japan). Monoclonal antibodies recognizing H were purchased from Chemicon International (Temecula, Calif.). Rabbit polyclonal antibodies directed against H and CD46 were produced in our laboratory as previously described (36). In addition, horseradish peroxidase-conjugated antibodies and fluorescein isothiocyanate (FITC)-conjugated antibodies were purchased from Jackson Laboratories (West Grove, Pa.). Endoglycosidase H (Endo H) and N-glycosidase F (PNGaseF) were purchased from New England Biolabs (Beverly, Mass.) and used according to the manufacturer's instructions. Rat anti-influenza hemagglutinin peptide tag (HA) affinity matrix and rat anti-HA-peroxidase antibodies were purchased from Roche Molecular Biochemicals. Rabbit anti-HA antibodies were purchased from Sigma-Aldrich (St. Louis, Mo.).

Flow cytometry analysis of SLAM, CD46, and H molecules. Infected or noninfected 1A2 and B95-8 cells (2 x 10⁶ cells/antibody incubation) were harvested, washed twice by centrifugation with fluorescence automated cell scanning (FACS) buffer (phosphate-buffered saline [PBS] containing 1% bovine serum albumin, 5 mM EDTA, and 0.1% sodium azide). The cells were then incubated with a 1:100 dilution of monoclonal antibodies against human SLAM, human CD46, or measles H protein for 30 min on ice. The cells were washed and incubated on ice with FITC-labeled goat anti-mouse immunoglobulin G (IgG; heavy plus light chains) secondary antibodies for 30 min. Just before analysis, the cells were washed and resuspended in 0.5 ml of FACS buffer, and the assays were performed on a Becton Dickinson analyzer using FACScaliber software. Intracellular staining of the measles virus H protein (MVH) was performed with a BD Cytofix/Cytoperm kit (BD Biosciences Pharmingen) by paraformaldehyde fixation and saponin permeabilization.

Production of recombinant vaccinia viruses. Edmonston F, Edmonston H, Montefiore 89 H, and Edmonston H_ER proteins were cloned into the vaccinia virus vector pSC11 by using the SmaI cloning site. The measles virus proteins were amplified by PCR from cDNA of Vero or B95-8 cells infected with either Edmonston or Montefiore 89, respectively. The resulting PCR products were digested with SmaI and inserted into pSC11. Edmonston H_ER was cut out of the plasmid pCG-H_ER, which was kindly provided by R. Cattaneo, with PacI and inserted into pSC11. To produce recombinant vaccinia viruses, 143B Tk- cells were transfected with one of the four respective plasmids, pSC11-EdF, pSC11-EdH, pSC11-wtH, or pSC11-H_ER, followed by infection with wild-type vaccinia virus. Resulting virus supernatants were plaque purified three times and then amplified in 143B Tk- cells in the presence of 0.015 mg of bromodeoxyuridine/ml. Expression of the F or H protein by the recombinant viruses was analyzed by immunoblotting or FACS analysis.

Virus infections. 1A2 or B95-8 cells (6 x 10⁶ cells for each time point) were infected with either Edmonston or Montefiore 89 measles virus at a multiplicity of infection (MOI) of 5. At specified time points postinfection (p.i.), the cells were harvested and subjected to FACS analyses. 1A2 cells were infected with either wild-type vaccinia virus or recombinant vaccinia virus expressing Edmonston F, Edmonston H, Montefiore 89 H, or Edmonston H_ER at a MOI of 10. The cells were harvested 24 h p.i., and FACS analyses were performed. Sf9 cells were infected at a MOI of 1 for 24 h.

Immunoprecipitation experiments. Transfected and infected 293Tad cells were harvested, washed once with PBS, and resuspended in cell lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.3% NP-40, and protease inhibitors). After a 30-min incubation period at 4°C, the lysed cells were centrifuged at 10,000 x g for 10 to 15 min at 4°C. The supernatant was precleared through incubation with previously washed protein G-conjugated beads for 1 to 2 h at 4°C. After a 5-min centrifugation at 10,000 x g, 30 µl of anti-HA affinity matrix (Roche) or 10 µl of anti-measles virus H monoclonal antibody (Chemicon) or 10 µl of an anti-CD46 monoclonal antibody (Seikagaku) was added to the supernatant, which was incubated overnight at 4°C. After incubation, protein G was added to the lysate, which contained the H or CD46 antibody, and incubated for 1 to 2 h at 4°C. Either the HA affinity tag matrix or the protein G-measles virus H or protein G-CD46 beads were centrifuged and washed with lysis buffer three to five times by centrifugation. The beads were then resuspended in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and boiled for 5 min. After a 5-min spin, the supernatant was subjected to PAGE. The primary antibody was either an anti-HA antibody conjugated to horseradish peroxidase (clone 3F10; Roche), a polyclonal H antibody, or a polyclonal CD46 antibody.

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD150 (SLAM) surface expression is down regulated during measles virus infection. Epstein-Barr virus (EBV)-transformed human B-cell line 1A2 and activated marmoset B-cell line B95-8 express SLAM on their surfaces and can be infected by the Montefiore 89 (wild-type) strain and the Edmonston strain (vaccine) of measles virus. To determine whether SLAM expression is modulated after infection, FACS analysis of SLAM surface expression was performed at 24 and 48 h p.i. Infection by Montefiore 89 measles virus (Fig. 1A and C) and Edmonston measles virus (Fig. 1B and D) caused a substantial decrease in SLAM cell surface expression in both marmoset and human B-cell lines. The decrease of SLAM was evident by at least 24 h p.i. and was substantial at 48 h p.i. The reduction of SLAM surface expression is characteristic and specific to measles virus infection, since vaccinia virus infection did not lead to a decrease in SLAM expression (data not shown). In a control experiment, CD46 surface expression on 1A2 cells, following infection with measles virus, was also studied. Expression of CD46 was assayed at 24 and 48 h p.i. by FACS analysis. As previously reported, only Edmonston measles virus infection led to reduction of CD46 on the surfaces of 1A2 cells (Fig. 1F) (23, 37, 38), but CD46 expression was not altered during Montefiore 89 infection (Fig. 1E).

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FIG. 1. The surface expression of SLAM/CD150 is down regulated by measles virus infection. (A) Activated marmoset B-cell line B95-8 was infected with Montefiore measles virus. At 24 and 48 h p.i., SLAM surface expression was analyzed by FACS. (B) Marmoset B95-8 cells were infected with Edmonston measles virus. At 24 and 48 h p.i., SLAM surface expression was analyzed by FACS. (C) EBV-transformed human B-cell line 1A2 was infected with Montefiore measles virus. At 24 and 48 h p.i., SLAM expression was analyzed by FACS. (D) Human 1A2 cells were infected with Edmonston measles virus. At 24 and 48 h p.i., SLAM expression was determined by FACS. (E) Human 1A2 cells were infected with Montefiore measles virus. At 24 and 48 h p.i., CD46 expression was analyzed by FACS. (F) Human 1A2 cells were infected with Edmonston measles virus. At 24 and 48 h p.i., CD46 expression was analyzed by FACS. Grey lines, mock-infected cells stained with the anti-SLAM antibody (A to D) or anti-CD46 antibody (E and F) and detected with the FITC-conjugated goat anti-mouse antibody; black lines, mock-infected cells incubated with the FITC-conjugated goat anti-mouse secondary antibody only; solid peaks, cells infected with Montefiore (A, C, and E) or Edmonston (B, D, and F) measles virus stained with the anti-SLAM antibody (A to D) or the anti-CD46 antibody (E and F), followed by an FITC-conjugated goat anti-mouse antibody. Insets, levels of H protein expression on the surfaces of B95-8 and 1A2 cells infected with Montefiore 89 and Edmonston strains of measles virus following 48 h of incubation. The cells were stained with anti-measles H antibody, followed by FITC-conjugated goat anti-mouse secondary antibody. The solid lines represent infected cells; the dashed lines represent mock-infected cells.

The expression of measles virus H protein is sufficient to mediate the down regulation of SLAM. To determine whether H by itself could cause the down regulation of SLAM and CD46, recombinant vaccinia viruses that expressed the H protein of either the Montefiore 89 (vaccinia-WtH) or the Edmonston strain (vaccinia-EdH) of measles virus were utilized in subsequent experiments. Wild-type vaccinia virus and recombinant vaccinia virus expressing the F protein (vaccinia-F) of measles virus were used as controls. 1A2 cells were infected with wild-type vaccinia virus, vaccinia-F, vaccinia-WtH, or vaccinia-EdH. At 24 h p.i., the cells were harvested and SLAM (Fig. 2A) or CD46 (Fig. 2B) surface expression was subjected to FACS analysis. Analogous to our results with wild-type measles virus, vaccinia-WtH infection caused the down regulation of SLAM on the surfaces of 1A2 cells but not the down regulation of CD46. Vaccinia-EdH infection resulted in the down regulation of both SLAM and CD46, which agreed with our results obtained using Edmonston measles virus. Furthermore, wild-type vaccinia virus or vaccinia-F did not down regulate SLAM or CD46. Together, these experiments indicated that H alone was sufficient to cause the decrease in surface expression of both cellular receptors during measles virus infections.

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FIG. 2. The expression of the measles virus H protein alone can down regulate surface expression of SLAM and CD46. Human 1A2 B cells were infected with vaccinia virus recombinants that expressed Edmonston F (Ed F), Edmonston H (Ed H), or Montefiore H (Wt H) proteins. FACS analyses of SLAM surface expression (A) and CD46 surface expression (B) were performed. Black line, 1A2 cells infected with wild-type vaccinia virus (VV) incubated with an FITC-conjugated goat anti-mouse secondary antibody; gray line, 1A2 cells infected with wild-type vaccinia virus stained with a mouse anti-SLAM antibody (A) or mouse anti-CD46 antibody (B) and detected with an FITC-conjugated goat anti-mouse antibody; solid peak, 1A2 cells infected with the indicated vaccinia virus recombinants, stained with a mouse anti-SLAM antibody (A) or mouse anti-CD46 antibody (B), and detected with an FITC-conjugated goat anti-mouse secondary antibody. Infected 1A2 cells were stained for surface expression of the F and H proteins (black line) with a rabbit polyclonal antibody specific for the F protein and a monoclonal antibody directed against measles virus H proteins (C). The dashed lines represent wild-type vaccinia virus-infected cells probed for expression of the F and H proteins.

Measles virus H protein and SLAM interact in the infected cell. To determine whether measles virus H and SLAM interact in an infected cell, immunoprecipitation experiments were performed. Since a commercial antibody that can recognize SLAM on immunoblots was not available, 293Tad cells transfected with a construct that expressed SLAM fused to an HA epitope were used. The presence of the HA tag at the C terminus of SLAM did not affect the protein's function as a measles virus receptor (data not shown). A chimeric protein consisting of the V region of CD48 and the C2, transmembrane, and cytoplasmic tail of SLAM was also tagged at its C terminus. Since the binding site of H is situated in the V region of SLAM, this chimera does not function as a measles virus receptor and was used as a negative control during the immunoprecipitation experiments (32). 293Tad cells were transfected with vector alone, chimeric SLAM-HA, or SLAM-HA. The transfected cells were infected with either vaccinia virus or Edmonston measles virus 24 h posttransfection. Approximately 1 day after infection, the cells were harvested and lysed. Cell lysates were divided into two samples and immunoprecipitated with either a monoclonal anti-measles virus H antibody or anti-HA affinity matrix. The immunoprecipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal anti-H or anti-HA tag antibody. As expected, the anti-HA tag was able to bind the chimeric SLAM-HA and SLAM-HA proteins in both vaccinia virus- and measles virus-infected cells (Fig. 3A). In measles virus-infected cells, H coprecipitated with SLAM-HA but not with chimeric SLAM-HA (Fig. 3A). Conversely, in measles virus-infected cell lysates that were incubated with anti-measles virus H antibody, SLAM-HA but not chimeric SLAM-HA coprecipitated with measles virus H (Fig. 3B). These results indicated that the H and SLAM proteins interact in cells infected with measles virus.

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FIG. 3. Measles virus H protein and its receptor SLAM interact within the infected cell. Human 293Tad kidney cells were transfected with pcDNA3.1-HA3, pcDNA3.1-CD48SLAM-HA3, and pcDNA3.1-SLAM-HA3 expression vectors. At 36 h posttransfection, cells were infected with vaccinia control virus or measles virus. Following a 24-h infection, the cells were harvested and lysed. Proteins were immunoprecipitated (IP) with either a rat anti-HA tag antibody (3F10) affinity matrix or a mouse monoclonal anti-measles virus H antibody (MVH). (A) The samples were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with a peroxidase-conjugated anti-HA tag antibody. (B) The samples were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with a rabbit polyclonal anti-measles virus H antibody. Arrows, measles virus H protein; asterisk, bands for rat IgG (3F10) heavy chain recognized by the goat anti-rabbit secondary antibody.

Expression of a mutant H protein that is expressed only in the ER results in the reduction of SLAM surface expression. There are two mechanisms by which H could reduce the surface expression of SLAM. The first process involves SLAM-H interaction on the surface of an infected cell to induce endocytosis of SLAM followed by its subsequent degradation in lysosomes. The second mechanism involves a specific interaction between SLAM and H in the ER that prevents the export of newly synthesized SLAM from the ER. A standard technique used to determine whether a glycosylated protein resides in the ER involves studying its sensitivity to Endo H digestion. Endo H is a glycosidase that can cleave only the high-mannose forms of a glycosylated protein, which are located solely in the ER. Thus, sensitivity of a protein to Endo H is indicative of its location in the ER. These digested glycoproteins migrate faster on SDS-PAGE gels than untreated, partially digested, or Endo H-resistant forms of the protein. Another glycosidase, PNGaseF, cleaves all N-linked glycosylated proteins and was used as a control for complete Endo H digestion.

To assess whether an ER retention mechanism contributes to the reduction of SLAM surface expression, a mutant Edmonston H protein that is translated and retained in the ER was expressed in SLAM⁺ cells. This mutant H (H_ER) contains five arginines directly after the initiating methionine and is retained in the ER (34). A recombinant vaccinia virus expressing H_ER was constructed (vaccinia-H_ER), and expression of H in cells infected with this virus was restricted to the ER (Fig. 4F and G). The surface expression of SLAM on 1A2 cells was monitored via FACS analysis, 40 h after vaccinia-H and vaccinia-H_ER infection, with monoclonal antibodies directed against SLAM and H. In the presence of either H or H_ER, SLAM surface expression was reduced (Fig. 4A and B). However, the decrease in surface expression of SLAM due to H_ER was less than that caused by expression of Edmonston H (Fig. 4A and B). A similar analysis of CD46 expression showed that Edmonston H can cause the down regulation of CD46 surface expression but that the ER-retained version did not (Fig. 4B and D). Although Edmonston H migrated normally to the host cell plasma membrane, there was no expression of H_ER on the surfaces of these cells. FACS analysis of intracellular staining with monoclonal antibodies directed against H shows that H_ER was expressed inside the cell, as expected (Fig. 4E and F). When H_ER is expressed, only the Endo H-sensitive form of H was evident, which is indicative of its location in the ER (Fig. 4G).

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FIG. 4. Expression of a measles virus H that is retained in the ER induces SLAM down regulation. Human 1A2 B cells were infected with normal vaccinia virus, the vaccinia-EdH recombinant virus (V-EdH), or the vaccinia-EdHER recombinant virus for 36 h. These samples were subsequently split into two samples and analyzed by FACS and immunoblot detection. (A) FACS analysis of SLAM expression on 1A2 cells infected for 36 h with vaccinia-EdH. (B) FACS analysis of SLAM expression on 1A2 cells infected for 36 h with vaccinia-EdHER. (C) FACS analysis of CD46 expression on 1A2 cells infected for 36 h with vaccinia-EdH. (D) FACS analysis of CD46 expression on 1A2 cells infected for 36 h with vaccinia-EdHER. (E) FACS analysis of H expression on 1A2 cells infected for 36 h with vaccinia-EdH. Inset, intracellular H staining of permeabilized cells with a monoclonal antibody specific for the H protein. (F) FACS analysis of H expression on 1A2 cells infected for 36 h with vaccinia-EdHER. Inset, intracellular H staining of permeabilized cells with a monoclonal antibody specific for the H protein. (G) Immunoblot analysis of measles virus H and HER expression in 1A2 cells. The 1A2 cells were lysed in SDS protein running buffer containing ß-mercaptoethanol and incubated with or without Endo H for 1 h at 37°C. The samples were then subjected to SDS-PAGE and probed with a rabbit polyclonal anti-measles virus H primary antibody and detected with a peroxidase-conjugated goat anti-rabbit secondary antibody by enhanced chemiluminescence. Arrows, protein products derived from Endo H-resistant and Endo H-sensitive bands. The H protein that is retained in the ER (HER) is completely sensitive to Endo H digestion.

To clarify these findings, the protein turnover rates of SLAM and CD46 on the surfaces of 1A2 cells were compared by using the drug tunicamycin. This reagent inhibits the glycosylation of newly synthesized protein and prevents proper expression of glycoproteins on the surface of the cell (1). 1A2 cells were treated with tunicamycin, and it was determined that the half-life of SLAM on the surfaces of 1A2 cells was between 24 and 36 h (Fig. 5). The half-life of CD46 on the cell surface was found to be greater than 36 h. This slower protein turnover rate could explain the lack of down regulation of CD46 that was evident at 40 h after infection with vaccinia-H_ER. To look at CD46 at a later time following expression with H_ER, 293Tad cells were transfected with H and H_ER. At 72 h posttransfection, CD46 was down regulated by H but not by H_ER, indicating that CD46 expression at the cell surface is not affected by H_ER expression (data not shown). Thus, SLAM but not CD46 expression is partially down regulated by a specific interaction between H and SLAM in the ER.

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FIG. 5. SLAM has a more rapid cell surface turnover rate than CD46. Human 1A2 B-cell lymphoma cells were treated with tunicamycin, which inhibits N-linked glycosylation and migration of newly synthesized SLAM to the cell surface. At the times indicated, FACS analyses of SLAM and CD46 expression were performed. The mean fluorescence intensity was measured by gating on live cells and was expressed as the relative percentage of steady-state receptor expression on 1A2 cells (0 h). The data shown are the averages of three independent experiments, with the error bars representing the standard deviations.

Measles virus H protein expression inhibits the formation of complex glycosylated variants of SLAM but not of CD46. To further describe the ER retention mechanism involved in SLAM down regulation, SLAM's ability to migrate from the ER to the surface of a cell in the presence or absence of H was studied. 293Tad cells were cotransfected with the plasmids pcDNA3.1-SLAM-HA and either pCG, pCG-H, or pCG-H_ER. Thirty-six hours after transfection, the cells were lysed and immunoprecipitated with anti-H, anti-CD46, or anti-HA tag. Subsequently, the samples were incubated with Endo H or PNGaseF and then subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an anti-H, anti-CD46, or anti-HA antibody. Expression of SLAM-HA resulted in variants that are Endo H resistant and Endo H sensitive. However, the Endo H-resistant form of SLAM was not apparent when SLAM was expressed in the presence of H and H_ER (Fig. 6A). While H_ER expression completely inhibited SLAM transport from the ER, expression of H led to a partially Endo H-resistant band that is not expressed on the cell surface. Furthermore, SLAM coprecipitated with both H and H_ER (Fig. 6C and D). These data indicate that SLAM migration from the ER is impaired in cells that express H or H_ER and that this contributes to the down regulation of SLAM. Conversely, transport of CD46 from the ER to the cell surface was not affected by the expression of H or H_ER (Fig. 6B). In the presence or absence of H or H_ER expression, CD46 remained resistant to Endo H digestion. In addition, CD46 did not coprecipitate with H or H_ER (Fig. 6E and F). These results suggest that H expression can inhibit SLAM but not CD46 transport to the cell surface and that it may act as a mechanism for SLAM down regulation.

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FIG. 6. The presence of the measles virus H (MVH) protein in the ER slows or prevents the complex glycosylation of SLAM but not CD46. Human 293Tad embryonic kidney cells were transfected with pcDNA3.1-SLAM-HA3 and either pCG, pCG-H, or pCG-HER. At 36 h posttransfection, cells were lysed and immunoprecipitated with anti-H, anti-CD46, or anti-HA tag antibodies. The resulting samples were left untreated (–) or treated with Endo H or PNGaseF (+). The samples were subjected to SDS-PAGE; transferred to nitrocellulose; probed with an anti-HA antibody, anti-H antibody, or anti-CD46 antibody; and detected with a peroxidase-conjugated secondary antibody by enhanced chemiluminescence. (A) Lysates were immunoprecipitated (IP) with an anti-HA antibody, and the blots were probed with an anti-HA tag antibody. Expression of H or HER inhibits complex glycosylation of SLAM and maintains its sensitivity to Endo H. (B) Lysates were immunoprecipitated with a mouse monoclonal anti-CD46 antibody that recognized endogenous CD46. The blots were probed with a rabbit polyclonal anti-CD46 antibody. Expression of H or HER did not inhibit the complex glycosylation of CD46, and the glycoprotein exhibited little or no sensitivity to Endo H. (C) Lysates were immunoprecipitated with a mouse monoclonal anti-MVH antibody, and the blots were probed with an anti-HA antibody. The SLAM receptor coprecipitates with MVH proteins, and HER expression prevents the complex glycosylation of SLAM and maintains its sensitivity to Endo H. (D) Lysates were immunoprecipitated with an anti-HA antibody, and the blots were probed with a rabbit polyclonal anti-MVH antibody. MVH coprecipitates with SLAM and is partially sensitive to Endo H. (E) 293Tad cells were transfected with only pCG, pCG-H, or pCG-HER. Lysates were immunoprecipitated with a mouse monoclonal anti-MVH antibody, and the blots were probed with a rabbit polyclonal anti-CD46 antibody as in panel B. Endogenous CD46 does not coprecipitate with MVH. (F) 293Tad cells were transfected with only pCG, pCG-H, or pCG-HER. Lysates were immunoprecipitated with a mouse monoclonal anti-CD46 antibody, and the blots were probed with a rabbit polyclonal anti-MVH antibody as in panel D. MVH does not coprecipitate with endogenous CD46.

Interactions between SLAM and H at the surfaces of cells can also reduce SLAM expression. After it was established that an ER retention mechanism was important in the process of SLAM down regulation, the role of SLAM-H interactions at the surfaces of SLAM-expressing cells was also considered. 1A2 cells were cocultured in trans with CHOP cells that expressed H, Edmonston H_ER, or vector alone. The surface expression of SLAM and CD46 was analyzed 24 h after coincubation of the two types of cells. The surface expression of both SLAM and CD46 was down regulated by coincubation with CHOP-H cells but not CHOP-Edmonston H_ER or CHOP-vector cells (Fig. 7A).

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FIG. 7. Down regulation of SLAM and CD46 from the surfaces of 1A2 cells results from coincubation with Sf9 insect and Chinese hamster ovary (CHOP) cells that express measles virus H on their surfaces. (A) CHOP cells were transfected with pcDNA1.1-H. At 24 h after transfection, the CHOP cells were washed once with PBS, and 5 x 105 1A2 cells were added. FACS analysis of SLAM and CD46 expression on 1A2 cells was performed at 0 (gray line) and 24 h (solid peak) after coincubation. FACS analysis of H expression on the CHOP cells was also performed in the right graph using a monoclonal antibody that recognizes measles virus H. Gray line, cells transfected with pcDNA1.1; solid peak, cells transfected with pcDNA1.1-H. 1A2 cells were stained and gated for CD21, a marker specific for B cells. CHOP cells that express Edmonston H on their surfaces down regulate expression of CD46 and SLAM from the surfaces of human 1A2 cells. (B) Sf9 insect cells were infected with wild-type baculovirus or a recombinant baculovirus that expresses the Edmonston H protein. At 18 h after infection, the Sf9 cells were added to 1A2 cells at the ratios indicated. FACS analysis of SLAM expression on the 1A2 cells and H expression on the Sf9 cells was performed 24 h after coincubation. In the left column, the black line represents 1A2 cells stained with a goat anti-mouse secondary antibody, the gray line represents 1A2 cells coincubated with Sf9 cells infected with wild-type baculovirus and stained with mouse anti-human SLAM, and the solid peak represents 1A2 cells coincubated with Sf9 cells expressing H and stained with mouse anti-human SLAM. In the right column, the solid peak represents Sf9 insect cells expressing H protein stained with mouse anti-measles virus H and the gray line represents Sf9 insect cells infected with wild-type baculovirus stained with mouse anti-measles virus H.

To determine whether SLAM down regulation was dependent on the levels of H on the cell surface, 1A2 cells were cocultured with Sf9 insect cells that expressed Edmonston H. Sf9 cells expressing H (Sf9-H) were added to 1A2 cells at ratios of 1:1, 3:1, and 5:1. After 36 h of incubation, the Sf9-H-1A2 cell mixtures were harvested and FACS analyses of SLAM and H expression were performed (Fig. 7B). SLAM expression was reduced in the presence of Sf9-H cells, and this decrease was dependent on the amount of Sf9-H cells added. The reduction of SLAM expression was similar to that seen on cells infected by vaccinia-EdH_ER but less than that observed on cells infected by vaccinia-EdH. These results seem to indicate that both mechanisms contribute to the down regulation of SLAM from the cell surface.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, the process of down regulation of CD150 (SLAM) from the cell surface by measles virus was characterized. We confirmed previous reports that measles virus induced down regulation of SLAM (11, 40) and demonstrated that expression of the H protein of measles virus is sufficient for this effect. We provided biochemical data that indicate that an ER retention mechanism is involved in this process and confirmed this finding through the use of H that is retained in the ER. We also showed that interactions between H and SLAM at the surface also contribute to SLAM down regulation.

Several different viruses including human immunodeficiency virus (HIV), EBV, and measles virus can down regulate the surface expression of their cellular receptors during the process of infection (9, 15, 27, 33, 37, 38, 40). There are two major mechanisms by which this phenomenon can occur. These viruses can induce changes in the cell that cause increased internalization of the receptor (8, 15, 27, 48). Alternatively, the cellular receptor can be retained in the ER of an infected cell, thereby inhibiting the efficient transport of the cellular receptor to the cell surface (9, 19). For HIV, the main purpose of CD4 down regulation seems to be to enhance HIV replication and prevent superinfection of the cell (22, 25, 33). As a result, the virus utilizes both mechanisms to down regulate CD4 during infection. Reports indicate that the HIV-encoded protein Nef causes the rapid endocytosis of CD4, resulting in the degradation of the receptor in lysosomes (22, 25-27). It has also been reported that CD4 interacts with the gp160 protein of HIV in the ER of HIV-infected cells and that this interaction can inhibit the transport of CD4 to the cell surface (9, 19).

Unlike what is found for HIV, viral accessory proteins are not involved in measles virus receptor down regulation. The H protein solely mediates CD46 and SLAM down regulation. For measles virus, it has been shown that the surface expression of the cellular receptor CD46 can be down regulated by the expression of the H protein on adjacent cells. This process seems to require a Tyr-X-X-Leu motif in the membrane-proximal region of CD46 (15, 48). To date, the role that an ER retention mechanism plays in down regulation of measles virus receptors has not been considered.

To determine whether an ER retention mechanism plays a role in SLAM down regulation, a vaccinia virus that expressed an ER-retained H protein (vaccinia-H_ER) was constructed. Expression of H protein by both vaccinia-EdH and vaccinia-H_ER reduced SLAM surface expression in 1A2 cells. Surprisingly, the decrease observed with vaccinia-H_ER was less than that seen when cells were infected with vaccinia-EdH. This could be explained by the fact that both ER retention and receptor-mediated endocytosis mechanisms are functioning with vaccinia-EdH, yielding more-efficient down regulation of SLAM. In a parallel experiment, CD46 expression was unaffected following vaccinia-H_ER infection but was reduced by vaccinia-EdH infection. In addition, CD46 expression in 293Tad kidney cells transfected with pcDNA1.1-H_ER was unchanged, even at 72 h posttransfection. H_ER also did not coprecipitate with CD46 or inhibit its transport from the ER to the cell surface. Taken together, these data demonstrate that an ER retention mechanism plays a more important role in SLAM down regulation than in CD46 down regulation during measles virus infections.

H protein expression on CHOP and Sf9 cells in trans to adjacent 1A2 cells also led to the down regulation of both SLAM and CD46. However, this process did not yield total removal of SLAM from the cell surface. These data indicate that both ER retention and receptor-mediated endocytosis are likely involved in the down regulation of SLAM expression during measles virus infections. On the other hand, CD46 expression was reduced to a greater extent than that of SLAM during incubations with CHOP cells expressing H protein. This again suggests that ER retention plays a less important role than receptor-mediated endocytosis in CD46 down regulation. Future experiments will investigate the role and type of endocytosis involved in surface-mediated down regulation of SLAM and CD46. The processes of micropinocystosis, macropinocystosis, and caveola-dependent, clathrin-dependent, or caveola- and clathrin-independent endocytosis will be considered. Drugs such as chlorpromazine and dominant negative mutants could be used to dissect this process.

A major consequence of SLAM down regulation is the prevention of superinfection of the host cell by the incoming virus. Based on SLAM's known contributions to immune responses, there are several potential consequences of SLAM down regulation for host immunity. SLAM, a self-ligand, is expressed primarily on activated B cells, T cells, and dendritic cells (28). It is associated with a T-cell response that produces gamma interferon (IFN-) and interleukin-12 (IL-12), also known to define a Th1-type cytokine profile (4, 6, 12). Engagement of SLAM on T cells in conjunction with T-cell receptor activation results in cellular proliferation and IFN- production. It has also been demonstrated that cytotoxic-T-cell activity is induced under similar conditions. In B cells, it has been reported that SLAM stimulation induces cell proliferation and the secretion of Ig (35). Taken together, measles-induced down regulation of SLAM surface expression may have consequences for T-cell and B-cell proliferation and function. Interactions of measles virus with SLAM may help to establish the Th1-to-Th2 cytokine shift that is characteristic of infections with this virus. During measles, T-cell responses including IFN- and IL-12 production are reduced and IL-4 levels are elevated (5, 14, 45). Although it has previously been reported that interaction of H with CD46 may impair the immune response by decreasing levels of IL-12 (20), an effect on SLAM⁺ cells by measles virus may also explain the impaired Th1 phenotype observed in infected patients. Additional in vivo studies and an increased knowledge of SLAM/CD150 signal transduction pathways are required to determine whether down regulation of this receptor contributes to the crippled immune response that is characteristic of measles virus infections (44).

 
We thank Roberto Cattaneo and Richard Plemper for the pCG-H and pCG-H_ER constructs. The technical support of Denis Bouchard during FACS analysis was much appreciated. We thank the entire Richardson lab for helpful discussion during the course of this project.

The work presented in this article was supported by Canadian Institutes of Health Research grant MT-10638.

 
* Corresponding author. Mailing address: Ontario Cancer Institute, 620 University Ave., Suite 706, Toronto, Ontario, Canada M5G 2C1. Phone: (416) 946-2849. Fax: (416) 204-2278. E-mail: chrisr{at}uhnres.utoronto.ca.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, September 2004, p. 9666-9674, Vol. 78, No. 18
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.18.9666-9674.2004
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