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Mouse mammary tumor virus (MMTV) is transmitted as an infectious viral particle from a lactating mother to a suckling offspring via milk (2). B lymphocytes in the draining lymph nodes have been shown to be the primary targets for MMTV infection (7-9). After infection, expression of the viral superantigen (Sag) at the surface of B cells in association with major histocompatibility complex class II molecules leads to the activation of Sag-reactive T cells and Sag-mediated T-cell help (reviewed in reference 14). Contradictory findings were obtained about the nature of the cellular receptor of the gp52 surface (SU) glycoprotein of MMTV. Utilization of pseudotyped murine leukemia virus, vesicular stomatitis virus, and Kirsten sarcoma virus particles in tissue culture gave complex results concerning the nature of the MMTV receptor (1, 6, 10, 19). Either a restricted presence on mouse and rat cells (23) or a broad distribution on mouse, rat, cat, and mink cells (12, 13, 21) of the MMTV receptor has been reported. Furthermore, somatic-cell genetic studies have mapped the gene for the MMTV receptor to chromosome 16 but chromosomes 7 and 17 have also been postulated to be implicated in susceptibility to the virus (10). Recently, a novel membrane protein has been proposed as the MMTV receptor. The corresponding gene has been mapped to chromosome 19 (6). Northern blot analyses showed that the mRNA coding for this protein is ubiquitously expressed (6). In contrast, MMTV has been shown to infect only a limited range of cells in vivo (7, 8; reviewed in reference 14). Variable levels of receptor protein, requirements for coreceptors, or events after virus entry could, individually or together, explain some of these discrepancies. The use of Polybrene in the different infection protocols in tissue culture might be an explanation for the variable results obtained. Indeed, this compound favors the fusion of membranes and could therefore stabilize otherwise weak interactions between the gp52 protein and a low-affinity receptor molecule. In addition, all groups were able to only partially inhibit infection with a neutralizing antiserum. A likely explanation for the results of studies using pseudotypes is the presence at the surface of some envelope molecules of the parental virus, as the pseudotypes were made by coinfections. Furthermore, unrelated molecules might be carried by the pseudotyped virions that could, in theory, mediate unspecific uptake and lead to infection. To address the question of receptor expression on different target cells, we analyzed env binding on fresh lymphocytes.

The gp52 SU glycoprotein of MMTV has been shown to mediate the binding of the virus to the cellular receptor (reviewed in reference 14). The coding sequence from the envelope gene of MMTV(GR) was subcloned (3) upon addition of BamHI linkers in the pQE10 bacterial expression vector (QIAGEN Inc.) (Fig. 1A). Western blotting (Fig. 1B) was used to monitor MMTV SU protein expression. A clear and strong signal was observed at 46 kDa (lane 3), which corresponded to the expected molecular mass of the gp52 polypeptide including the histidine tag. No signal was observed in noninduced bacteria (lane 4) or in bacteria containing the plasmid with an insert in the reverse orientation (lanes 1 and 2). A second specific band was visible below 18.5 kDa. It could either result from internal initiation, as it did not carry the histidine tag (data not shown), or result from partial degradation of the main product. Since the attempts to purify the recombinant envelope protein under native conditions failed, purification done under denaturing conditions by following the manufacturer's (QIAGEN Inc.) instructions was achieved. To use the protein in our experiments, it was recovered from a sodium dodecyl sulfate (SDS)-11% polyacrylamide gel electrophoresis (PAGE) preparative gel, electroeluted at 4°C in TBT buffer (192 mM glycine, 25 mM Tris-HCl [pH 8], 0.005% SDS) for 4 h, buffer exchanged to phosphate-buffered saline (PBS), and concentrated by using a Centricon 30 filtration unit (Amicon Inc.). Two milligrams of purified recombinant envelope protein was consistently recovered from the initial bacterial lysate. Furthermore, the purification procedure was highly reproducible.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   (A) Scheme of the MMTV envelope coding sequence, composed of the leader sequence (ls), the surface glycoprotein gp52 (SU, gp52), and the transmembrane glycoprotein gp36 (TM, gp36). Also shown is the protein expressed in bacteria, i.e., the SU envelope protein gp52 fused at the N terminus to a histidine tag (6xHis). (B) Expression of gp52 in bacteria. Colonies containing the insert in the antisense (lanes 1 and 2) or sense (lanes 3 and 4) orientation were grown, induced (i) with IPTG or not induced, resolved by SDS-12% PAGE under reducing conditions, electrotransferred to nitrocellulose, and immunoblotted with a mouse monoclonal anti-gp52 antibody (22). The gp52 signal is indicated by the arrow. (C) Purified bacterial gp52. Two different batches of purified bacterial gp52 in PBS were resolved by SDS-12% PAGE under nonreducing conditions and stained with Coomassie blue.

We used carboxylate-modified fluorescein isothiocyanate (FITC)-labeled latex fluospheres (FS; Molecular Probes, Inc.) coupled to different proteins, such as fetal calf serum proteins (FCS), anti-immunoglobulin antibodies (Ig), anti-complement receptor antibodies (7G6; 11), or bacterial recombinant envelope gp52 (env), to perform binding studies (Fig. 2). One million ex vivo BALB/c mouse spleen cells were incubated in Dulbecco modified Eagle medium with either 3 × 10¹⁰ FS or the indicated amounts of biotinylated virus particles [see below; MM(MMTV) = 3.7 × 10⁸ g/mol (16)] in a total volume of 60 µl for 2 h on ice. The cells were washed twice in PBS supplemented with 3% FCS and stained with a mixture containing phycoerythrin (PE)-conjugated anti-CD4 and anti-CD8 PE antibodies (H129.19 and 53-6.7, respectively; Boehringer Mannheim) and an anti-B220 tricolor-conjugated antibody (RA3-6B2; Caltag) in 50 µl for 30 min on ice. FITC-conjugated streptavidin (Caltag) was added to the mixture when biotinylated virus was used, and then a washing step was performed (see below). FS have a diameter of 200 nm, similar to the diameter of MMTV particles (120 nm). They provide multivalent binding sites for potential target molecules, thereby increasing binding avidity. They display very strong fluorescence when analyzed by flow cytometry. This makes it possible to detect a single FS bound to target cells by fluorescence-activated cell sorter (FACS) analysis. Figure 2A shows the binding of protein-coated FS to mouse ex vivo spleen cells in a representative FACS analysis. The weakest FITC signal represents a single FS bound to a lymphocyte. Figure 2B shows the quantification of a representative experiment. Preferential and dose-dependent binding of env-coated FS to B cells (B220⁺) was seen with up to 35% positive B cells at the highest FS concentration (1/60 dilution). Sevenfold lower binding was observed on T cells (CD4⁺ and CD8⁺) at the same dilution, reflecting either a lower abundance of receptor molecules on T cells, the presence of a lower-affinity receptor on T cells, or background staining. The binding of FCS-coated FS to both types of cells was low (2.2 and 0.4%; Fig. 2B) and was used to determine the background of the method. Furthermore, the binding of either Ig- or 7G6-coated FS demonstrated the sensitivity of the method since at the highest dilution of FS tested (1/540), more than 65% of the B cells were stained (Fig. 2B). When higher doses of FS were used, nearly all of the B cells turned positive but the background with nonexpressing T cells started to increase (data not shown). The background observed with T cells which express neither surface immunoglobulins nor the complement receptors was very low (2.4 and 1.3%, respectively), indicating again the high specificity of the binding observed with those types of FS. Binding experiments using the same type of FS performed on sorted spleen cells confirmed the strong preference of env-, Ig-, or 7G6-coated FS for binding to B cells under the same experimental conditions (data not shown). Indeed, even when 98% pure T cells were used, the binding was never higher than 7%.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   (A) Representative FACS profiles obtained upon binding of env-coated FS to B and T cells. The percentage of positive cells based on marker 1 (M1) is indicated. (B) Preferential binding of gp52-coated FS to mouse ex vivo B cells. Various protein-coated FITC-FS were incubated at dilutions of 1/60, 1/180, and 1/540 with 106 ex vivo BALB/c mouse spleen cells. The cells were then stained with tricolor-conjugated anti-B220, PE-conjugated anti-CD4, and PE-conjugated anti-CD8 antibodies and analyzed by flow cytometry. FCS, FCS-coated FS; Ig, Ig-coated FS; 7G6, anti-complement receptor antibody-coated FS; gp52, gp52-coated FS. The results are representative of four experiments, and those of one experiment are shown (mean of three independent measurements ± the standard error of the mean).

To demonstrate that binding of the env-coated FS was, indeed, env mediated, we preincubated the different protein-coated FS either with a rabbit polyclonal serum obtained against the gp52 SU of MMTV (env, Fig. 3) (15) or with the preimmune serum (p.i.) of the immunized rabbit. For binding, neither the negative control FS (FCS) nor the positive control FS (7G6) was affected by the preincubation steps with the two sera. In addition, no influence on binding efficiency was seen upon preincubation of the env-coated FS with the p.i. serum. On the contrary, dose-dependent diminution of the binding to B cells (B220⁺) of those same beads was observed upon incubation with the env serum. These results clearly show that the binding of the FS to B lymphocytes is envelope mediated.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Evidence for specific gp52-mediated binding of gp52-coated FS. The protein-coated FS (FCS, 7G6, and gp52) described in the legend to Fig. 2B were incubated at a dilution of 1/180 with no antibody (), rabbit anti-gp52 polyclonal serum (env) (1, 1 µl; 101, 0.1 µl; 102, 0.01 µl; 103, 0.001 µl), or the p.i. serum of the same animal for 20 min on ice, washed, and processed as described in the legend to Fig. 2B. The results are representative of three experiments, and those of one experiment are shown (mean of three independent measurements ± the standard error of the mean.

To confirm and extend the results obtained with the env-coated FS, biotinylated MMTV(GR) particles were prepared. GR cells (18) were grown in Dulbecco modified Eagle medium with 10% FCS to 80% confluence. Fresh culture medium containing 10⁶ M dexamethasone was added and collected twice daily. The combined supernatant was clarified (10 min, 1,500 × g) and ultracentrifuged (2 h at 95,000 × g and 4°C), and the virus pellet was recovered in PBS. The virus was further purified on a linear 20 to 60% sucrose gradient (2 h at 95,000 × g and 4°C) and pelleted again (2 h at 95,000 × g and 4°C). The final viral pellet was resuspended in PBS at a concentration of 1 mg/ml. For biotinylation of the purified MMTV(GR) particles, 20 µl of biotinylation reagent (biotinamidocaproate N-hydroxysuccinamide ester; Sigma) was added to 1 ml of the viral preparation, incubated for 1 h on a test tube rotator at room temperature, diluted in 30 ml of PBS, ultracentrifuged (2 h at 95,000 × g and 4°C), and resuspended in PBS at 1 mg/ml. The biotinylated MMTV was used in binding studies with mouse ex vivo spleen cells (see above; Fig. 4). Figure 4A shows a representative FACS analysis of the binding of biotinylated MMTV to mouse ex vivo spleen cells with a dose of 0.6 µg of particles per million cells. Again, preferential and dose-dependent binding to B cells (B220⁺) was observed (Fig. 4B), with up to 25% of the B cells being positive at the highest dose of virus used (3 µg). The binding to T cells (CD4⁺ and CD8⁺) remained very low (~1%) at all of the concentrations of MMTV used (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   (A) Representative FACS profiles obtained upon binding of 0.6 µg of biotinylated MMTV particles to B cells (left profile) and T cells (right profile). The percentage of positive cells based on marker 1 (M1) is indicated. (B) Preferential binding of MMTV to mouse ex vivo B cells. Increasing doses of MMTV were incubated with 106 ex vivo BALB/c mouse spleen cells for 2 h on ice, washed, stained as described in the legend to Fig. 2B, and analyzed by flow cytometry. The results are representative of four experiments, and those of one experiment are shown (mean of three independent measurements ± the standard error of the mean.

Specific env-mediated binding was tested by incubating the biotinylated MMTV particles with either an isotype-matched control antibody (Mel-14) (4) or a neutralizing anti-gp52 mouse monoclonal antibody (H141) (20) (Fig. 5). A 60% reduction in binding was achieved with the specific antibody, and no significant influence was seen with the unrelated antibody. The same experiments were performed with the rabbit sera mentioned above, and similar results (data not shown) were obtained, demonstrating the envelope-mediated binding of biotinylated MMTV particles to B cells. We also performed competition studies by preincubation of the cells with bacterially produced gp52 (1 µg), followed by binding of biotinylated MMTV (0.6 µg). Weak but consistent inhibition (~20%) of binding of biotinylated MMTV to B cells was measured (data not shown), with no effect on T cells. Higher concentrations of bacterially produced gp52 turned out to be toxic for the cells. This result shows that both of the reagents used in these experiments bind to the same factor(s) at the surface of B cells. Taken together, our results show clear preferential binding of either env-coated FS or biotinylated MMTV to freshly isolated B lymphocytes.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Evidence for specific gp52-mediated binding of MMTV to mouse ex vivo B cells. One microgram of MMTV was incubated with 3 µg of either an anti-gp52 monoclonal antibody (H141; mouse immunoglobulin G2a) or an unrelated, isotype-matched monoclonal antibody (Mel-14; mouse immunoglobulin G2a) or with no antibody () for 30 min on ice, washed, and processed as described in the legend to Fig. 4B. The results are representative of four experiments, and those of one experiment are shown (mean of three independent measurements ± the standard error of the mean.

Infection with human immunodeficiency virus (HIV) has recently been understood in much more detail. In order to infect a cell, HIV requires the presence of both CD4 and a seven-transmembrane domain G protein-coupled receptor on the cell surface (reviewed in reference 17). To date, at least 10 seven-transmembrane domain receptors have been identified that can serve as HIV type 1 or simian immunodeficiency virus entry coreceptors but the chemokine receptors CCR5 and CXCR4 are the most widely utilized coreceptors in that all of the viruses tested use either one or both for entry (reviewed in reference 5). Similarly, MMTV could use a coreceptor or several different coreceptors for entry into different cell types. The identification and characterization of MMTV coreceptors will give insight into its interaction with and infection of different cell types in vivo.

In conclusion, the results reported here indicate that T cells have a much lower avidity for virus binding than do B cells. The question of whether this reflects either a lower abundance of MMTV receptor molecules or the presence of a lower-affinity MMTV receptor on T cells needs further investigation.