The endoplasmic reticulum (ER) was investigated as the initial oligomerization site for the envelope glycoproteins H and F of measles virus (MV), a clinically relevant member of the Paramyxoviridae family, and consequences of this interaction for viral replication were studied. Both proteins were tagged at their cytosolic tails with RRR and KKXX motifs, respectively, resulting in their efficient retention in the ER. Cotransfection of the retained constructs with transport competent MV glycoproteins revealed a dominant negative effect on their biological activity indicating intracellular complex formation and thus retention. Pulse-chase analysis and co-immunoprecipitation experiments demonstrated that this effect is based on both homo-and hetero-oligomerization in the ER. Recombinant viruses additionally expressing ERretained F showed an altered cytopathic phenotype accompanied by greatly reduced particle release. Similar mutant viruses additionally expressing ERretained H could not be rescued indicating an even greater negative effect of this protein on virus viability. Our study suggests that both homo-and heterooligomerization of MV glycoproteins occur in the ER and that these events are of significance for early steps of particle assembly.

To understand the molecular determinants of measles virus (MV) cytopathicity, we have characterized mutant viruses exhibiting a more-extensive cell-to-cell fusion while maintaining efficient replication to high titers. A virus which is modified by the addition of an 8-amino-acid Flag epitope tag at the cytoplasmic tail of its H (for MV hemagglutinin) envelope glycoprotein replicates efficiently, has an increased cytopathicity, possesses a greater infectivity per particle, and has an altered protein composition compared with that of unmodified MV. The mutant phenotype is not specifically linked to the epitope sequence, since an alternatively added HA (for influenza virus-derived hemagglutinin) epitope tag caused similar effects. We demonstrate that both epitope tags weaken the interaction between the H and fusion (F) glycoproteins in virus-infected cells. This reduction in strength of H/F interaction is independent of the presence of the viral matrix (M) protein. Viruses with this less stable complex are more sensitive to neutralization by a soluble octameric form of the CD46 receptor, consistent with their increased fusogenicity. Similar analyses of glycoproteins derived from MV strains with reduced cytopathicities confirm that the strength of H and F glycoprotein interaction is a modulator of viral fusogenicity.Enveloped viruses that enter target cells through membrane fusion at neutral pH spread through a cell culture both by producing infectious particles and by lateral cell-to-cell fusion. The latter mechanism occurs through binding of viral envelope glycoproteins present at the infected cell surface to a viral receptor(s) on surrounding uninfected cells, thereby inducing membrane fusion, recruiting many cells into a multinucleated syncytium which ultimately dies (16).The determinants of viral infectivity and the balance between lateral spread and release are complex and incompletely understood. Syncytium formation may be important for viral pathogenesis in vivo. For measles virus (MV), a clinically relevant member of the Paramyxoviridae, syncytia have been reported in cells from infected humans (27), primates (29), and transgenic mice (19) expressing one of the virus receptors, CD46 (7, 21). However, syncytia are not produced in every MV-infected tissue, and some primary MV isolates are poorly or nonfusogenic in many cell lines (35). Repeated passaging of the MV-Edmonston (MV-Edm) original isolate on chicken embryo fibroblasts provided the basis for the generation of the MV-Edm B line and the currently used vaccine strains (8, 22, 28), which are no longer pathogenic in vivo. Further adaptation by laboratory passage on cultured primate cells often results in the selection of more fusogenic variants.Recombinant MVs lacking matrix (M) or the cytoplasmic tail of the fusion (F) protein, mutations found in MV variants causing the rare and fatal complication, subacute sclerosing panencephalitis (17), spread more extensively by cell-to-cell fusion but achieve a significantly lower titer, particularly of released vi...

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the V H and V L domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.

Attachment of measles virus (MV) to its cellular receptor is mediated by the viral envelope glycoprotein hemagglutinin (H). H exists at the viral surface as a disulfide-linked dimer which may associate into a tetramer. We aimed to define regions of H essential for its homo-oligomerization. To delineate these more precisely, we have generated a series of H ectodomain truncation mutants and studied their abilities to form both homotypic complexes and heterotypic complexes with full-length H. We define a "minimal unit" which is sufficient for MV H dimerization as that encompassing residues 1 to 151. This unit forms both homodimers and heterodimers with full-length H protein, although neither is transported to the cell surface even in the presence of other MV proteins. We show that cysteine residues at positions 139 and 154 are both critical in mediating covalent dimerization, not only of the truncated H mutants but also of full-length MV H protein. Even those cysteine mutants unable to form covalent intermolecular interactions are biologically active, mediating the formation of syncytia, albeit at a reduced rate. We demonstrate that this impaired capacity to mediate cell-to-cell fusion is based mainly on a reduced transport rate of the mutant molecules to the cell surface, indicating a role for covalent intermolecular interactions in efficient transport of MV H dimers to the cell surface.

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