We generated constructs expressing three forms of the αCEA MFE-23 scAb as C-terminal fusions of H (Fig. A). The scAb forms differed in the length of the linker separating the VH and VL domains and were designated zero (0), short (S), and long (L), corresponding to linker lengths of 0, 6, and 16 amino acids, respectively. In each construct, the C terminus of H was separated from the scAb by an 8-amino-acid spacer, including a factor Xa cleavage site to facilitate removal of the displayed ligand. The chimeric H-αCEA proteins were designated HX0, HXS, and HXL, accordingly.
FIG. 1 (A) Schematic diagram showing the relative positions of MV H (shaded), the 8-amino-acid spacer incorporating the factor Xa cleavage site (open), and the appended scAb domain (hatched). The linker sequences separating VH and VL in the three forms of the (more ...) Expression, stability, oligomerization, and cell surface localization of chimeric H-αCEA proteins.
Expression of HX0, HXS, and HXL proteins at the expected molecular weights in all cell lines used was confirmed by Western blot analysis (shown in Fig. B for Vero cells; data not shown for HeLa, HeLa-CEA, MC38, and MC38-CEA cells). Furthermore, pulse-chase analyses demonstrated similar stabilities of expression for the chimeric H-αCEA proteins and for unmodified H (Fig. A). The half-lives for all proteins were greater than 3 h, as determined by band densitometry with a phosphorimager.
FIG. 2 (A) All H-αCEA proteins are as stable as unmodified MV H. Pulse-chase analysis of Vero cells transfected with the indicated H constructs is shown. Antigenic material was precipitated after the indicated chase times (in minutes). (B) HXL protein (more ...)
The H-like conformation of the HXL protein was verified by its ability to form covalently linked dimers with itself and with unmodified H. Following cotransfection of Flag-tagged HXL with unmodified, untagged H, or with empty plasmid or Flag-tagged H as a control, cells were metabolically labeled. Using an αFlag antibody, tagged proteins and any interacting untagged H proteins were immunoprecipitated, and the dimerization status was analyzed by gel electrophoresis under nonreducing conditions (Fig. B). Both homotypic dimers (HXL-HXL) and heterotypic dimers (HXL-H) were identified. In addition, and consistent with the postulated tetrameric nature of MV H, we observed the presence of untagged H-H dimers in the presence of tagged HXL, presumably resulting from the interaction of H-H with HXL-HXL dimers. Under conditions in which both homotypic and heterotypic complexes could form, the heterotypic HXL-H complex predominated, suggesting that dimerization of HXL with unmodified H was more efficient than that with itself. However, we cannot formally exclude the possibility that an excess of H was expressed compared to HXL and that this may bias the efficiency of dimer formation. Assuming that this is not the case, the efficiencies of both HXL-HXL and HXL-H complex formation were reduced compared with that of H-H dimerization. Thus, display of the scAb on H may reduce the efficiency of, but does not prevent, dimerization of the underlying H molecule.
Since the formation of covalently linked dimers is a prerequisite for efficient H transport (22
), our data suggested that the HXL protein should be efficiently transported. Indeed, cell surface expression of not only HXL but all H-αCEA proteins was confirmed by FACS analysis of transfected cells to be similar to that of unmodified H (Fig. C), indicating efficient transport for all H-αCEA proteins.
HXL supports syncytium formation in both CD46-positive and CD46-negative, CEA-positive cells.
Although display of a scAb on MV H did not affect its proper folding or transport, its receptor-binding and fusion support functions may have been disrupted. We assessed the functionality of the H-αCEA proteins by measuring syncytium formation following coexpression with MV F.
In three CD46-positive cell lines (HeLa, HeLa-CEA, and Vero [Fig. A]), expression of HXL with F induced extensive syncytium formation, to a degree similar to that with unmodified H. The HXS protein supported syncytium formation at a lower level, while no cell-cell fusion was observed in cells expressing HX0. No significant difference was observed in the numbers of syncytia in HeLa versus HeLa-CEA cells expressing any of the chimeric proteins. Thus, addition of the long-linker form of the scAb did not impair MV H-induced cell-cell fusion via CD46. We found these three cell lines to be negative for surface SLAM by FACS analysis (data not shown); thus, receptor usage was limited in this case to CD46 and, where expressed, CEA.
FIG. 3 (A) HXL protein efficiently induces syncytium formation in CD46-positive cells. Vero, HeLa, and HeLa-CEA cells were cotransfected with MV F and the indicated H constructs, and syncytia were scored 48 h posttransfection. The error bars indicate standard (more ...)
To assess the contribution of the scAb-CEA interaction in the fusion process, we analyzed CEA-dependent syncytium formation in a CD46-negative background. For this we used MC38-CEA cells, a mouse cell line stably expressing high levels of cell surface human CEA (25
), and their CEA-negative parent cell line, MC38. FACS analysis (Fig. B) demonstrated levels of CD46 to be similarly high on Vero, HeLa, and HeLa-CEA cells and undetectable on MC38 and MC38-CEA cells. As expected, expression of CEA was high only on HeLa-CEA and MC38-CEA cells. Fusions in MC38, MC38-CEA, and Vero cells were compared (Fig. C and D). In MC38 and MC38-CEA cells, coexpression of F with unmodified H induced a low level of syncytium formation, presumably reflecting an inefficient, CD46-independent fusion mechanism for MV H. Strikingly, coexpression of F with chimeric HXL in MC38-CEA cells led to extensive syncytium formation. Although at a reduced level compared with that in Vero cells, the numbers of syncytia were over 100-fold greater than those seen with unmodified H. The HXS protein supported a reduced level of syncytium formation in both Vero and MC38-CEA cells, while coexpression of HX0 with F yielded no detectable cell-cell fusion in any cell line. Thus, MV H displaying the long-linker form of the scAb initiated cell-cell fusion via a novel receptor.
Recovery of replication-competent MV containing chimeric HXL protein in place of H.
The ability of chimeric HXL to functionally replace unmodified H in the context of replicating virus was assessed. In a full-length infectious MV Edmonston cDNA, the H gene was replaced with that encoding HXL, and using the MV recovery protocol (24
), virus was isolated from individual syncytia formed in Vero cells.
The authenticity of the recovered MV-HXL was confirmed by Western blot analysis of purified particles (Fig. A). Consistent with the sizes of transiently expressed HXL and H proteins (Fig. B), purified MV-HXL particles expressed an H protein of ~110 kDa, in contrast to that of ~80 kDa expressed from unmodified MV. Furthermore, treatment of purified MV-HXL virious with factor Xa protease demonstrated specific cleavage of the appended scAb, generating an 80-kDa protein corresponding to unmodified H (Fig. B). As expected, factor Xa treatment of unmodified MV did not affect the size of the antigenic material detectable as H.
FIG. 4 (A) Recombinant MV-HXL expresses an H protein of the expected molecular weight. The protein compositions of MV and MV-HXL were revealed by Western blotting with an MV-specific antiserum. The numbers refer to molecular weights in thousands. (B) The displayed (more ...) MV-HXL binds to the surfaces of CD46-positive and CD46-negative, CEA-positive cells.
The abilities of MV and MV-HXL to bind cells expressing either CD46 or CEA at the surface were next compared by flow cytometry (Fig. C). Neither virus was able to bind the surface of CD46-negative, CEA-negative MC38 cells. In contrast, both viruses bound CD46-positive, CEA-negative Vero cells, with small but significant shifts observed. Thus, the addition of the scAb did not negate the interaction of MV-HXL with cell surface CD46, consistent with the ability of the HXL protein to induce cell-cell fusion in CD46-positive cells. Importantly, MV-HXL bound the surface of the CD46-negative, CEA-positive MC38-CEA cell line, while binding of unmodified MV was negligible.
MV-HXL replicates in CEA-positive cells in the absence of CD46.
Given that MV-HXL bound cells expressing CEA, we assessed its ability to infect both CD46-positive and CD46-negative, CEA-positive cells. We first compared the infectivities of MV and MV-HXL for Vero, MC38, and MC38-CEA cells by observing syncytium formation in the inoculated cells (Fig. A). Consistent with our previous results, Vero cells were infectable by either virus. Significantly, infection of MC38-CEA cells with MV-HXL resulted in extensive syncytium formation. In contrast, infection of MC38-CEA cells with unmodified MV and infection of MC38 cells with either virus were undetectable.
FIG. 5 (A) MV-HXL infects and induces cell-cell fusion in CD46-negative, CEA-positive cells. MV, MV-HXL, or no virus was incubated with Vero, MC38, and MC38-CEA cells at an MOI of 3. Representative fields of view were photographed 72 h postinfection. (B) MV-HXL (more ...)
We next compared the replicative ability of MV-HXL with that of parental MV by determining the viral titers achieved in Vero, HeLa, HeLa-CEA, MC38, and MC38-CEA cells by TCID50 assays using each of the cell lines as a target (Fig. B shows one typical example). MV-HXL replicated to titers almost indistinguishable from those obtained with parental MV in all three CD46-positive cell lines tested (7 × 105 to 5 × 107 PFU/ml, depending on the cell line). Thus, the ability of MV-HXL to replicate in a CD46-dependent manner was not affected by display of the scAb, consistent with our previous data.
Remarkably, MV-HXL reached similar titers on CD46-negative, CEA-positive MC38-CEA cells and on CD46-positive cells (from independent experiments, an average of 6.2 × 105 ± 1.3 × 105 PFU/ml), demonstrating that its ability to replicate in a CEA-dependent manner was about as efficient as its CD46-dependent replication. Negligible infection (titers of <102 PFU/ml) was detected in MC38-CEA cells with parental MV and in CEA-negative MC38 cells with either virus. The fact that parental MV H induced a low level of syncytium formation in MC38 cells while viral titers were negligible may be accounted for by differences in the requirements for cell-cell and virus-cell fusion.
We also measured the infectivities of MV and MV-HXL for all cell lines by infecting each at an MOI of 3 and quantifying cell-associated virus by TCID50 titration 72 h postinfection using Vero cells as targets. Since no intrinsic differences in replication of the two viruses in Vero cells existed (Fig. B), no bias would be introduced by using this approach. This method gave a pattern of virus titers reproducibly similar to that from the previous assay, but the absolute values were 1 to 2 log units lower, with the titer of MV-HXL on MC38-CEA cells reaching 3.1 × 104 ± 5.1 × 103 PFU/ml (data not shown). This assay was used for subsequent blocking experiments.
Replication of MV-HXL in CD46-negative, CEA-positive cells depends on a specific interaction between the displayed scAb and CEA.
Our data suggested a specific interaction between MV-HXL and CEA. To test this, we assessed the infectivities of MV and MV-HXL for MC38-CEA and Vero cells following incubation of the virus with factor Xa protease to cleave the displayed scAb or pretreatment of the cells with an αCEA MAb (COL1) to block cell surface CEA. In both cases, virus was quantified from the cells 72 h postinfection by TCID50 titration using Vero cells as targets (Fig. ).
FIG. 6 MV-HXL infectivity for MC38-CEA cells is inhibited by cleavage of the displayed scAb or antibody preadsorption of cell surface CEA. Vero cells (as a control) and MC38-CEA cells were infected with MV or MV-HXL (MOI, 1), untreated or pretreated with 10 (more ...)
On Vero cells, neither treatment significantly affected the titer of either MV or MV-HXL; similarly, on MC38-CEA cells, the titer of unmodified MV was unaffected by either treatment. Strikingly, cleavage of the displayed scAb by factor Xa protease reduced the titer of MV-HXL on MC38-CEA cells by greater than 100-fold (from independent experiments, an average of 177-fold ± 2-fold). Inhibition of MV-HXL by pretreating MC38-CEA cells with the αCEA MAb COL1 was less drastic but still significant, with an inhibition of greater than 10-fold. The less pronounced inhibition seen with COL1 may be explained by multivalent virus attachment being inefficiently inhibited by monomeric ligands. The inability of COL1 to inhibit MV infectivity for either cell line demonstrates the specificity of its inhibition for MV-HXL. These data confirm that the ability of MV-HXL to infect CEA-positive cells independently of CD46 depends on a specific interaction between the displayed scAb and the targeted antigen.