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Infectious disease has long been recognised as a potent selective force. Malaria provides us with our best examples of recent human evolution, through the changes it has wrought within human globin genes. Mutations responsible for genetic blood disorders, such as the thalassaemias and sickle cell anaemia, reach high frequencies in malarious regions due to the protection they offer against death from malaria. However, evidence is growing that malaria protective haemoglobin mutations interact with one another in surprising ways. Alpha and beta thalassaemia can alleviate one another's blood disorders when co-inherited; alpha thalassaemia seems to cancel out the malaria protection offered by sickle cell trait. In the first half of this thesis I explore the population genetic consequences of these intracellular interactions, and show that they help account for two observations: (i) alpha thalassaemia frequencies do not exceed 50% in sub-Saharan Africa, and (ii) the relative rarity of the sickle cell gene in the Mediterranean. Another human genomic region where intriguing patterns emerge is the major histocompatibility complex. This region contains the Human Leukocyte Antigen (HLA) genes that encode the molecules responsible for displaying antigenic fragments to the adaptive arm of the immune system; HLA genes display a high degree of haplotypic structuring. In the second half of this thesis I explore the interplay between antigenic variation in the pathogen and recognition heterogeneity in the host. I demonstrate that host heterogeneity can significantly affect the type of strain structuring that is possible in the pathogen, and show that strain structuring in the pathogen can generate linkage disequilibrium between recognition loci in the host. Finally, I demonstrate that feedbacks between host and pathogen can generate a new type of co-evolutionary cycling, in which different strain structures periodically emerge in the pathogen, precipitating changes in the host population.

Murine infection with the natural mouse pathogen S. typhimurium is the most widely accepted model for studying immunity to typhoid fever. Attenuated strains of Salmonella have been developed for use as live vaccines, and there is an extensive body of evidence demonstrating their efficacy both in human and animal studies [1]. Several attenuated strains of S. typhimurium were engineered to express defined cytokines, including IL- 10, IL-2, IL-4, IL-6, TNF-o or IFN-γ. Moreover these recombinant strains have been shown to be efficient vectors for the delivery of cytokines in experimental disease models [2],[3], Thus targeted delivery of cytokines can potentially be used as a therapeutic approach in treatment of autoimmune conditions, infectious disease and cancer. The main aim of this research was to develop animal models to facilitate the investigation of the role of cytokines by assessing their potential of modulating the immune response to microbial infections, namely using an attenuated strain of S. typhimurium, which expresses IFN gamma (IFN-γ). For this purpose, two attenuated strains of S. typhimurium were compared for their efficacy in inducing protective immune responses in a number of different genetically-defined inbred mouse lines differing in their genetic susceptibility profile; mice expressing wild type allele of Nramp1 gene (Nramp1 n), but differing in their expression of TLR4 gene, key genes controlling mouse susceptibility to Salmonella infection, C3H/HeN (TLRn/Nrampn), and C3H/HeJ (TLRd/Nrampn) mice. Mice on C57BLl6 background with defective Nramp1 gene (TLRn/Nrampd), the other critical determinant of susceptibility to Salmonella infection In order to characterise further the potential mechanism of the enhanced influence of the IFNγ-expressing Salmonella strain, two additional recombinant mouse strains, on the C57BLl6 background with a second gene defect in CD154 (CD40L) or MyD88 protein, a critical adaptor molecule essential for signalling through most of the TLRs were also used. It was hypothesised that host immune responses may be amenable to manipulation by cytokine-expressing bacterial strains. The nature of the immune response to infection with a parental attenuated strain of S. typhimurium (designated BRD509) compared with its recombinant derivative that has been engineered to express IFN-γ (designated GIDIFN). The two bacterial strains used in this study were the aroA aroD double auxotrophic strain (designated BRD509,) and its recombinant derivative that has been genetically engineered to express murine IFN-γ (designated GIDIFN). The investigations focused on assessment of the immunoregulatory influence of bacteria-expressed IFN-γ on the Immune response to Salmonella infection by following in vivo bacterial survival in peritoneal cavity (site of inoculation). The role of IFN-γ as a mediator at the different stages of infection, including the early innate response, plateau clearance stages, as well as the developments of protective memory responses were defined. The level of bacterial load in the peritoneal cavity was assessed during the first 48 hrs post i.p infection with -1x106 BRDq09 or GIDIFN organisms per mouse. Rapid clearance of IFNγ-expressing Salmonella in vivo host target organs, mice were inoculated i.p. with -0.5-1 x 106 CFUs/mouse and at the indicated time points were sacrificed and the bacterial load in spleens and livers enumerated, as well as bacterial shedding in faeces. Outcome of the infection on animal morbidity and mortality were assessed, Production of proinflammatory cytokines (IL-6, IL-12p40, and TNF-a) and nitric oxide by ex vivo cultured splenocytes and peritoneal exudates cells, the levels of IFN-y in the serum was determined. Phenotype of cellular infiltrates in the peritoneal cavity was assessed using flow cytometry. While infiltrates in spleens and livers of infected mice were assessed by histology and immunohistochemistry at different time points after inoculation. The potential influence of bacteria-expressed IFN-γ on the production of systemic/mucosal Salmonella-specific antibodies (lgM, IgG and slgA isotypes was determined titre and isotype of anti-Salmonella antibodies up to 2 months following inoculation. Efficacy of BRD509 and GIDIFN strains in affording protection against virulent challenge using titrated doses for vaccination was assessed and efficacy of the Salmonella strains as vaccines was demonstrated through their capacity to protect against virulent challenge. The results of this investigation demonstrated that IFNγ-expressing Salmonella strain (GIDIFN) has immunopotentiating effects on the host's immune response, in comparison to non-IFNγ-expressing stain (BRD509). The data obtained demonstrated that the GIDIFN strain was able to induce more vigorous innate and adaptive immune responses and, hence, was cleared more rapidly from the infected host than the parental BRD509 strain in all mouse strains tested. The major experimental findings that GIDIFN strain enhanced the early, proinflammatory cytokine response to infection, including IL-6, IL-12, TNF-a and IFN-y, improved inflammatory cell recruitment into infected tissues/organs, increased bacterial shedding in the faeces and lead to a stronger mucosal IgA response. GIDIFN enhanced the Th1-driven Immunoglobulin isotype response in susceptible mice. The GIDIFN-induced immune responses resulted in significantly enhanced host survival following infection in susceptible mouse strains. Moreover, there was a small but significant enhancement in the survival of immunised animals following lethal challenge with virulent Salmonella. The present study demonstrates that IFNγ-expressing Salmonella strains has immunopotentiating effects on the host's immune response and provide direct evidence for the utility of IFNγ-expressing attenuated Salmonella in enhancement of immune responsiveness in immune-deficient hosts. Thus, the expression of IFN-y by attenuated S. typhimurium renders the recombinant strain safer and more immunogenic to use, particularly in immunocompromised hosts, and open the way for further fine-tuning of immune responses. These findings point to the possibility of constructing more efficacious vaccines for protection against typhoid fever in humans and in animals. Moreover, IFNγ-expressing Salmonella may well be useful as bacterial vectors that act as effectors to manipulate unwanted immune responses in chronic diseases such as cancer and autoimmune conditions.

Les maladies infectieuses émergentes ont façonné l'histoire de l'espèce humaine. Encore aujourd'hui, l'émergence de nouveaux pathogènes menace la santé publique. Deux axes principaux ont été abordés : la dynamique épidémique des maladies infectieuses émergentes (MIEs) dans les populations humaines et l'impact du comportement humain sur le contrôle des infections. La dynamique épidémique des MIEs est peu connue car elle reste souvent étudiée sans prendre en compte l'effet de leurs caractéristiques, à savoir leur maintien dans un réservoir et leur capacité à émerger chez plusieurs espèces animales. Nous avons modélisé la dynamique des MIEs et mis en évidence que la transmission via réservoir et les populations intermédiaires est aussi importante que la transmission inter humaine pour comprendre les nombreuses et imprévisibles épidémies que l'on peut observer. Par la suite, l'impact du comportement humain sur le contrôle des infections a été étudié en considérant deux aspects, la prise de décision de vaccination et les pratiques culturelles. La considération de biais cognitifs liés à la prise de décision de vaccination et l'interaction entre le comportement et l'épidémiologie peut aboutir aux fluctuations de la couverture vaccinale observées empiriquement. Enfin, l'étude des pratiques culturelles a montré que, bien que souvent considérées comme à l'origine de la propagation de pathogènes dans la population, certaines pratiques peuvent en limiter la transmission. L'ensemble de ces résultats suggère que la prise en compte de l'écologie permet de faire de meilleures prédictions sur l'influence de l'environnement sur l'émergence et la réémergence des maladies infectieuses. / Infectious diseases have shaped the history of the human species. Nowadays, the emergence of new pathogens threatens public health. Understanding the interaction between pathogen ecology and human behaviour can help understanding the dynamics observed in human populations. In this thesis, two main axes were studied: the epidemic dynamics of emerging infectious diseases (EID's) in human populations and the impact of human behaviour on the control of infectious diseases. The epidemic dynamics of emerging pathogens is poorly understood because it is often studied without taking into account the effect of their characteristics, namely their persistence in a reservoir population and their ability to emerge in a broad range of species. For the first time, we modeled the dynamics of EID's and highlighted that transmission from both the reservoir and intermediate populations are critically important to consider in order to understand the many and unpredictable outbreaks that can be observed. Thereafter, the impact of human behaviour on infectious diseases control was studied by considering two aspects, vaccination decision-making and cultural practices. We show that consideration of cognitive biases related to vaccination decision-making and the interaction between behaviour and epidemiology can lead to the fluctuations observed in vaccination coverage. Finally, the study of cultural practices has shown that, although often assumed to favour the spread of pathogens in a population, certain practices can limit disease transmission. The results taken together suggest that an ecological approach is key for predicting the dynamics underpinning the emergence and re-emergence of infectious diseases and adapt control strategies.

The hypotheses challenged in this project were (1) people are the source for meticillin-resistant Staphylococcus aureus (MRSA) in pets, (2) risk factors for MRSA infection and carriage mirror those described in humans, (3) S. aureus continues to evolve on animals, (4) MRSA is carried by a substantial number of companion animals and (5) pets can be a reservoir for MRSA. Risk factors for MRSA pet infection were determined in a UK-wide case-control study enrolling dogs and cats with S. aureus infection (138 MRSA; 122 MSSA), their veterinary staff and owners. MRSA were typed and 12 paired human-animal isolates were compared by whole genome microarrays. MRSA carriage was examined in selected populations of dogs, cats and horses (n=1692) in the Greater London area and dog-to-dog transmission of MRSA was examined during an outbreak in a rescue kennel. Key findings were (a) an occupational risk for MRSA carriage in UK first opinion veterinary staff (9%), (b) antimicrobial therapy, surgery and admission to veterinary hospitals as major risk factors for pet MRSA infection; (c) human healthcareassociated lineages predominated amongst animals but (d) host-specific variation occurred within the same lineage, (e) MRSA carriage in the studied animal populations was low «1.5%), (f) "classical" risk factors were not involved in animal carriage but co-carriage of other staphylococci was protective against MRSA, (g) decolonisation occurred naturally and (h) dog-to-dog transmission was not observed. MRSA ST398 was identified from one horse, the first isolation from an animal in the UK. These findings support the concept that pets acquire MRSA primarily from people but are unlikely natural hosts for healthcare-associated MRSA. Therefore, rigorous personal and environmental hygiene combined with conscientious use of antimicrobial agents should be highly effective in veterinary clinics. Bacterial interference should be further investigated as a preventative measure. Vigilance is warranted as new strains may evolve on and spread between companion animals.

MSc (Microbiology) / Department of Microbiology

Francisella tularensis, l’agent étiologique de la tularémie, est une bactérie à multiplication intracellulaire facultative capable d’infecter de nombreux types cellulaires avec un tropisme particulier pour les macrophages. Cette bactérie est responsable d’infections graves chez de nombreuses espèces animales mais aussi chez l'homme. En particulier, la sous-espèce F. tularensis ssp tularensis a été classée comme agent de bioterrorisme de type A du fait de son pouvoir pathogène extrêmement élevé avec une faible dose infectieuse. Des approches de mutagénèse aléatoire et de criblage de banques de mutants ont suggéré l’importance des gènes impliqués dans les fonctions métaboliques et nutritionnelles dans le cycle intracellulaire de Francisella. Parmi ces gènes, on retrouve de très nombreux systèmes de transport d’acides aminés dont la sous-famille de transporteurs amino-polyamine-organocation (APC). Dans un premier temps, nous nous sommes intéressés à un transporteur APC codé par le gène FTN_0571, que nous avons appelé GadC. Pour comprendre l’importance de GadC dans la virulence de F. tularensis, nous avons réalisé un mutant chromosomique, délété du gène gadC, chez la sous-espèce novicida. Nous avons démontré que GadC est un importeur de glutamate et qu’il est nécessaire à la multiplication intracellulaire et à la virulence de Francisella, en assurant une sortie normale de la bactérie du phagosome. Ce phénomène s’explique par l’implication de GadC dans la résistance au stress oxydant généré dans le phagosome. De façon remarquable, la multiplication du mutant gadC est restaurée dans un contexte gp91phox-/-, incapable de générer des espèces réactives de l’oxygène, aussi bien in vitro qu’in vivo. Enfin, nous avons montré que l’activité de GadC modifie la production de certains intermédiaires du cycle de Krebs, et la transcription de l’enzyme qui leur est associée, démontrant un lien étroit entre la résistance au stress oxydant, le métabolisme du glutamate et la virulence de F. tularensis. Ces résultats nous ont conduits à nous intéresser à un autre transporteur appartenant à la sous-famille APC, présentant une homologie de 33% avec GadC, et que nous avons nommé ArgP. Nous montrons qu’un mutant argP présente un défaut de multiplication intracellulaire et de virulence résultant d’un retard sévère de sortie du phagosome. Ce phénotype s’explique par un défaut d’import d’arginine. L’inactivation du gène argP dans la sous-espèce holarctica LVS provoque des défauts de multiplication intracellulaire similaires à ceux observés dans la sous-espèce novicida, suggérant un rôle conservé du transporteur ArgP dans les différentes sous-espèces de F. tularensis. Comme l’arginine constitue un acide aminé essentiel pour la bactérie, nous nous sommes posés la question de l’importance de cet acide aminé durant la phase phagosomale. Une analyse du protéome bactérien du mutant argP de F. novicida, dans des conditions mimant les conditions nutritionnelles phagosomales, révèle que l’arginine joue un rôle prépondérant dans la traduction des protéines en affectant la synthèse des protéines ribosomales. L’ensemble des travaux réalisés au cours de cette thèse constitue la première démonstration de l’importance de l’acquisition d’acides aminés durant la phase phagosomale du cycle intracellulaire de F. tularensis. / Francisella tularensis, the etiologic agent of the zoonotic disease tularemia, is a facultative intracellular bacterium which can infect multiple cell types with specific tropism for macrophages. This bacterium is responsible for severe infections in numerous animal species and in humans. Of note, F. tularensis subsp. tularensis has been classified as a type A bioterrorism agent because of its high infectivity and very low infectious dose. Genome sequence analyses and genome-scale genetic studies have revealed the importance of genes involved in metabolic functions throughout the bacterial intracellular cycle. Among these genes, several amino acid transporter where found to belong to the amino-acid-polyamine organocation subfamily (APC), prompting us to address the role of these transporters in bacterial virulence. We first focused on the APC transporter encoded by gene FTN_0571 in F. tularensis subsp. novicida and named GadC. We showed that GadC was a genuine glutamate importer, necessary for Francisella intracellular multiplication and virulence. gadC inactivation completely blocked bacterial phagosomal escape. Remarkably, multiplication of a gadC mutant was restored in gp91phox-/- macrophages that are unable to generate reactive oxygen species. Altogether, our study revealed that glutamate uptake was critical in bacterial oxidative stress resistance in the phagosomal compartment and highlighted possible links between glutamate utilization and the tricarboxylic acid (TCA) cycle. These results prompted us to address the role of a second APC transporter sharing 33 % amino acid identity with GadC and named ArgP. argP inactivation severely delayed bacterial phagosomal escape, thus impairing intracellular multiplication and virulence. We demonstrated that ArgP was a high affinity arginine transporter, suggesting that impaired phagosomal escape might be directly linked to an arginine import defect. argP inactivation in the F. tularensis subsp. holarctica Live vaccine strain also leads to a severe intracellular multiplication defect, consistent with a conserved role among all F. tularensis subspecies. Arginine is an essential amino acid for F. tularensis. To understand the importance of this amino acid during the phagosomal phase of the Francisella intracellular life cycle, a proteomic analysis of the bacteria, in conditions of arginine limitation, was carried out. This analysis revealed that arginine limitation affected in the argP mutant the expression of a series of proteins and in particular of all the ribosomal proteins. One may imagine that intracellular bacteria could also sense nutrient limitations in the phagosome as a subcellular localization signal. Altogether, these studies constitute the first demonstration of the importance of amino acid acquisition during F. tularensis phagosomal escape.

Dans la mucoviscidose (CF), Pseudomonas aeruginosa colonise les voies respiratoires, conduisant à une inflammation chronique de l’épithélium bronchique. Une analyse transcriptomique antérieure nous a permis d’identifier CHAC1 comme un gène différentiellement exprimé entre les cellules épithéliales bronchiques primaires de patients CF et non-CF, au niveau basal et au cours de l’infection à P. aeruginosa. CHAC1 est une protéine dégradant le glutathion et associée au stress du réticulum endoplasmique et à l’apoptose. L’objectif principal de ce travail était de comprendre la contribution de CHAC1, en particulier dans la réponse inflammatoire et l’apoptose des cellules épithéliales pulmonaires. Nous avons donc, dans un premier temps, confirmé que CHAC1 est surexprimé au niveau ARNm dans les cellules épithéliales bronchiques primaires non-CF par rapport aux cellules CF. Nous avons observé que P. aeruginosa et deux de ses facteurs de virulence, le LPS et la flagelline, induisent l’expression de CHAC1 dans les cellules non-CF. L’expression de CHAC1 induite par le LPS est indépendante de PERK mais implique ATF4. De plus, nous avons observé qu’une réduction de l’expression de CHAC1 est associée, après stimulation par du LPS et de la flagelline, à une modulation des marqueurs inflammatoires notamment l’IL-8, l’IL-6, CCL2 et PGE2. Enfin, nous avons montré que P. aeruginosa n’est pas capable d’induire de l’apoptose dans la lignée de cellules épithéliales bronchiques NCI-H292. Ces résultats suggèrent que la régulation de l’expression de CHAC1 dans les cellules CF pourrait contribuer à la réponse inflammatoire excessive et chronique observée chez les patients atteints de mucoviscidose. / In cystic fibrosis (CF), Pseudomonas aeruginosa colonizes the airways, leading to chronic inflammation of the bronchial epithelium. A previous transcriptomic analysis allowed us to identify CHAC1 as a gene differentially expressed between primary bronchial epithelial cells of CF and non-CF patients at the basal level and during P. aeruginosa infection. CHAC1 is a glutathione-degrading protein associated with endoplasmic reticulum stress and apoptosis. The main objective of this work was to understand the contribution of CHAC1, particularly in the inflammatory response and apoptosis of pulmonary epithelial cells. We therefore first confirmed that CHAC1 is overexpressed at the mRNA level in non-CF primary bronchial epithelial cells relative to CF cells. We observed that P. aeruginosa and two of its virulence factors, LPS and flagellin, induce CHAC1 expression in non-CF cells. The expression of CHAC1 induced by LPS is independent of PERK but involves ATF4. Moreover, we have observed that a reduction in the expression of CHAC1 is associated, after stimulation by LPS and flagellin, with a modulation of the inflammatory markers, in particular IL-8, IL-6, CCL2 and PGE2. Finally, we have shown that P. aeruginosa is not capable of inducing apoptosis in the NCI-H292 bronchial epithelial cell line. These results suggest that CHAC1 is involved in the regulation of bronchial cell inflammation during P. aeruginosa infection and the regulation of CHAC1 expression in CF cells may contribute to the observed excessive and chronic inflammatory response in patients with cystic fibrosis.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.

La protéine auxiliaire Vpr du VIH-1 est exprimée tardivement au cours de la réplication virale. Toutefois, du fait de son encapsidation dans les particules virales, elle joue un rôle important dès les étapes initiales du cycle de réplication viral. Cette protéine de 96 acides aminés intervient en effet au cours de la rétrotranscription du génome viral puis de la translocation de l’ADN viral vers le noyau de la cellule hôte. Parallèlement, elle provoque un arrêt du cycle cellulaire et l’apoptose des lymphocytes T infectés. Alors qu’il a été établi que Vpr participait au contrôle de la fidélité de la rétrotranscription via le recrutement au sein des particules virales de l’uracile DNA glycosylase 2 (UNG2), enzyme impliquée dans les processus de réparation de l’ADN, certaines études ont ensuite remis en question l’impact positif de l’encapsidation de l’UNG2 sur la réplication virale. Les travaux présentés ici permettent de confirmer le rôle de l’UNG2 dans le contrôle du taux de mutations au sein de l’ADN synthétisé à partir de l'ARN viral par un mécanisme indépendant de son activité enzymatique, mais lié à des déterminants situés dans la partie N-terminale de la protéine engagée dans le recrutement de la sous-unité p32 du complexe RPA (Replication protein A) (RPA32). Nous avons montré, dans un premier temps, que la production de virus dans des cellules dont les niveaux d'expression de l'UNG2 et de RPA32 étaient diminués se traduisait par une réduction significative du pouvoir infectieux des particules virales et de la synthèse de l’ADN viral. Nous avons ensuite montré que la protéine Vpr est capable de former un complexe tri-moléculaire avec les protéines UNG2 et RPA32, et confirmé l’importance de ces deux protéines cellulaires pour permettre une réplication virale optimale aussi bien dans des lignées cellulaires T que dans les cellules primaires cibles du VIH-1. Même si les macrophages et les PBMCs (cellules mononucléaires du sang périphérique), cellules cibles du VIH-1, expriment des niveaux faibles d’UNG2 et de RPA32, ces protéines cellulaires semblent requises pour permettre une synthèse d'ADN virale suffisante à la réplication optimale du virus dans ces cellules primaires. L’ensemble de ces résultats suggère que le contrôle de la rétrotranscription par Vpr a lieu via le recrutement de deux protéines cellulaires UNG2 et RPA32 permettant la dissémination efficace du VIH-1 dans les cellules cibles primaires. / The HIV-1 auxiliary protein Vpr is expressed during the late steps of the viral replication. However, Vpr is incorporated into HIV-1 viral particles and plays a key role during the initial steps of the viral replication cycle. This 96 amino acids protein is involved in viral genome reverse transcription as well as in viral DNA translocation into the nucleus of the host cell. In parallel, Vpr provokes cell cycle arrest and apoptosis of infected T cells. Previously, it has been well established that Vpr participates in the control of the fidelity of the reverse transcription through the recruitment of the Uracil DNA Glycosylase 2 (UNG2) into the viral particles. UNG2 is an enzyme involved in different DNA repair pathway. However some studies have challenged the positive impact of UNG2 encapsidation for HIV-1 replication. Here, our studies confirm the important role of UNG2 for the control of the mutation rate in the newly synthesized viral DNA by a mechanism independent of its enzymatic activity but dependent to determinants located in the N-terminal domain that is involved in the recruitment of the p32 subunit of the RPA (Replication Protein A) complex (RPA32). First we showed that viruses produced in UNG2 or RPA32 depleted cells present a defect of infectivity and that the reverse transcription step is impaired during the course of infection of these viruses. Then we reported that the Vpr protein is able to form a trimolecular complex with UNG2 and RPA32 and we confirmed the importance of both UNG2 and RPA32 for optimal virus replication in a T cell line as well as in HIV-1 primary target cells. Even though macrophages and PBMCs (Peripheral Blood Mononuclear Cells), target cells of HIV-1, express low level of UNG2 and RPA32, these cellular proteins seem to be required for an efficient viral DNA synthesis leading to an optimal virus replication in primary cells. All these results suggest that Vpr controls the reverse transcription step through the recruitment of two cellular proteins UNG2 and RPA32 which allow the efficient dissemination of HIV-1 in the primary target cells.