A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells.

As we have shown previously, release of measles virus (MV) from polarized epithelial cells is not determined by the viral envelope proteins H and F. Although virus budding is restricted to the apical surfaces, both proteins were abundantly expressed on the basolateral surface of Madin-Darby canine kidney cells. In this report, we provide evidence that the basolateral expression of the viral proteins is of biological importance for the MV infection of polarized epithelial cells. We demonstrate that both MV glycoproteins possess a basolateral targeting signal that is dependent upon the unique tyrosine in the cytoplasmic tails. These tyrosines are shown to be also part of an endocytosis signal. In MV-infected cells, internalization of the glycoproteins was not observed, indicating that recognition of the endocytosis signals is disturbed by viral factors. In contrast, basolateral transport was not substantially hindered, resulting in efficient cell-to-cell fusion of polarized Madin-Darby canine kidney cells. Thus, recognition of the signals for endocytosis and polarized transport is differently regulated in infected cells. Mutation of the basolateral sorting signal in one of the MV glycoproteins prevented fusion of polarized cells. These results suggest that basolateral expression of the MV glycoproteins favors virus spread in epithelia.

Proteolytic cleavage of the fusion protein but not membrane fusion is required for measles virus-induced immunosuppression in vitro.

Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F(0) protein by cellular proteases, such as furin, into the F(1)-F(2) subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F(0) proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F(0) proteins was not due to an impaired F(0)-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F(0) protein.

Recombinant measles virus requiring an exogenous protease for activation of infectivity.

Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.

Polarized budding of measles virus is not determined by viral surface glycoproteins.

For viruses that mature by a budding process, the envelope glycoproteins are considered the major determinants for the site of virus release from polarized epithelial cells. Viruses are usually released from that membrane domain where the viral surface glycoproteins are transported to. We here report that measles virus has developed a different maturation strategy. Measles virus was found to be released from the apical membrane domain of polarized epithelial cells, though the surface glycoproteins H and F were transported in a nonpolarized fashion and to the basolateral membrane domain, respectively.

Membrane cofactor protein (CD46) is a basolateral protein that is not endocytosed. Importance of the tetrapeptide FTSL at the carboxyl terminus.

Membrane cofactor protein (MCP) is a widely distributed complement regulatory protein that is expressed on the basolateral surface of polarized epithelial cells. The basolateral targeting of the BC1 isoform of MCP was analyzed by generating deletion mutants and point mutants within the cytoplasmic tail of 16 amino acids. A sequence of four amino acids, FTSL, was found to be indispensable for the basolateral transport of MCP. This tetrapeptide has two unique features compared with the targeting motifs of other basolateral proteins: (i) it contains a phenylalanine rather than a tyrosine at position 1; (ii) it is located at the very COOH-terminal end. Replacement of the phenylalanine or the leucine by an alanine resulted in a nonpolarized delivery to the cell surface. On the other hand, substitution of a tyrosine for the phenylalanine did not affect the basolateral transport of MCP. The latter mutant, however, was efficiently internalized, whereas the wild type protein was not subject to endocytosis. Our results indicate that the targeting signal YXX-large aliphatic that is involved in various sorting events has been modulated in MCP in such a way that it allows basolateral transport but not endocytosis.

Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Madin-Darby canine kidney cells (type I).

gp40 has been recently identified as a major apical cell-surface sialoglycoprotein of type-I Madin-Darby canine kidney cells, a cell line widely used for the study of polarized transport. The determination of two internal amino acid sequences of the purified glycoprotein by Edman degradation enabled us to isolated the cDNA encoding the 18.6 kDa protein backbone of gp40. Sequence analysis revealed that gp40 is a type-I membrane protein which has several characteristics in common with glycophorin A and other mucin-type glycoproteins. At least 14 serine/threonine residues were found to be used for O-glycosylation. No potential sites for N-glycosylation were detected. gp40 turned out to represent the canine homologue of a cell-surface antigen expressed by various epithelial and non-epithelial cells in rat and mouse. Potential O-glycosylation sites, transmembrane and cytoplasmic domains were found to be highly conserved in the three species. gp40 was detected in canine lung, intestine, kidney, brain and heart but not in liver and spleen. The subline II of Madin-Darby canine kidney cells was found not to express gp40. Stable expression of gp40 in transfected type-II cells revealed that gp40 is predominantly delivered to the apical plasma membrane. N-Glycans and a glycosylphosphatidylinositol anchor, both proposed apical targeting signals, are absent from gp40, indicating that other determinants are responsible for its polarized transport.

The N-glycan of the SCR 2 region is essential for membrane cofactor protein (CD46) to function as a measles virus receptor.

Membrane cofactor protein (MCP) (CD46), a complement-regulatory protein, serves as a cellular receptor for measles virus. Its amino-terminal portion is composed of four short consensus repeats (SCR), three of which (SCR1, SCR2, and SCR4) carry an N-linked oligosaccharide. In order to determine the importance of the three N-glycans for the function of MCP as a measles virus receptor, we established Chinese hamster ovary (CHO) cell lines that stably express mutant MCPs lacking one of the three motifs for N glycosylation (NQ1, NQ2, and NQ4). In an additional mutant (NQ1-2), two glycosylation motifs were altered, allowing the addition of an N-linked oligosaccharide only in SCR4. The abilities of the mutant MCPs to function as measles virus receptors were analyzed with three different assays: (i) binding of measles virus hemagglutinin to MCP immobilized on nitrocellulose; (ii) binding of measles virus to CHO cells expressing wild-type or mutant MCP; and (iii) infection of the transfected CHO cells by measles virus. In all three assays, the abilities of the NQ2 and NQ1-2 mutants to serve as measles virus receptors were drastically impaired. The NQ1 and NQ4 mutants were recognized by measles virus almost as efficiently as the wild-type protein. These results indicate that the N-glycan attached to SCR2 is essential for MCP to serve as a measles virus receptor, while the oligosaccharides attached to SCR1 and SCR4 are of only minor importance.

Two different cytoplasmic tails direct isoforms of the membrane cofactor protein (CD46) to the basolateral surface of Madin-Darby canine kidney cells.

Membrane cofactor protein (MCP; CD46), a widely distributed regulatory protein of the complement system, was analyzed for expression in polarized epithelial cells. Both a human and a simian (Vero C1008) cell line were found to contain endogenous MCP mainly on the basolateral surface. Transfected Madin-Darby canine kidney cells stably expressing human MCP delivered this protein also predominantly to the basolateral surface. A deletion mutant lacking the cytoplasmic tail was transported in a nonpolarized fashion, indicating that the targeting signal for the basolateral transport is located in the cytoplasmic domain. A characteristic feature of MCP is the presence of various isoforms that contain either of two different cytoplasmic tails as a consequence of alternative splicing. Two isoforms differing only in the cytoplasmic tail (tail 1 or 2) were analyzed for polarized expression in Madin-Darby canine kidney cells. Surface biotinylation, as well as confocal immunofluorescence microscopy, indicated that both proteins were transported to the basolateral surface. Because no sequence similarity has been observed, the two tails contain different basolateral targeting signals. A deletion mutant lacking the only tyrosine residue in tail 1 retained the polarized expression indicating that, in contrast to most basolateral sorting signals, the transport signal of the tail 1 isoform is not dependent on tyrosine. The maintenance of a targeting motif in two distinct cytoplasmic tails suggests that the basolateral expression of MCP in polarized epithelial cells is of physiological importance.

Membrane cofactor protein with different types of N-glycans can serve as measles virus receptor.

Membrane cofactor protein (MCP) has been shown to act as a cellular receptor for measles virus. In previous binding studies we demonstrated a direct interaction between the measles virus H protein and MCP. The binding was shown to be independent of the O-glycans but dependent on the N-glycans of MCP. To elucidate the role of N-glycans for the receptor function of MCP, the effect of the glycosylation inhibitors tunicamycin (TM) and 1-deoxymannojirimycin (DMJ) was analyzed. TM which prevents N-glycosylation has been reported to inhibit the expression of functional measles virus receptors. Here we show that MCP lacking all N-glycans was detectable on the surface of Vero cells, although in a reduced amount. Therefore, the lack of receptor activity cannot be explained by intracellular degradation or defective transport. In the presence of DMJ, a mannosidase I inhibitor, MCP is synthesized with N-glycans of the high-mannose type in contrast to the complex oligosaccharides present on MCP of untreated cells. Both MCP with mannose-rich and MCP with complex N-glycans were recognized by measles virus H protein in an in vitro binding assay. They both could also serve as receptors for the infection of cultured Vero cells, arguing against a direct binding of virus to a carbohydrate moiety within the N-glycans of MCP. We propose that N-linked oligosaccharides are required to maintain a conformation-dependent receptor determinant of MCP.

Binding of measles virus to membrane cofactor protein (CD46): importance of disulfide bonds and N-glycans for the receptor function.

Two cellular proteins, membrane cofactor protein (MCP) and moesin, were reported recently to be functionally associated with the initiation of a measles virus infection. We have analyzed the interaction of measles virus with cell surface proteins, using an overlay binding assay with cellular proteins immobilized on nitrocellulose. Among surface-biotinylated proteins from a human rectal tumor cell line (HRT), measles virus was able to bind only to a 67-kDa protein that was identified as MCP. The virus recognized different isoforms of MCP expressed from human (HRT and HeLa) and simian (Vero) cell lines. The binding of measles virus to MCP was abolished after cleavage of the disulfide bonds by reducing agents as well as after enzymatic release of N-linked oligosaccharides. By contrast, removal of sialic acid or O-linked oligosaccharides did not affect the recognition of MCP measles virus. These data indicate that the receptor determinant of MCP is dependent on a conformation of the protein that is maintained by disulfide bonds and N-glycans present in the complement binding domains. Our results are consistent with a role of MCP as primary attachment site for measles virus in the initial stage of an infection. The functional relationship between MCP and moesin in a measles virus infection is discussed.