Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family that includes Dengue, West ... more Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family that includes Dengue, West Nile, Yellow Fever and Japanese encephalitis viruses, causing a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here, we determine the importance of the human skin in the entry of ZIKV and its contribution to the induction of anti-viral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, among which DC-SIGN, AXL, TYRO3, and to a lesser extent, TIM-1, permitted ZIKV entry with a major role for the TAM receptor AXL. ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing Ab and specific RNA silencing. ZIKV induced the transcription of TLR-3, RIG-I and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15 and MX1, characterized by a strongly enhanced interferon-β gene expression. ZIKV was found to be sensitive to the antiviral effect of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy or the specific autophagy inhibitor 3-Methyladenine. The results presented herein permit to gain better insight in the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging Flavivirus. Zika virus (ZIKV) is an arbovirus belonging to Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor, and cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide for the first time a general insight into the interaction between ZIKV and its mammalian host.
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, Jan 3, 2015
Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bit... more Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bite of infected arthropods. Early transmission to vertebrates is initiated by skin puncture and deposition of virus in this organ. However, events at the bite site remain largely unknown. Here, we report that Chikungunya virus (CHIKV) and West Nile virus (WNV), despite belonging to distinct viral families, elicit a common antiviral signature in primary human dermal fibroblasts, attesting for the up regulation of interferon signalling pathways and leading to an increased expression of IFN-β, interleukins and chemokines. Remarkably, CHIKV and WNV enhance IL-1β expression and induce maturation of caspase-1, indicating the capacity of these pathogens to elicit activation of the inflammasome program in resident skin cells. CHIKV and WNV also induce the expression of the inflammasome sensor AIM2 in dermal fibroblasts, whereas inhibition of caspase-1 and AIM2 with siRNA interferes with both CHIKV...
Due to climate change and the propagation of competent arthropods worldwide, arboviruses have bec... more Due to climate change and the propagation of competent arthropods worldwide, arboviruses have become pathogens of major medical importance. Early transmission to vertebrates is initiated by skin puncture and deposition of virus together with arthropod saliva in the epidermis and dermis. Saliva components have the capacity to modulate skin cell responses by enhancing and/or counteracting initial replication and establishment of systemic viral infection. Here, we review the nature of the cells targeted by arboviruses at the skin level and discuss the type of cellular responses elicited by these pathogens in light of the immunomodulatory properties of arthropod vector-derived salivary factors injected at the inoculation site. Understanding cutaneous arbovirus-host interactions may provide new clues for the design of future therapeutics.
We report the diversity and polymorphism of phospholipase A(2) (PLA(2)) transcripts from snakes b... more We report the diversity and polymorphism of phospholipase A(2) (PLA(2)) transcripts from snakes belonging to nine European viper subspecies. This diversity results in the expression of a combination of six PLA(2) species--ammodytin I1, ammodytin I2, ammodytin L, ammodytoxin, vaspin A and vaspin B--with 19 known isoforms of the first five of these species. Most of the European viper venoms studied contained either a myotoxin or a neurotoxin, and all contained ammodytin I1 and ammodytin I2. There is no evidence that a given pattern of PLA(2) species constitutes a taxonomic criterion, and isoform analysis would be required for such discrimination. Analysis of the phylogenetic relationships between PLA(2) species from European vipers and those of other members of the Viperinae revealed a strong correlation between the geographical source of the viper and the clustering seen for the different isoforms, for each PLA(2) species. The K(a)/K(s) values calculated for the mature protein-coding region of paralogous genes showed that ratios for pairs including vaspin B or one ammodytoxin isoform were greater than 1.09, whereas those for most of the remaining pairs were less than 1. Different patterns of mutation were observed in comparisons of the different PLA(2) isoforms. The mechanisms directing a mutation toward a precise exon remain unresolved.
ABSTRACT Trimeresurus stejnegeri venom plasminogen activator (TSV-PA), a serine protease sharing ... more ABSTRACT Trimeresurus stejnegeri venom plasminogen activator (TSV-PA), a serine protease sharing 63% sequence identity with batroxobin, a thrombin-like enzyme from Bothrops atrox venom, and 23% sequence identity with tissue type plasminogen activator (t-PA) protease domain, specifically converts human plasminogen to plasmin but does not act on fibrinogen (Zhang et al., 1994). TSV-PA, which contains 12 cysteins in the form of six disulfide bonds, was overexpressed in E. coli as inclusion bodies, then refolded and purified with a final yield of 10-15 mg from 1 litre of cell culture. The recombinant TSV-PA had the same specific activity as the natural enzyme, when assayed on a variety of substrates including plasminogen. The three regions of TSV-PA, located in the surface loops around the catalytic site, which differed the most with batroxobin, were replaced by corresponding batroxobin residues: region A, residues SNNGF (60-64, chymotrypsin numbering system) was replaced by RRMFR; region B, residues DDE (96a-98), by NVI; and region C, residues SWRQV (173-176), by NGFPL. The replacement in TSV-PA of DDE (96a-98) by NVI abolished its plasminogen activation activity without affecting the activity on small chromogenic substrates. Furthermore, three monomodified variants were constructed: D(96a)-N, D(97)-V and E(98)-1. All three mutants have increased Km value and decreased kcat value, replacement D(97)-V being much more critical than the two other modifications. None of these substitutions generated a fibrinogen clotting activity or direct fibrinogenolytic activity. A molecular model of TSV-PA and its interaction with plasminogen is proposed.
The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurot... more The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (beta-neurotoxins): single-chain beta-neurotoxins, such as agkistrodotoxin and ammodytoxin-A, and dimeric beta-neurotoxins, which, in the case of the best studied ones, crotoxin-like toxins, consist of the non-covalent association of a phospholipase A2 (CB) and a non-enzymatic chaperon (CA). Possible evolutionary relationships of these beta-neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex. CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti-CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone. A 10-fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA.agkistrodotoxin. Ammodytoxin A did not compete with the anti-CA mAb. These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).
Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family that includes Dengue, West ... more Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family that includes Dengue, West Nile, Yellow Fever and Japanese encephalitis viruses, causing a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here, we determine the importance of the human skin in the entry of ZIKV and its contribution to the induction of anti-viral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, among which DC-SIGN, AXL, TYRO3, and to a lesser extent, TIM-1, permitted ZIKV entry with a major role for the TAM receptor AXL. ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing Ab and specific RNA silencing. ZIKV induced the transcription of TLR-3, RIG-I and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15 and MX1, characterized by a strongly enhanced interferon-β gene expression. ZIKV was found to be sensitive to the antiviral effect of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy or the specific autophagy inhibitor 3-Methyladenine. The results presented herein permit to gain better insight in the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging Flavivirus. Zika virus (ZIKV) is an arbovirus belonging to Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor, and cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide for the first time a general insight into the interaction between ZIKV and its mammalian host.
Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, Jan 3, 2015
Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bit... more Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bite of infected arthropods. Early transmission to vertebrates is initiated by skin puncture and deposition of virus in this organ. However, events at the bite site remain largely unknown. Here, we report that Chikungunya virus (CHIKV) and West Nile virus (WNV), despite belonging to distinct viral families, elicit a common antiviral signature in primary human dermal fibroblasts, attesting for the up regulation of interferon signalling pathways and leading to an increased expression of IFN-β, interleukins and chemokines. Remarkably, CHIKV and WNV enhance IL-1β expression and induce maturation of caspase-1, indicating the capacity of these pathogens to elicit activation of the inflammasome program in resident skin cells. CHIKV and WNV also induce the expression of the inflammasome sensor AIM2 in dermal fibroblasts, whereas inhibition of caspase-1 and AIM2 with siRNA interferes with both CHIKV...
Due to climate change and the propagation of competent arthropods worldwide, arboviruses have bec... more Due to climate change and the propagation of competent arthropods worldwide, arboviruses have become pathogens of major medical importance. Early transmission to vertebrates is initiated by skin puncture and deposition of virus together with arthropod saliva in the epidermis and dermis. Saliva components have the capacity to modulate skin cell responses by enhancing and/or counteracting initial replication and establishment of systemic viral infection. Here, we review the nature of the cells targeted by arboviruses at the skin level and discuss the type of cellular responses elicited by these pathogens in light of the immunomodulatory properties of arthropod vector-derived salivary factors injected at the inoculation site. Understanding cutaneous arbovirus-host interactions may provide new clues for the design of future therapeutics.
We report the diversity and polymorphism of phospholipase A(2) (PLA(2)) transcripts from snakes b... more We report the diversity and polymorphism of phospholipase A(2) (PLA(2)) transcripts from snakes belonging to nine European viper subspecies. This diversity results in the expression of a combination of six PLA(2) species--ammodytin I1, ammodytin I2, ammodytin L, ammodytoxin, vaspin A and vaspin B--with 19 known isoforms of the first five of these species. Most of the European viper venoms studied contained either a myotoxin or a neurotoxin, and all contained ammodytin I1 and ammodytin I2. There is no evidence that a given pattern of PLA(2) species constitutes a taxonomic criterion, and isoform analysis would be required for such discrimination. Analysis of the phylogenetic relationships between PLA(2) species from European vipers and those of other members of the Viperinae revealed a strong correlation between the geographical source of the viper and the clustering seen for the different isoforms, for each PLA(2) species. The K(a)/K(s) values calculated for the mature protein-coding region of paralogous genes showed that ratios for pairs including vaspin B or one ammodytoxin isoform were greater than 1.09, whereas those for most of the remaining pairs were less than 1. Different patterns of mutation were observed in comparisons of the different PLA(2) isoforms. The mechanisms directing a mutation toward a precise exon remain unresolved.
ABSTRACT Trimeresurus stejnegeri venom plasminogen activator (TSV-PA), a serine protease sharing ... more ABSTRACT Trimeresurus stejnegeri venom plasminogen activator (TSV-PA), a serine protease sharing 63% sequence identity with batroxobin, a thrombin-like enzyme from Bothrops atrox venom, and 23% sequence identity with tissue type plasminogen activator (t-PA) protease domain, specifically converts human plasminogen to plasmin but does not act on fibrinogen (Zhang et al., 1994). TSV-PA, which contains 12 cysteins in the form of six disulfide bonds, was overexpressed in E. coli as inclusion bodies, then refolded and purified with a final yield of 10-15 mg from 1 litre of cell culture. The recombinant TSV-PA had the same specific activity as the natural enzyme, when assayed on a variety of substrates including plasminogen. The three regions of TSV-PA, located in the surface loops around the catalytic site, which differed the most with batroxobin, were replaced by corresponding batroxobin residues: region A, residues SNNGF (60-64, chymotrypsin numbering system) was replaced by RRMFR; region B, residues DDE (96a-98), by NVI; and region C, residues SWRQV (173-176), by NGFPL. The replacement in TSV-PA of DDE (96a-98) by NVI abolished its plasminogen activation activity without affecting the activity on small chromogenic substrates. Furthermore, three monomodified variants were constructed: D(96a)-N, D(97)-V and E(98)-1. All three mutants have increased Km value and decreased kcat value, replacement D(97)-V being much more critical than the two other modifications. None of these substitutions generated a fibrinogen clotting activity or direct fibrinogenolytic activity. A molecular model of TSV-PA and its interaction with plasminogen is proposed.
The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurot... more The venoms from Crotalinae and Viperinae snakes contain only two kinds of phospholipase A2 neurotoxins (beta-neurotoxins): single-chain beta-neurotoxins, such as agkistrodotoxin and ammodytoxin-A, and dimeric beta-neurotoxins, which, in the case of the best studied ones, crotoxin-like toxins, consist of the non-covalent association of a phospholipase A2 (CB) and a non-enzymatic chaperon (CA). Possible evolutionary relationships of these beta-neurotoxins have been investigated by analyzing whether CA could behave as a chaperon toward agkistrodotoxin and ammodytoxin, as it does in the crotoxin complex. CA increased the lethal potency of agkistrodotoxin and modified its pharmacological effect on Torpedo synaptosomes. Sedimentation experiments proved that CA can form an heterocomplex with agkistrodotoxin. Agkistrodotoxin prevented the binding to CA of an anti-CA mAb which recognizes an epitope at the zone of interaction between crotoxin subunits, suggesting the association of CA and agkistrodotoxin implicated the same zone. A 10-fold molar excess of CA over ammodytoxin modified the effect of ammodytoxin on acetylcholine release but did not increase the lethal potency of ammodytoxin. Sedimentation experiments showed CA and ammodytoxin can form an heterocomplex which is less stable than CA.agkistrodotoxin. Ammodytoxin A did not compete with the anti-CA mAb. These observations are in good agreement with the sequence similarities between CB and agkistrodotoxin (80%) and ammodytoxin A (60%).
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Papers by Valerie Choumet