The aim of this study was to investigate the epidemiology of Rift Valley fever (RVF) in South Africa. The first part of the study consisted of the compilation of a full history of RVF in South Africa. This was done by compiling all references to outbreaks of the disease in South Africa from all available literature, annual reports, disease reports and animal disease databases. The geographic location and temporal occurrence of each outbreak was recorded as accurately as allowed by the available records. The result was a better and more complete picture than has hitherto been available of the spatial and temporal distribution of RVF for the period 1950, when the disease was first recognised in South Africa, to 2010. Several smaller outbreaks not mentioned in the literature were found. It emerged that large outbreaks occur in the Free State Province, Eastern Cape Province and Northern Cape Province with long periods of absence and smaller outbreaks occur in KwaZulu-Natal, Mpumalanga and Gauteng at more frequent intervals.The second part of the study used the data collected during the first part of the study to determine which climatic and other environmental factors could have played a role in the occurrence of RVF in South Africa. Multiple logistic regression analysis was used to estimate associations between the various potential risk factors and the occurrence of Rift Valley fever.The study found that the El Niño/Southern Oscillation influence on rainfall in South Africa has an effect on the occurrence of RVF in South Africa which is opposite to the effect that has been described for Kenya. A positive Southern Oscillation Index (La Niña) increases the likelihood of a RVF outbreak in South Africa.The study also found that very high rainfall during the summer months (December to February) is an important risk factor for the occurrence of RVF and it confirmed the increased risk of an outbreak where pans and wetlands are present as reported in several articles and disease reports on past outbreaks. Several other factors, such as minimum and maximum temperature were also found to have a statistically significant effect on the occurrence of Rift Valley fever. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Production Animal Studies / unrestricted

No description available.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus that is classified as an overlap select agent by the USDA and the HHS. It was first discovered in the Rift Valley of Kenya in the early 1930s. RVFV is an arbovirus that is transmitted by mosquitoes and infects ruminants and humans. RVFV in humans causes an acute self-limiting febrile illness but in a small percentage of cases, a severe version is noted by ocular disease, hepatitis, hemorrhagic fever, and death. In ruminants, the disease is similar with young livestock being the most susceptible. RVFV is also known to cause "abortion storms" where infected pregnant ruminants abort their fetuses with a near 100% fatality rate. Viruses are obligate intracellular parasites utilizing host-factors to replicate. This study identified three host-protein interactors of the viral Gn and L proteins that aid in viral replication. UBR4 was determined to be an interactor of Gn via immunoprecipitation followed by either LC/MS/MS or western blot analysis. Its inhibition via siRNA or CRISPR-Cas9 knockout showed a reduction of viral titers and viral RNA production. It was determined that UBR4 specifically affects viral RNA production and not entry or egress. Conversely, CK1α and PP1α were identified as binding partners of the L protein using similar methods. CK1α, a kinase, and PP1α, a phosphatase, were chosen for further verification due to data demonstrating the L protein is phosphorylated on at least one serine residue, in addition to PP1α already being shown to impact RVFV replication. Inhibition of CK1 and PP1 via small molecule inhibitors, D4476 and 1E7-03, respectively, showed a decrease in viral titers and RNA production. Strand-specific RT-qPCR demonstrates that CK1 and PP1 impact genomic replication. Upon treatment with D4476 a decrease in L protein phosphorylation was observed. Additionally, it has already been shown that treatment with 1E7-03 increases L protein phosphorylation. These data indicate that CK1 and PP1 modulate L protein phosphorylation, contributing to changes in RVFV replication. This study identifies three host-proteins that affect viral replication, which could be used as a foundation for host-based therapeutic and vaccine development. / Doctor of Philosophy / Rift Valley fever virus (RVFV) is a major biological threat due to its ability to infect both livestock and humans and be passed by mosquito bite. RVFV was first discovered in Africa in the early 1930s. To date, there is no approved therapeutic or vaccine. RVFV usually causes very mild disease but in a small percentage of cases, it progresses to include liver disease, vision loss, swelling of the brain, bleeding, and death. A virus itself is not alive; it needs a living host in order to replicate. To do this, it utilizes things naturally occurring inside the host. The purpose of this study is to identify host-factors that the virus uses in order to efficiently make more viruses. The first viral protein of interest is the glycoprotein, Gn, which is important for viral entry and assembly of the viral particles. It was determined that the host-protein UBR4 is an interactor of Gn and that the inhibition of UBR4 decreases the amount of infectious virus being produced. Similarly, the host-proteins, CK1α and PP1α, were found to be interactors of the viral L protein. The L protein is responsible for synthesizing the building blocks of the virus. It was determined that when CK1 and PP1 are inhibited, the L protein is less efficient at making these building blocks. Understanding the host-factors the virus utilizes is important to the basic understanding of how RVFV infects the host and the development of therapeutics to combat an outbreak.

Le Virus du Nil Occidental (VNO) et le Virus de la Fièvre de la Vallée du Rift (VFVR) sont deux arbovirus transmis par le moustique Culex pipiens comprenant deux biotypes: pipiens et molestus. Au cours de ce projet, nous avons évalué la circulation du VNO au Liban dans des populations de moustiques, des humains, des chevaux et des poulets. Nous avons aussi évalué la compétence vectorielle des populations locales de Cx. pipiens à transmettre le VNO et le VFVR.Des moustiques ont été récoltés et testés pour la présence d’un gène spécifique du VNO. En plus, des sérums humains, de chevaux et de poulets ont été analysés pour rechercher des anticorps spécifiques par ELISA puis confirmés par neutralisation. En outre, des spécimens de Cx. pipiens ont été infectés avec la lignée 1 du VNO ou la souche de VFVR Clone 13. Ensuite, les taux d’infection, de dissémination et de transmission ont été déterminés à différents jours après infection des moustiques. La compétence vectorielle a été comparée entre les différents biotypes.Les résultats entomologiques ont révélé que Cx. pipiens est dominant (87.2%). Tous les moustiques analysés étaient négatifs pour le VNO. Les taux de séroprévalence étaient de 1.01% et 1.98% parmi les humains et les chevaux respectivement. De plus, Cx. pipiens s’est révélé bien plus compétent pour transmettre le VNO que le VFVR. Le biotype molestus est capable de transmettre le VNO plus tôt que celui de pipiens. Cette étude présente des preuves sur une faible circulation du VNO au Liban. Cx. pipiens s’est révélé compétent pour assurer cette transmission. Ainsi, il est essentiel d'établir des programmes de surveillance pour prévenir les éventuelles épidémies. / West Nile virus (WNV) and Rift Valley Fever virus (RVFV) are two emerging arboviruses that have never been reported in Lebanon. They can be transmitted by Culex pipiens mosquito species including two biotypes: pipiens and molestus. During this project, we assessed the circulation of WNV among mosquitoes, human, horse and chicken populations in Lebanon. Moreover, we evaluated, under experimental conditions, the capacity of local Cx. pipiens biotypes to transmit both viruses.Adult mosquitoes were collected, identified and tested to detect WNV RNA. Besides, human, horse and chicken blood samples were collected and screened for WNV antibodies using an in-house ELISA and then confirmed by neutralization assay. Moreover, local Cx. pipiens specimens were experimentally infected with WNV lineage 1 or RVFV Clone 13 strain. The viral infection, dissemination and transmission were then estimated at different days post infection.The vector competence was compared between Cx. pipiens biotypes.Entomological results revealed that 87.2% of collected adult mosquitoes were Cx. pipiens. Screened mosquitoes were negative for WNV. Seroprevalence rates were 1.01% and 1.98% among humans and horses respectively. Besides, local Cx. pipiens were highly competent for WNV transmission and to a lesser extent to RVFV. The molestus biotype was able to transmit WNV earlier than pipiens biotype.The present study provides new evidence of a low circulation of WNV among human and horses in Lebanon. Cx. pipiens is the suspected vector and is experimentally competent to ensure transmission. Therefore, there is a need to establish surveillance program to predict and prevent potential outbreaks.

La fièvre de la Vallée du Rift (FVR) est une arbovirose zoonotique décrite pour la première fois en 1930 au Kenya. Transmise principalement entre ruminants par des moustiques des genres Aedes, Culex et Anopheles, elle peut aussi se transmettre à l'homme par contact direct avec des produits d'avortement ou des fluides corporels d'animaux virémiques. Cette maladie a été décrite dans de nombreux pays d'Afrique ainsi que sur la péninsule arabique et dans l'Océan Indien. La circulation du virus de la FVR (VFVR) a été décrite dans 3 écosystèmes distincts : (i) les zones semi-arides sud- et est-africaines, (ii) les mares temporaires des zones arides, (iii) les zones irriguées adjacentes à de grands fleuves. Au sein de chacun de ces écosystèmes, le rôle des mouvements d'animaux dans l'introduction du VFVR et des moustiques vecteurs dans sa transmission ont fortement été mis en avant. Malgré l'existence de modèles statistiques pouvant prédire l'émergence du VFVR en Afrique de l'est notamment grâce au niveau de pluviométrie, les mécanismes de transmissions en jeu dans les autres régions sont encore incertains. L'apparition de foyers dans un écosystème tempéré et montagneux de Madagascar, où les espèces et abondances vectorielles ne sont pas favorable à la persistance du VFVR, suscite des interrogations quant à ces mécanismes. L'objectif de cette thèse est d'identifier les facteurs et les processus épidémiologiques permettant la circulation récurrente du VFVR dans un écosystème tempéré de Madagascar. L'étude a pris en considération les pratiques socio-économiques ainsi que les principaux moustiques vecteurs du virus de la zone tempérée des hautes terres malgaches. Les principaux modes de diffusion du virus entre bovins ont été étudiés au cours de 3 années de suivi sérologique. Un modèle mathématique calibré sur la base de nombreuses données empiriques collectées dans cette zone est proposé. Deux pratiques commerciales ont pu être distinguées : le commerce classique et une pratique traditionnelle de troc. Les résultats de l'analyse suggèrent une implication différente de ces deux pratiques dans la circulation du VFVR. Alors que le commerce pourrait permettre l'introduction du virus, la pratique du troc serait quant à elle le support de la circulation au sein de la zone. Un modèle déterministe est construit afin de comparer quatre scénarios pouvant expliquer la circulation virale dans cet écosystème peu favorable. Les résultats suggèrent que, si la transmission vectorielle reste la principale voie de transmission dans cet écosystème inhabituel, la transmission directe lors du vêlage de vaches virémiques pourrait également jouer un rôle. La circulation du VFVR est de plus favorisée par les pratiques socio-économiques de la zone qui permettent, malgré la saison sèche, l'exposition des bovins introduits et potentiellement virémiques aux moustiques vecteurs. / Rift Valley fever (RVF) is a zoonotic disease first described in 1930 in Kenya. Primarily transmitted between ruminant by mosquitoes of Aedes, Culex and Anopheles genus, it can also be transmitted to humans by direct contact with abortion products or body fluids of viraemic animals. This disease has been described in many African countries, in the Arabian Peninsula and the Indian Ocean. The circulation of RVF virus (RVFV) has been reported in three ecosystems: (i) semi-arid areas of South and East Africa, (ii) temporary ponds in arid areas, (iii) irrigated areas near large rivers. Within each of these ecosystems, the role of animal movements in the introduction of RVFV and that of mosquitoes in virus transmission has been strongly emphasized. Despite the existence of statistical models that predict the emergence of RVFV in East Africa based on rainfall level, the transmission mechanisms involved in other areas are still uncertain. The occurrence of an outbreak in a temperate and mountainous ecosystem of Madagascar, where the species and vector abundances are unfavorable to the persistence of RVFV, raises questions about these mechanisms.The objective of this thesis is to identify the factors and the epidemiological processes that support the RVFV recurrent circulation in a temperate ecosystem of Madagascar. The study took into account the socio-economic practices as well as major mosquito vectors of the area. The main modes of virus spread between cattle were studied during a three years serological follow-up. A mathematical model was elaborated and calibrated using empirical and field data collected in the area. Two cattle exchange practices could be distinguished: the usual trade and a traditional practice of barter. The results of the analysis suggest a different impact of these two practices in RVFV circulation. While trade may allow virus introduction, the barter practice would support its spread within the area. A deterministic model was built to compare four scenarios that could explain the recurrent virus circulation in this unfavorable ecosystem. Results suggest that, if the vector-based transmission remains the main transmission mode, direct transmission from viremic cows at calving could also play a role. RVFV circulation is favored by socio-economic practices of the area that led, despite the dry season, to the exposure of introduced, and potentially viremic, cattle to vectors.

Transmis par les moustiques, le virus de la fièvre de la vallée du Rift (vFVR) est un virus zoonotique qui affecte principalement les ruminants en Afrique et conduit à des pertes économiques importantes. Il n’existe actuellement pas de traitements et les seuls vaccins disponibles sont à usage vétérinaire. Le développement de nouveaux vaccins plus sûrs contre le vFVR est une priorité de l’OMS en raison du risque d’émergence de cet arbovirus dans d’autres continents. Dans cette étude, nous avons développé une vaccination à ADN optimisée contre le vFVR qui consiste à administrer par voie cutanée un plasmide codant pour l’ectodomaine de la glycoprotéine de surface Gn du vFVR (eGn) en présence d’un plasmide adjuvant codant le GM-CSF et combinée avec une électroporation. De plus, nous avons également optimisé la vaccination à ADN en l’associant à la stratégie de ciblage des cellules dendritiques (DCs) via un plasmide qui code des fragments d’anticorps scFv fusionnés avec l’eGn dirigés contre les récepteurs DEC205 et CD11c exprimés à la surface des DCs. Les vaccins ont été testés chez le mouton, hôte naturel du virus et dans le modèle murin pour étudier les mécanismes de protection. Dans nos deux modèles d’études, l’immunisation par le plasmide codant l’eGn confère une meilleure protection après une épreuve virale ainsi qu’une forte production d’anticorps non neutralisants par rapport au ciblage des DCs. En revanche, le ciblage d’eGn vers des récepteurs de DCs protège partiellement contre une épreuve virale et induit une immunogénicité différente dans les deux espèces. Nous avons confirmé le rôle protecteur de ces anticorps anti-eGn par un transfert passif dans le modèle murin et le mécanisme d’action de ces anticorps protecteurs reste encore à être déterminé. Notre étude montre pour la première fois la protection par un vaccin à ADN contre le vFVR chez le mouton. / The Rift valley fever virus (RVFV) is a mosquito-borne virus that mainly affect ruminants in Africa, resulting in economic burden. There is currently no treatment and only vaccine for veterinary use against the RVFV are available. The development of new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus to other continents. In the present work, we developed an optimized DNA vaccination against RVFV using a plasmid encoding the ectodomain of surface glycoprotein Gn (eGn) of RVFV into the skin with plasmid adjuvant encoding GM-CSF and electroporation in sheep. We further optimized the DNA vaccination using dendritic cell targeting strategy with a plasmid encoding a single chain fragment variable (scFv) fused with eGn directed to two DC receptors, DEC205 and CD11c. The efficacy of the vaccines were tested in the sheep, the natural host and in the mouse model to investigate the mechanism of protection. In both models non-targeted eGn vaccine confer a better clinical protection and higher non-neutralizing antibody production than DC-targeted vaccine. However, in both models eGn targeting to DEC205 differentially affected the immune response and induced a partial protection after a challenge. We further demonstrated that non-neutralizing antibodies induced by native eGn protect mice by passive transfer. The mechanism mediated by these antibodies remains to be investigated. Overall, this work indicates the proof of concept that DNA vaccine can confer protection against the RVFV in the sheep.

Rift Valley fever (RVF) is an emerging viral zoonosis that causes frequent outbreaks in east Africa and on the Arabian Peninsula. The likelihood of RVF global expansion due to climate change and human anthropogenic factors is an important issue. The causative agent, RVF virus, is an arbovirus that is transmitted by several mosquito species and is able to infect a wide range of livestock as well as people. The infection leads to mass abortions and death in livestock and a potentially deadly hemorrhagic fever in humans. RVF has severe socio-economic consequences such as animal trade bans between countries, disruption of food security, and economic disaster for farmers and pastoralists as well as for countries. Human behavior such as direct contact with infected animals or their fluids and exposure to mosquito bites increases the risk for contracting the disease. To better understand the challenges associated with RVF outbreaks and to explore prevention and control strategies, we used the One Health approach. The local community had to be involved to understand the interaction between the environment, animals, and humans. We focused on Sudan, Saudi Arabia, and Kenya. First, we systematically reviewed the literature and then we performed cross sectional community-based studies using a special One Health questionnaire. Climatic and remote sensing data were used in combination with statistics to develop a sub-region predictive model for RVF. For both Saudi Arabia and Sudan, the ecology and environment of the affected areas were similar. These areas included irrigation canals and excessive rains that provide an attractive habitat for mosquito vectors to multiply. The surveillance systems were unable to detect the virus in livestock before it spread to humans. Ideally, livestock should serve as sentinels to prevent loss of human lives, but the situation here was reversed. Differences between countries regarding further spread of RVF was mainly determined by better economic and infrastructure resources. In Sudan, there was a lack of knowledge and appropriate practices at the studied community regarding RVF disease symptoms and risk factors for both animals and humans. The community was hesitant in notifying the authorities about RVF suspicion in livestock due to the lack of a compensation system. The perceived role of the community in controlling RVF was fragmented, increasing the probability of RVF transmission and disease. In Kenya, our study found that better knowledge about RVF does not always translate to more appropriate practices that avoid exposure to the disease. However, the combination of good knowledge, attitudes, and practices may explain why certain communities were less affected. Strategies to combat RVF should consider socio-cultural and behavioral differences among communities. We also noticed that RVF outbreaks in Kenya occurred in regions with high livestock density exposed to heavy rains and wet soil fluxes, which could be measured by evapotranspiration and vegetation seasonality variables. We developed a RVF risk map on a sub-regional scale. Future outbreaks could be better managed if such relevant RVF variables are integrated into early warning systems. To confront RVF outbreaks, a policy is needed that better incorporates ecological factors and human interactions with livestock and environment that help the RVF pathogen spread. Early detection and notification of RVF is essential because a delay will threaten the core of International Health Regulations (IHR), which emphasizes the share of information during a transboundary disease outbreak to avoid unnecessary geographical expansion.

La Fièvre de la Vallée du Rift (FVR) est une zoonose observée pour la première fois au Kenya en 1930 qui s'est peu à peu propagée à la plupart des pays d'Afrique. La FVR est une maladie à transmission vectorielle dont le virus appartient au genre Phlebovirus de la famille des Bunyaviridæ. En Afrique de l'Est, l'émergence de foyers est prédite par des modèles statistiques, ce qui n'est pas le cas en Afrique de l'Ouest où les facteurs et les mécanismes en jeu sont encore mal définis. L'objectif de cette thèse est d'identifier les facteurs et les processus épidémiologiques expliquant l'émergence de foyers de FVR au Sénégal, en mettant en œuvre une approche éco-épidémiologique centrée sur les principaux moustiques vecteurs du virus. Par l'étude de variables environnementales et climatiques et par leur exploitation dans des modèles mathématiques, nous avons tenté de répondre à deux questions épidémiologiques majeures : (1) quelles sont les zones potentiellement à risque, et (2) quelles sont les périodes favorables à l'apparition de foyers. L'étude a été menée à l'échelle locale, dans une zone d'environ 10 km2 autour du village de Barkedji situé dans la région sylvo-pastorale du Ferlo.Pour localiser les zones à risque de transmission du virus, nous utilisons la télédétection et l'analyse paysagère afin de caractériser l'environnement favorable aux deux principaux candidats vecteurs du virus, Aedes vexans et Culex poicilipes. Pour identifier les périodes à risque, nous avons développé un modèle d'abondance de populations de moustique des deux espèces vectrices prenant en compte la dynamique des gîtes larvaires (les mares), et dont les simulations ont été validées avec des données de terrain de capture de moustiques. Pour se faire, nous avons dû préalablement développé un modèle dynamique de hauteur d'eau des mares temporaires, modèle calibré et validé à partir de données de terrain et de données d'observation de la Terre.Les résultats de l'analyse paysagère ont confirmé que les milieux favorables aux vecteurs de la maladie pouvaient être caractérisés par télédétection. Ils ont aussi mis en évidence l'importance des mares et de la densité de végétation environnante, et ont abouti à une cartographie de l'hétérogénéité spatiale du risque de circulation de la FVR. Les résultats de l'analyse temporelle ont montré que les années de circulation active du virus coïncidaient avec les années pour lesquelles les deux espèces de moustiques étaient présentes en forte quantité. On observe ainsi deux années à très forte densité des deux moustiques vecteurs, en 1987 et en 2003, correspondant aux années d'épidémie/épizootie les plus importantes dans la région. / The Rift Valley fever (RVF) is an arboviral zoonosis, first identified in Kenya in 1930, which has spread over many African countries. The RVF virus (RVFV) is a mosquito-borne virus member of the family Bunyaviridae, genus Phlebovirus. Statistical models that are used for predicting RVF outbreaks in East Africa do not work in West Africa where the factors and processes involved are still not well described. The aim of this thesis is to identify the factors and epidemiological processes that explain the emergence of RFV outbreaks in Senegal. To achieve that, we have chosen an eco-epidemiological approach targeted on the main candidate mosquito vectors of the RFV virus. By using the environmental and climatic variables and by their exploitation in mathematical models, we tried to answer two major epidemiological questions: (1) where are the potential zones at risk? And, (2) when are the periods favourable to RFV outbreaks?The study has been carried out at a local scale, in an area of about 10 km2 centred on the village of Barkedji in the pastoral Ferlo region in northern Senegal.When identifying risk areas for virus transmission, we used remote sensing and landscape analysis to characterize favourable environments fot the two main candidate vectors, Aedes vexans and Culex poicilipes (Diptera: Culicidae). For predicting risk of RVF outbreaks, we developed a mosquito population model for the two vector species taking into account the dynamics of the ponds as breeding sites. The results of the simulation have been validated with captured mosquito field data. This required the prior development of a temporary pond dynamics model that was calibrated and validated with field and remote sensing data.The results of the landscape analysis confirmed that favourable environments for the mosquito vectors of the RVF could be characterised by remote sensing. The importance of the ponds and the surrounding vegetation density was also highlighted, allowing to map the spatial heterogeneity of RVF circulation risk. The results of the mosquito model simulations showed that years of active virus circulation matched the years when both vector species were densely present. Indeed, the simulations showed high mosquito densities in 1987 and 2003, which correspond to the most important epidemic and epizootic events in that region.

L'autophagie maintient l'homéostasie cellulaire en dégradant les composants cellulaires. Chez l'humain, les protéines LC3 jouent un rôle central dans l'autophagie en interagissant avec d'autres facteurs contenant des régions d'interaction LC3 (LIR). Cette thèse porte sur le rôle de différents facteurs contenant des LIR, tels que le facteur nucléaire DOR et la protéine NSs du virus de la fièvre de la vallée du Rift (VFVR). Les protéines LC3 sont principalement présentes dans le noyau des cellules au repos normales, et leur passage au cytosol en réponse au stress nécessite une interaction avec DOR. Récemment, il a été démontré que cette interaction entre DOR et LC3B dépend de la désacétylation de deux résidus lysine conservés (K49/K51 de LC3A et K46/K48 de GABARAP). Cependant, les détails mécanistiques du rôle des résidus lysine individuels dans le transfert d'autres protéines LC3 demeurent inconnus. De plus, la caractérisation de l'interaction NSs-LC3 ainsi que son impact sur l'autophagie lors de l'infection par le RVFV demeurent évasives. Par conséquent, l'objectif de ces études est d'investiguer les différences structurelles et fonctionnelles des protéines humaines LC3 à différents stades de l'autophagie via leur interaction avec DOR et NSs. Nos études biophysiques et structurales ont permis d’identifier des éléments clés déterminant la spécificité de la région d'interaction LC3 de DOR (DORLIR) pour GABARAP. Nos études structurales ont défini une conformation en feuillet  chez DORLIR lorsqu'elle est en complexe avec GABARAP, ce qui joue un rôle important dans l'établissement de cette spécificité. Les études structurales ont également montré que l'acétylation de la deuxième Lys de GABARAP ou LC3A perturbe des interactions clés du W35 de DORLIR, ce qui conduit à une diminution de l'affinité qui est cohérente avec nos résultats ITC. Ces résultats ont été confirmés grâce à des expériences cellulaires en utilisant des substitutions K-en-Q pour imiter l'acétylation des Lys. En cellules, les substitutions K-en-Q à la deuxième Lys ont entravé le transfert cytoplasmique de GABARAP et de LC3A, ainsi que leur colocalisation avec DOR, tandis que les substitutions K-en-Q à la première Lys se comportent comme des protéines de type sauvage. Dans l'ensemble, la désacétylation de la deuxième Lys conservée est cruciale pour le transfert cytoplasmique de GABARAP et LC3A lors de l'autophagie, ce qui diffère de ce qui a été observé auparavant avec LC3B, où la désacétylation des deux Lys était nécessaire. Cette étude fournit également des informations sur les interactions entre la protéine NSs du VFVR et les protéines LC3, ainsi que l'impact de NSs sur l'autophagie lors de l'infection par le VFVR. Nous avons identifié quatre motifs potentiels d'interaction LC3 (NSs1-4) dans la protéine NSs, et des études d’ITC ont démontré que NSs4 interagit avec une affinité sous micromolaire-micromolaire avec les protéines LC3 humaines. De plus, nous avons confirmé que les protéines LC3 interagissent avec NSs dans les cellules, et que chez les cellules infectées par le RVFV, LC3A colocalise avec NSs. Dans l'ensemble, les résultats indiquent que la protéine NSs joue un rôle clé dans la modification de l'autophagie lors des infections par le VFVR. / Autophagy maintains cellular homeostasis through catabolism of cellular components including organelles, proteins, and pathogens. In humans, the six LC3 (Microtubule-associated protein 1 light chain 3) protein (LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in autophagy through interactions with other factors that contain LC3-interacting regions (LIRs). This study focuses on the role of different factors that contain LIRs such as the nuclear factor DOR and the NSs protein from the RVFV. LC3 proteins are predominantly present in the nucleus of normal resting cells and their shuttling to the cytosol in response to stress requires interaction with DOR. Recently, this interaction between DOR and LC3B was shown to depend on the deacetylation of two conserved Lys residues (K49/K51in LC3 subfamily proteins and K46/K48 in GABARAP subfamily proteins). However, the mechanistic details of the role of the individual Lys residues in the shuttling other LC3 proteins is unknown. In addition, the characterization of NSs-LC3 interaction as well as its impact on RVFV (Rift Valley fever virus) infection on autophagy remains elusive. Therefore, the goal of these studies is to investigate the structural and the functional differences of the six human LC3 proteins in different stages of autophagy through their interaction with DOR and NSs. Our biophysical and structural studies identified key elements determining the specificity of the LIR from DOR (DORLIR) for the GABARAP subfamily. Our structural studies defined a -sheet conformation in DORLIR when complexed with GABARAP, which is important role for establishing this specificity. ITC studies with acetylated versions of LC3A and GABARAP demonstrated that acetylation of the second Lys significantly decreases binding to the DORLIR whereas acetylation at the first Lys has little to no effect. Our structural studies also demonstrate that acetylation at the second Lys of either GABARAP or LC3A disrupts key interactions between W35 of the DORLIR, which leads to the decreased affinity. The in vitro results were verified in cellular experiments using K-to-Q substitutions to mimic Lys acetylation. In cells, K-to-Q substitutions at the second Lys impaired the cytoplasmic shuttling of both GABARAP and LC3A from the nucleus as well as their colocalization with DOR, whereas K-to-Q substitutions at the first Lys behaved like wild-type proteins. Taken together, the deacetylation of the second conserved Lys is critical for the cytoplasmic shuttling of GABARAP and LC3A during autophagy, which is in contrast to what was observed with LC3B where deacetylation of both Lys was required. This study also provides insights into interactions between the NSs protein of RVFV and LC3 proteins and the impact of NSs on autophagy during RVFV infection. We identified four potential LIR motifs (NSs1-4) in the NSs protein and ITC studies demonstrated that NSs4 interacts with submicromolar-micromolar affinity with the human LC3 proteins. In addition, we confirmed that LC3 proteins interact with NSs in cells and that in RVFV infected cell LC3A colocalizes with NSs. Taken together, the results indicate that the NSs protein plays a key role in altering autophagy during RVFV infections.

No description available.