Noël Tordo

Identification of four conserved motifs among the RNA-dependent polymerase encoding elements.

Four consensus sequences are conserved with the same linear arrangement in RNA‐dependent DNA polymerases encoded by retroid elements and in RNA‐dependent RNA polymerases encoded by plus‐, minus‐ and double‐strand RNA viruses. One of these motifs corresponds to the YGDD span previously described by Kamer and Argos (1984). These consensus sequences altogether lead to 4 strictly and 18 conservatively maintained amino acids embedded in a large domain of 120 to 210 amino acids. As judged from secondary structure predictions, each of the 4 motifs, which may cooperate to form a well‐ordered domain, places one invariant amino acid in or proximal to turn structures that may be crucial for their correct positioning in a catalytic process. We suggest that this domain may constitute a prerequisite ‘polymerase module’ implicated in template seating and polymerase activity. At the evolutionary level, the sequence similarities, gap distribution and distances between each motif strongly suggest that the ancestral polymerase module was encoded by an individual genetic element which was most closely related to the plus‐strand RNA viruses and the non‐viral retroposons. This polymerase module gene may have subsequently propagated in the viral kingdom by distinct gene set recombination events leading to the wide viral variety observed today.

Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

Host Switching in Lyssavirus History from the Chiroptera to the Carnivora Orders

ABSTRACT Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora and Chiroptera orders. We studied 36 carnivoran and 17 chiropteran lyssaviruses representing the main genotypes and variants. We compared their genes encoding the surface glycoprotein, which is responsible for receptor recognition and membrane fusion. The glycoprotein is the main protecting antigen and bears virulence determinants. Point mutation is the main force in lyssavirus evolution, as Sawyers test and phylogenetic analysis showed no evidence of recombination. Tests of neutrality indicated a neutral model of evolution, also supported by globally high ratios of synonymous substitutions (dS ) to nonsynonymous substitutions (dN ) (>7). Relative-rate tests suggested similar rates of evolution for all lyssavirus lineages. Therefore, the absence of recombination and similar evolutionary rates make phylogeny-based conclusions reliable. Phylogenetic reconstruction strongly supported the hypothesis that host switching occurred in the history of lyssaviruses. Indeed, lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies, very likely following spillovers from bats. Using dated isolates, the average rate of evolution was estimated to be roughly 4.3 × 10−4 dS /site/year. Consequently, the emergence of carnivoran rabies from chiropteran lyssaviruses was determined to have occurred 888 to 1,459 years ago. Glycoprotein segments accumulating more dN than dS were distinctly detected in carnivoran and chiropteran lyssaviruses. They may have contributed to the adaptation of the virus to the two distinct mammal orders. In carnivoran lyssaviruses they overlapped the main antigenic sites, II and III, whereas in chiropteran lyssaviruses they were located in regions of unknown functions.

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity.

ABSTRACT The genetic diversity of representative members of theLyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), theEuropean bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and theAustralian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.

Taxonomy of the order Mononegavirales: update 2016

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Cytoplasmic Dynein LC8 Interacts with Lyssavirus Phosphoprotein

ABSTRACT Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon “retrograde transport” and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.

PCR technique as an alternative method for diagnosis and molecular epidemiology of rabies virus

We have investigated the PCR amplification technique of viral nucleic acids as an alternative protocol for diagnosis and epidemiological studies of rabies virus. A primer set mapping in the nucleoprotein cistron allowed a specific and sensitive amplification of infected brain material, fulfilling the diagnosis requirements. One hundred samples checked by Southern or dot-blot analysis using both radioactive and non-radioactive probes showed identical results in parallel with routine techniques. For molecular epidemiological studies we selected another set of conserved primers flanking the highly evolutive pseudogene (psi gene) region. This set was found to be efficient for all tested fixed rabies virus strains or wild rabies virus isolates as well as the rabies-related Mokola virus. We describe a progressive characterization of the strain that could be extended from rapid typing by a limited panel of restriction enzymes, to the ultimate identification of the nucleotide sequence by an original direct sequencing technique of amplified segments.

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Targeting of incoming retroviral Gag to the centrosome involves a direct interaction with the dynein light chain 8.

The role of cellular proteins in the replication of retroviruses, especially during virus assembly, has been partly unraveled by recent studies. Paradoxically, little is known about the route taken by retroviruses to reach the nucleus at the early stages of infection. To get insight into this stage of virus replication, we have studied the trafficking of foamy retroviruses and have previously shown that incoming viral proteins reach the microtubule organizing center (MTOC) prior to nuclear translocation of the viral genome. Here, we show that incoming viruses concentrate around the MTOC as free and structured capsids. Interestingly, the Gag protein, the scaffold component of viral capsids, targets the pericentrosomal region in transfected cells in the absence of any other viral components but in a microtubule- and dynein/dynactin-dependent manner. Trafficking of Gag towards the centrosome requires a minimal 30 amino acid coiled-coil motif in the N-terminus of the molecule. Finally, we describe a direct interaction between Gag and dynein light chain 8 that probably accounts for the specific routing of the incoming capsids to the centrosome prior to nuclear import of the viral genome.

DNA-based immunization for exploring the enlargement of immunological cross-reactivity against the lyssaviruses

DNA-based immunization was used for studying the cross-reactivity of lyssavirus neutralizing antibodies and for exploring the induction of a wider range of protection against lyssaviruses. In order to immunize mice with homogeneous and chimeric genes of glycoproteins (G) from two divergent lyssaviruses, we used for the first time a new plasmid (pCI-neo) known to be a highly efficient vector for in vitro expression. The homogeneous plasmids pGPV and pGMok encoded the Pasteur virus (PV: genotype 1-GT-) and Mokola virus (Mok: GT 3) G, respectively. The chimeric pGMokPV encoded the NH2 part of GMok and the COOH part of GPV. These plasmids elicited full protection against intracerebral challenges with various lyssaviruses and a range of antigen-specific and non-specific immune responses. Virus neutralizing antibody (VNAb) levels were dose dependent and a single intramuscular (i.m.) injection of plasmids was sufficient to induce continuous high levels of VNAb. Production of antigen-specific T helper (Th), cytotoxic T cells (Tc) and non-specific natural killer cells was observed. Cross-reactivity studies showed that VNAb are obtained by immunizing with: (i) pGPV against GT 1 (classical rabies), GT 4 (Duvenhage: Duv), GT 5 (European Bat Lyssavirus: EBL-1) and GT 6 (European Bat Lyssavirus: EBL-2); (ii) pGMok against GT 2 (Lagos Bat: LB) and GT 3 (Mokola: Mok); (iii) pGMokPV against all GTs except GT 4 which is weakly neutralized. Therefore, the DNA-based immunization with the chimeric pGMokPV, could be very interesting to enlarge protection to all the lyssaviruses. According to the cross-reactivity of VNAb induced by the G genes, the lyssavirus GTs could be classified into two groups: the first including GT 1, 4, 5 and 6; the second including GT 2 and 3.