Regulatory Role of the Morbillivirus Attachment Protein Head-to-Stalk Linker Module in Membrane Fusion Triggering.

Morbillivirus (e.g., measles virus [MeV] and canine distemper virus [CDV]) host cell entry is coordinated by two interacting envelope glycoproteins, namely, an attachment (H) protein and a fusion (F) protein. The ectodomain of H proteins consists of stalk, connector, and head domains that assemble into functional noncovalent dimer-of-dimers. The role of the C-terminal module of the H-stalk domain (termed linker) and the connector, although putatively able to assume flexible structures and allow receptor-induced structural rearrangements, remains largely unexplored. Here, we carried out a nonconservative mutagenesis scan analysis of the MeV and CDV H-linker/connector domains. Our data demonstrated that replacing isoleucine 146 in H-linker (H-I146) with any charged amino acids prevented virus-mediated membrane fusion activity, despite proper trafficking of the mutants to the cell surface and preserved binding efficiency to the SLAM/CD150 receptor. Nondenaturing electrophoresis revealed that these charged amino acid changes led to the formation of irregular covalent H tetramers rather than functional dimer-of-dimers formed when isoleucine or other hydrophobic amino acids were present at residue position 146. Remarkably, we next demonstrated that covalent H tetramerization per se was not the only mechanism preventing F activation. Indeed, the neutral glycine mutant (H-I146G), which exhibited strong covalent tetramerization propensity, maintained limited fusion promotion activity. Conversely, charged H-I146 mutants, which additionally carried alanine substitution of natural cysteines (H-C139A and H-C154A) and thus were unable to form covalently linked tetramers, were fusion activation defective. Our data suggest a dual regulatory role of the hydrophobic residue at position 146 of the morbillivirus head-to-stalk H-linker module: securing the assembly of productive dimer-of-dimers and contributing to receptor-induced F-triggering activity.IMPORTANCE MeV and CDV remain important human and animal pathogens. Development of antivirals may significantly support current global vaccination campaigns. Cell entry is orchestrated by two interacting glycoproteins (H and F). The current hypothesis postulates that tetrameric H ectodomains (composed of stalk, connector, and head domains) undergo receptor-induced rearrangements to productively trigger F; these conformational changes may be regulated by the H-stalk C-terminal module (linker) and the following connector domain. Mutagenesis scan analysis of both microdomains revealed that replacing amino acid 146 in the H-linker region with nonhydrophobic residues produced covalent H tetramers which were compromised in triggering membrane fusion activity. However, these mutant proteins retained their ability to traffic to the cell surface and to bind to the virus receptor. These data suggest that the morbillivirus linker module contributes to the folding of functional pre-F-triggering H tetramers. Furthermore, such structures might be critical to convert receptor engagement into F activation.

Measles virus receptors.

Measles virus (MV) has two envelope glycoproteins, the hemagglutinin (H) and fusion protein, which are responsible for attachment and membrane fusion, respectively. Signaling lymphocyte activation molecule (SLAM, also called CD150), a membrane glycoprotein expressed on immune cells, acts as the principal cellular receptor for MV, accounting for its lymphotropism and immunosuppressive nature. MV also infects polarized epithelial cells via an as yet unknown receptor molecule, thereby presumably facilitating transmission via aerosol droplets. Vaccine and laboratory-adapted strains of MV use ubiquitously expressed CD46 as an alternate receptor through amino acid substitutions in the H protein. The crystal structure of the H protein indicates that the putative binding sites for SLAM, CD46, and the epithelial cell receptor are strategically located in different positions of the H protein. Other molecules have also been implicated in MV infection, although their relevance remains to be determined. The identification of MV receptors has advanced our understanding of MV tropism and pathogenesis.

Structural disorder and modular organization in Paramyxovirinae N and P.

The existence and extent of disorder within the replicative complex (N, P and the polymerase, L) of Paramyxovirinae were investigated, drawing on the discovery that the N-terminal moiety of the phosphoprotein (P) and the C-terminal moiety of the nucleoprotein (N) of measles virus are intrinsically unstructured. We show that intrinsic disorder is a widespread property within Paramyxovirinae N and P, using a combination of different computational approaches relying on different physico-chemical concepts. Notably, experimental support that has often gone unnoticed for most of the predictions has been found in the literature. Identification of disordered regions allows the unveiling of a common organization in all Paramyxovirinae P, which are composed of six modules defined on the basis of structure or sequence conservation. The possible functional significance of intrinsic disorder is discussed in the light of experimental data, which show that unstructured regions of P and N are involved in numerous interactions with several protein and protein-RNA partners. This study provides a contribution to the rather poorly investigated field of intrinsically disordered proteins and helps in targeting protein domains for structural studies.