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.

Identification and mapping of a region on the mRNA of Morbillivirus nucleoprotein susceptible to RNA interference.

The morbillivirus genus includes important pathogens such as measles virus (MV), peste des petits ruminants virus (PPRV), and rinderpest virus (RPV) and forms a group of antigenically related viruses. The viral nucleoprotein (N) is a well-conserved protein among the genus and plays a central role in the replication of the virus. Using a comprehensive approach for siRNA screening of the conserved sequences of the N gene, including sequence analysis and functional in vitro tests, we have identified a region for the design of siRNA effective for the control of PPRV, RPV, and MV replication. Silencing of the N mRNA efficiently shuts down the production of N transcripts, the expression of N protein, and the indirect inhibition of matrix protein, resulting in the inhibition of PPRV progeny by 10,000-fold. These results suggest that siRNA against this region should be further explored as a therapeutic strategy for morbillivirus infections.

Structure and sequence motifs of siRNA linked with in vitro down-regulation of morbillivirus gene expression.

The most challenging task in RNA interference is the design of active small interfering RNA (siRNA) sequences. Numerous strategies have been published to select siRNA. They have proved effective in some applications but have failed in many others. Nonetheless, all existing guidelines have been devised to select effective siRNAs targeting human or murine genes. They may not be appropriate to select functional sequences that target genes from other organisms like viruses. In this study, we have analyzed 62 siRNA duplexes of 19 bases targeting three genes of three morbilliviruses. In those duplexes, we have checked which features are associated with siRNA functionality. Our results suggest that the intramolecular secondary structure of the targeted mRNA contributes to siRNA efficiency. We also confirm that the presence of at least the sequence motifs U13, A or U19, as well as the absence of G13, cooperate to increase siRNA knockdown rates. Additionally, we observe that G11 is linked with siRNA efficacy. We believe that an algorithm based on these findings may help in the selection of functional siRNA sequences directed against viral genes.

Search for morbillivirus proteins in multiple sclerosis brain tissue.

We investigated brain samples of patients with multiple sclerosis (MS) and controls with immunohistochemistry using monoclonal antibodies (MoAbs) against canine distemper virus (CDV) and measles virus (MV) proteins. All stained negative except for MoAb F3-5, which recognises a conserved epitope on the fusion protein of morbilliviruses. F3-5 immunostaining was found in 8/9 MS plaques and 2/5 herpes simplex virus encephalitis brain samples, but not in six controls or four patients with ischaemic stroke. Using RT-PCR we found no evidence for the presence of MV in MS plaques. The F3-5 epitope may represent a protein that is upregulated during inflammation or point to a yet unrecognised morbillivirus in the human central nervous system that might be implicated in MS 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.

Genomic characterization of wild-type measles viruses that circulated in different states in Brazil during the 1997 measles epidemic.

Despite the marked reduction in the incidence of measles in Brazil, a measles epidemic occurred in this country in 1997. The measles cases observed during this epidemic began to reappear in large numbers in São Paulo, and spread to Rio de Janeiro and other Brazilian states. In the present study molecular biology techniques were used for the detection and genomic characterization of measles viruses from clinical samples such as urine and nasopharyngeal secretions collected in the states of Rio de Janeiro, Minas Gerais and Paraná, during the 1997 epidemic. RT-PCR and nucleotide sequence analysis of part of the carboxyl-terminal region of the nucleoprotein gene of measles viruses obtained directly from clinical samples or from infected cell cultures during this epidemic classified all as wild-type of genotype D6. As the genotype D6 was identified in different Brazilian states, this study demonstrated that this genotype was circulating in Brazil during the 1997 epidemic.

Genetic characterization of wild-type measles viruses isolated during the 1998 measles epidemic in Argentina.

This study reports on genomic characterization of six measles virus (MV) isolates obtained from a measles epidemic in Argentina in 1998. Reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of the carboxyl-terminal region of the nucleoprotein (N) gene of these isolates classified all of them as wild type MV of D6 genotype. MVs of D6 genotype with identical nucleotide sequences in the region analyzed were also identified during the 1997 measles epidemic in Brazil and the 1999 measles outbreak in Uruguai. These results suggest that the MVs associated with the 1998 measles epidemic in Argentina might have originated from Brazil. As the D6 genotype is also widely distributed in Europe, it is possible that this genotype was brought to South America from Europe.

Sequence and structure alignment of Paramyxoviridae attachment proteins and discovery of enzymatic activity for a morbillivirus hemagglutinin.

On the basis of the conservation of neuraminidase (N) active-site residues in influenza virus N and paramyxovirus hemagglutinin-neuraminidase (HN), it has been suggested that the three-dimensional (3D) structures of the globular heads of the two proteins are broadly similar. In this study, details of this structural similarity are worked out. Detailed multiple sequence alignment of paramyxovirus HN proteins and influenza virus N proteins was based on the schematic representation of the previously proposed structural similarity. This multiple sequence alignment of paramyxovirus HN proteins was used as an intermediate to align the morbillivirus hemagglutinin (H) proteins with neuraminidase. Hypothetical 3D structures were built for paramyxovirus HN and morbillivirus H, based on homology modelling. The locations of insertions and deletions, glycosylation sites, active-site residues, and disulfide bridges agree with the proposed 3D structure of HN and H of the Paramyxoviridae. Moreover, details of the modelled H protein predict previously undescribed enzymatic activity. This prediction was confirmed for rinderpest virus and peste des petits ruminants virus. The enzymatic activity was highly substrate specific, because sialic acid was released only from crude mucins isolated from bovine submaxillary glands. The enzymatic activity may indicate a general infection mechanism for respiratory viruses, and the active site may prove to be a new target for antiviral compounds.

Morbillivirus infection in cetaceans of the western Atlantic.

We report serologic evidence of morbillivirus infection in eleven of fifteen species of odontocete cetaceans from the western Atlantic since 1986. Blood samples were obtained both from free-ranging and stranded animals. Virus neutralizing titers were higher against porpoise and dolphin morbilliviruses than against peste des petits ruminants virus, phocine distemper virus or canine distemper virus (CDV). Serum from five species, tested in a heterologous immunoprecipitation assay using radiolabelled CDV, precipitated the nucleocapsid (N) protein. Clinical morbillivirus infection may potentially impact already threatened species such as the harbour porpoise and precipitate mass strandings of socially cohesive odontocetes.

Editing of morbillivirus P gene transcripts in infected animals.

RNA editing in the Morbillivirus genus in vivo was investigated by applying a polymerase chain reaction-based primer extension technique to measure the edited and non-edited mRNA transcripts. In this genus of the Paramyxoviridae the P gene transcript is altered by the co-transcriptional addition of one extra G residue to produce the mRNA for the V non-structural protein. Using tissues of phocine distemper virus (PDV) infected seals, canine distemper virus (CDV) infected dogs and rinderpest virus (RPV) infected cattle, it was demonstrated that editing occurs in vivo. The P:V mRNA ratios were generally similar to those found in tissue culture infections with the same virus and a minor fraction of transcripts had 2-4 extra G residues. In one seal brain infected with PDV the ratio of P:V mRNA was reversed but no differences were found in the levels of mRNA editing in different tissues from the same animal infected with CDV or RPV. However, variation was seen between animals infected with different isolates of RPV and even between animals infected with the same isolate of RPV.