The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been dete... more The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.
Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuro... more Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer
The family Rhabdoviridae includes 20 genera and 144 species of viruses with negative-sense, singl... more The family Rhabdoviridae includes 20 genera and 144 species of viruses with negative-sense, single-stranded RNA genomes of approximately 10–16 kb (Table 1.Rhabdoviridae). Virions are typically enveloped with bullet-shaped or bacilliform morphology but nonenveloped filamentous virions have also been reported. The genomes are usually (but not always) single RNA molecules with partially complementary termini. Almost all rhabdovirus genomes have 5 genes encoding the structural proteins (N, P, M, G and L); however, many rhabdovirus genomes encode other proteins in additional genes or in alternative open reading frames (ORFs) within the structural protein genes. The family is ecologically diverse with members infecting plants or animals including mammals, birds, reptiles or fish. Rhabdoviruses are also detected in invertebrates, including arthropods some of which may serve as unique hosts or may act as biological vectors for transmission to other animals or plants. Rhabdoviruses include i...
The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible ... more The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH 2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against ...
A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed ... more A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed to identify the major agents of bronchiolitis in infants: respiratory syncytial viruses A and B (RSVA and RSVB) and parainfluenzavirus 3 (PIV3). Two primer sets (P1-P2 and P1-P3) were selected in a conserved region of the polymerase L gene. In infected cell cultures, this method detected RSVA ( n = 14), RSVB ( n = 13), and PIV3 ( n = 13), with the exclusion of PIV1 ( n = 4), PIV2 ( n = 3), measles virus ( n = 6), mumps virus ( n = 4), influenza A virus ( n = 11), and influenza B virus ( n = 4). The differentiation of the amplicons by restriction fragment length polymorphism (RFLP) showed a Pvu II site for PIV3 strains and an Ava II site for RSV strains, with RSVA distinguished from RSVB by Bgl II. The hybridization-EIA, using three internal probes specific for each virus, correlated with the immunofluorescence assay (IFA) and RFLP results. Clinical aspirates from 261 infants hospitalized...
Truncated and chimeric lyssavirus glycoprotein (G) genes were used to carry and express non-lyssa... more Truncated and chimeric lyssavirus glycoprotein (G) genes were used to carry and express non-lyssavirus B and T cell epitopes for DNA-based immunization of mice, with the aim of developing a multivalent vaccine prototype. Truncated G (GPVIII) was composed of the C-terminal half (aa 253–503) of the Pasteur rabies virus (PV: genotype 1) G containing antigenic site III and the transmembrane and cytoplasmic domains. The chimeric G (GEBL1-PV) was composed of the N-terminal half (aa 1–250) of the European bat lyssavirus 1 (genotype 5) G containing antigenic site II linked to GPVIII. Antigenic sites II and III are involved in the induction of virus-neutralizing antibodies. The B cell epitope was the C3 neutralization epitope of the poliovirus type 1 capsid VP1 protein. The T cell epitope was the H2dMHC I-restricted epitope of the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) involved in the induction of both cytotoxic T cell (CTL) production and protection against LCMV. Truncat...
To understand the mutations and genetic rearrangements that allow rabies virus infections of new ... more To understand the mutations and genetic rearrangements that allow rabies virus infections of new hosts and adaptation in nature, the quasispecies structure of the nucleoprotein and glycoprotein genes as well as two noncoding sequences of a rabies virus genome were determined. Gene sequences were obtained from the brain and from the salivary glands of the original host, a naturally infected European fox, and after serial passages in mice, dogs, cats and cell culture. A relative genetic stasis of the consensus sequences confirmed previous results about the stability of rabies virus. At the quasispecies level, the mutation frequency varies, in the following order: glycoprotein region (21.9 x 10(-4) mutations per bp), noncoding sequence nucleoprotein-phosphoprotein region (7.2-7.9 x 10(-4) mutations per bp) and nucleoprotein gene region (2.9-3.7 x 10(-4) mutations per bp). These frequencies varied according to the number, type of heterologous passages and the genomic region considered. ...
The Onderstepoort journal of veterinary research, 1993
Currently, the Lyssavirus genus is composed of four serotypes and the ungrouped European bat lyss... more Currently, the Lyssavirus genus is composed of four serotypes and the ungrouped European bat lyssaviruses (EBL). Using previously described PCR amplification and direct sequencing methods, the N genes of 70 representative lyssaviruses collected from 40 different countries were examined. From the results, a phylogenetic tree with six branches (genotypes) was constructed: genotypes 1-4 correlated with the classification of serotypes 1-4 and in addition EBL 1 and EBL 2 were identified as two further genotypes (5 and 6). Analysis of the 51 rabies (genotype 1) viruses in the study identified nine distinct groups which could be correlated with their geographical origins. A comparison of the six genotypes with four principal Vesiculovirus serotypes showed that their relationship was closer than that of even the two most closely related Vesiculovirus serotypes. Antigenic site sequences of isolates from vaccine/treatment failures were similar to vaccine and field strains, suggesting that fai...
The Onderstepoort journal of veterinary research, 1993
The replication strategy, genome organization and extent of variation within the genome of the ge... more The replication strategy, genome organization and extent of variation within the genome of the genus Lyssavirus is briefly reviewed. Strategies used in the approach to genome studies are discussed.
The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N-RNA ... more The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N-RNA complex is the template for viral transcription and replication. The viral phosphoprotein (P) has two functions during the infection process: it binds through its carboxy-terminus to N in the N-RNA complex and at the same time with an amino-terminal domain to the polymerase and in this way fixes the polymerase to its template. The second function of P is to bind to newly produced N in the infected cell in order to prevent that N binds non-specifically and irreversibly to cellular RNA. In order to identify the part of the phosphoprotein that binds to N and keeps the latter soluble, we isolated the N-P complex, performed sequential protease digestions, and determined the identity of the remaining N and P peptides in the purified digested complex. Although the digestion steps removed short sequences of N, most of N remained intact and soluble, indicating that the overall structure was not affected. Most of P, including the carboxy-terminal N-RNA-binding domain, was removed during the first digestion step. N-terminal sequencing and mass spectrometry analysis identified a P peptide containing residues 4-40 that remained associated with N. Coexpression and coimmunoprecipitation experiments and yeast two-hybrid experiments showed that this peptide alone could bind to N in vivo.
Given the growing number of diseases caused by emerging or endemic viruses, original strategies a... more Given the growing number of diseases caused by emerging or endemic viruses, original strategies are urgently required: (1) for the identification of new drugs active against new viruses and (2) to deal with viral mutants in which resistance to existing antiviral molecules has been selected. In this context, antiviral peptides constitute a promising area for disease prevention and treatment. The identification and development of these inhibitory peptides require the high-throughput screening of combinatorial libraries. Phage-display is a powerful technique for selecting unique molecules with selective affinity for a specific target from highly diverse combinatorial libraries. In the last 15 years, the use of this technique for antiviral purposes and for the isolation of candidate inhibitory peptides in drug discovery has been explored. We present here a review of the use of phage display in antiviral research and drug discovery, with a discussion of optimized strategies combining the strong screening potential of this technique with complementary rational approaches for identification of the best target. By combining such approaches, it should be possible to maximize the selection of molecules with strong antiviral potential.
We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus tr... more We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to i...
Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora an... more 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 Sawyer's 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 ( d S ) to nonsynonymous substitutions ( d N ) (>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 suppor...
Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8... more 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 ...
We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies vi... more We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, a...
Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. Th... more Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein–reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained...
The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been dete... more The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.
Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuro... more Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer
The family Rhabdoviridae includes 20 genera and 144 species of viruses with negative-sense, singl... more The family Rhabdoviridae includes 20 genera and 144 species of viruses with negative-sense, single-stranded RNA genomes of approximately 10–16 kb (Table 1.Rhabdoviridae). Virions are typically enveloped with bullet-shaped or bacilliform morphology but nonenveloped filamentous virions have also been reported. The genomes are usually (but not always) single RNA molecules with partially complementary termini. Almost all rhabdovirus genomes have 5 genes encoding the structural proteins (N, P, M, G and L); however, many rhabdovirus genomes encode other proteins in additional genes or in alternative open reading frames (ORFs) within the structural protein genes. The family is ecologically diverse with members infecting plants or animals including mammals, birds, reptiles or fish. Rhabdoviruses are also detected in invertebrates, including arthropods some of which may serve as unique hosts or may act as biological vectors for transmission to other animals or plants. Rhabdoviruses include i...
The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible ... more The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH 2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against ...
A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed ... more A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed to identify the major agents of bronchiolitis in infants: respiratory syncytial viruses A and B (RSVA and RSVB) and parainfluenzavirus 3 (PIV3). Two primer sets (P1-P2 and P1-P3) were selected in a conserved region of the polymerase L gene. In infected cell cultures, this method detected RSVA ( n = 14), RSVB ( n = 13), and PIV3 ( n = 13), with the exclusion of PIV1 ( n = 4), PIV2 ( n = 3), measles virus ( n = 6), mumps virus ( n = 4), influenza A virus ( n = 11), and influenza B virus ( n = 4). The differentiation of the amplicons by restriction fragment length polymorphism (RFLP) showed a Pvu II site for PIV3 strains and an Ava II site for RSV strains, with RSVA distinguished from RSVB by Bgl II. The hybridization-EIA, using three internal probes specific for each virus, correlated with the immunofluorescence assay (IFA) and RFLP results. Clinical aspirates from 261 infants hospitalized...
Truncated and chimeric lyssavirus glycoprotein (G) genes were used to carry and express non-lyssa... more Truncated and chimeric lyssavirus glycoprotein (G) genes were used to carry and express non-lyssavirus B and T cell epitopes for DNA-based immunization of mice, with the aim of developing a multivalent vaccine prototype. Truncated G (GPVIII) was composed of the C-terminal half (aa 253–503) of the Pasteur rabies virus (PV: genotype 1) G containing antigenic site III and the transmembrane and cytoplasmic domains. The chimeric G (GEBL1-PV) was composed of the N-terminal half (aa 1–250) of the European bat lyssavirus 1 (genotype 5) G containing antigenic site II linked to GPVIII. Antigenic sites II and III are involved in the induction of virus-neutralizing antibodies. The B cell epitope was the C3 neutralization epitope of the poliovirus type 1 capsid VP1 protein. The T cell epitope was the H2dMHC I-restricted epitope of the nucleoprotein of lymphocytic choriomeningitis virus (LCMV) involved in the induction of both cytotoxic T cell (CTL) production and protection against LCMV. Truncat...
To understand the mutations and genetic rearrangements that allow rabies virus infections of new ... more To understand the mutations and genetic rearrangements that allow rabies virus infections of new hosts and adaptation in nature, the quasispecies structure of the nucleoprotein and glycoprotein genes as well as two noncoding sequences of a rabies virus genome were determined. Gene sequences were obtained from the brain and from the salivary glands of the original host, a naturally infected European fox, and after serial passages in mice, dogs, cats and cell culture. A relative genetic stasis of the consensus sequences confirmed previous results about the stability of rabies virus. At the quasispecies level, the mutation frequency varies, in the following order: glycoprotein region (21.9 x 10(-4) mutations per bp), noncoding sequence nucleoprotein-phosphoprotein region (7.2-7.9 x 10(-4) mutations per bp) and nucleoprotein gene region (2.9-3.7 x 10(-4) mutations per bp). These frequencies varied according to the number, type of heterologous passages and the genomic region considered. ...
The Onderstepoort journal of veterinary research, 1993
Currently, the Lyssavirus genus is composed of four serotypes and the ungrouped European bat lyss... more Currently, the Lyssavirus genus is composed of four serotypes and the ungrouped European bat lyssaviruses (EBL). Using previously described PCR amplification and direct sequencing methods, the N genes of 70 representative lyssaviruses collected from 40 different countries were examined. From the results, a phylogenetic tree with six branches (genotypes) was constructed: genotypes 1-4 correlated with the classification of serotypes 1-4 and in addition EBL 1 and EBL 2 were identified as two further genotypes (5 and 6). Analysis of the 51 rabies (genotype 1) viruses in the study identified nine distinct groups which could be correlated with their geographical origins. A comparison of the six genotypes with four principal Vesiculovirus serotypes showed that their relationship was closer than that of even the two most closely related Vesiculovirus serotypes. Antigenic site sequences of isolates from vaccine/treatment failures were similar to vaccine and field strains, suggesting that fai...
The Onderstepoort journal of veterinary research, 1993
The replication strategy, genome organization and extent of variation within the genome of the ge... more The replication strategy, genome organization and extent of variation within the genome of the genus Lyssavirus is briefly reviewed. Strategies used in the approach to genome studies are discussed.
The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N-RNA ... more The genomic RNA of rabies virus is always complexed with the viral nucleoprotein (N). This N-RNA complex is the template for viral transcription and replication. The viral phosphoprotein (P) has two functions during the infection process: it binds through its carboxy-terminus to N in the N-RNA complex and at the same time with an amino-terminal domain to the polymerase and in this way fixes the polymerase to its template. The second function of P is to bind to newly produced N in the infected cell in order to prevent that N binds non-specifically and irreversibly to cellular RNA. In order to identify the part of the phosphoprotein that binds to N and keeps the latter soluble, we isolated the N-P complex, performed sequential protease digestions, and determined the identity of the remaining N and P peptides in the purified digested complex. Although the digestion steps removed short sequences of N, most of N remained intact and soluble, indicating that the overall structure was not affected. Most of P, including the carboxy-terminal N-RNA-binding domain, was removed during the first digestion step. N-terminal sequencing and mass spectrometry analysis identified a P peptide containing residues 4-40 that remained associated with N. Coexpression and coimmunoprecipitation experiments and yeast two-hybrid experiments showed that this peptide alone could bind to N in vivo.
Given the growing number of diseases caused by emerging or endemic viruses, original strategies a... more Given the growing number of diseases caused by emerging or endemic viruses, original strategies are urgently required: (1) for the identification of new drugs active against new viruses and (2) to deal with viral mutants in which resistance to existing antiviral molecules has been selected. In this context, antiviral peptides constitute a promising area for disease prevention and treatment. The identification and development of these inhibitory peptides require the high-throughput screening of combinatorial libraries. Phage-display is a powerful technique for selecting unique molecules with selective affinity for a specific target from highly diverse combinatorial libraries. In the last 15 years, the use of this technique for antiviral purposes and for the isolation of candidate inhibitory peptides in drug discovery has been explored. We present here a review of the use of phage display in antiviral research and drug discovery, with a discussion of optimized strategies combining the strong screening potential of this technique with complementary rational approaches for identification of the best target. By combining such approaches, it should be possible to maximize the selection of molecules with strong antiviral potential.
We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus tr... more We wanted to develop a therapeutic approach against rabies disease by targeting the lyssavirus transcription/replication complex. Because this complex (nucleoprotein N-RNA template processed by the L polymerase and its cofactor, the phosphoprotein P) is similar to that of other negative-strand RNA viruses, we aimed to design broad-spectrum antiviral drugs that could be used as a complement to postexposure vaccination and immunotherapy. Recent progress in understanding the structure/function of the rabies virus P, N, and L proteins predicts that the amino-terminal end of P is an excellent target for destabilizing the replication complex because it interacts with both L (for positioning onto the N-RNA template) and N (for keeping N soluble, as needed for viral RNA encapsidation). Thus, peptides mimicking various lengths of the amino-terminal end of P have been evaluated, as follows: (i) for binding properties to the N-P-L partners by the two-hybrid method; (ii) for their capacity to i...
Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora an... more 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 Sawyer's 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 ( d S ) to nonsynonymous substitutions ( d N ) (>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 suppor...
Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8... more 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 ...
We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies vi... more We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i) an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii) a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, a...
Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. Th... more Lyssaviruses, the causative agents of rabies encephalitis, are distributed in seven genotypes. The phylogenetically distant rabies virus (PV strain, genotype 1) and Mokola virus (genotype 3) were used to develop a strategy to identify functional homologous interactive domains from two proteins (P and N) which participate in the viral ribonucleoprotein (RNP) transcription-replication complex. This strategy combined two-hybrid and green fluorescent protein–reverse two-hybrid assays in Saccharomyces cerevisiae to analyze protein-protein interactions and a reverse genetic assay in mammalian cells to study the transcriptional activity of the reconstituted RNP complex. Lyssavirus P proteins contain two N-binding domains (N-BDs), a strong one encompassing amino acid (aa) 176 to the C terminus and a weak one in the 189 N-terminal aa. The N-terminal portion of P (aa 52 to 189) also contains a homomultimerization site. Here we demonstrate that N-P interactions, although weaker, are maintained...
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Papers by Noël Tordo