La protéine Tat du VIH-1 est principalement connue pour son rôle majeur dans l’élongation de la transcription des gènes viraux. Cependant, plusieurs études ont montré que Tat pouvait être impliquée dans la rétrotranscription, étape précoce du cycle de réplication du VIH-1 et antérieure à l’intégration du génome viral dans la cellule hôte. Si Tat est impliquée dans la rétrotranscription, sa présence dans la particule virale pourrait procurer au virus un avantage cinétique. Pourtant, malgré une étude de protéomique l’ayant mis en évidence dans des virions purifiés, Tat n’est toujours pas considérée comme étant incorporée aux particules virales. Récemment, nous avons montré une interaction entre Tat et la cyclophiline A (CypA), une prolyl isomérase cellulaire. Cette protéine est connue pour être incorporée dans les particules virales via son interaction avec le domaine capside (CA) de la protéine Gag virale.Les données sur l’interaction de Tat avec CypA et l’implication de Tat dans l’étape précoce de rétrotranscription, nous ont conduits à formuler l’hypothèse suivante : L’interaction de Tat avec CypA permettrait son incorporation dans les particules virales et Tat serait donc présente dans les toutes premières étapes de l’infection des cellules hôtes par le VIH-1.Dans les travaux présentés, nous avons confirmés par GST pulls down et co-immunoprécipitation l’existence d’un complexe CA-CypA-Tat. Le complexe a pu être purifié par gel filtration. Puis, nous avons montré la présence de Tat dans les particules virales par deux approches complémentaires : une approche par western blot sur des particules virales purifiées, l’autre par microscopie à force atomique couplée à la microscopie à fluorescence. Grâce à un test fonctionnel, nous avons montré que Tat, associée au VIH-1, est fonctionnelle et délivrée dans le cytoplasme des cellules nouvellement infectées. Enfin, les interactions entre CA, CypA et Tat ont été caractérisées par différentes approches biochimiques faisant appel à des protéines purifiées. Les données de thermophorèse indiquent que l’affinité de Tat pour le complexe CA-CypA est plus forte que pour la CypA seule. Donc, dans une cellule infectée, Tat se fixerait préférentiellement sur la CypA déjà complexée à CA. Le complexe CA-CypA étant majoritairement retrouvé au niveau du site d’assemblage du virus, cela pourrait favoriser l’incorporation de Tat dans les virus.La mise en évidence de Tat dans les particules virales permettra sans doute d’apporter un angle de vue nouveau sur la réplication du VIH-1, en particulier sur les étapes précoces du cycle viral. Ces résultats pourraient initier le développement d’inhibiteurs de l’incorporation de Tat pour une application thérapeutique. / HIV-1 protein Tat is essentially known for its key role in the transcription elongation of the viral genes. However, several studies showed that Tat could favor reverse transcription. This early and essential step of HIV-1 replication cycle takes place before the integration of viral genome into cellular DNA and therefore prior to the production of the viral proteins. If Tat is involved in reverse transcription, its presence in viral particles might give a kinetic advantage to the virus. Yet, Tat is still not considered as incorporated into the virus, even though a proteomic study identified Tat in purified viral particles. Our team recently demonstrated an interaction between Tat and cyclophilin A (CypA), a cellular prolylisomerase. The latter is known for being incorporated into HIV-1 virions via its binding to the capsid (CA) domain of HIV-1 Gag protein.The evidences for Tat positive effect on reverse transcription and Tat-CypA interaction led us to formulate the following hypothesis: the Tat-CypA interaction enables HIV-1 Tat protein incorporation into viral particles.In the present work, we confirmed, using both co-immunoprecipitation and GST-pull down assays, the existence of a CA-CypA-Tat complex. In addition, this complex could be purified by gel filtration. We accordingly demonstrated the presence of Tat inside viral particles by two complementary methods: the western blot analysis of purified virions and atomic force microscopy coupled with fluorescence microscopy. Through a functional test, we found that Tat, incorporated by HIV-1, is functional and delivered to the cell cytoplasm of newly infected cells. Additionally, CA, CypA and Tat interactions were characterized by several biochemical techniques using purified proteins. Thermophoresis results indicated that Tat affinity is stronger for the complex CA-CypA than for CypA alone. Hence, in infected cells, Tat will preferably bind a CypA protein already complexed with CA compared to cytosolic CypA, thereby favoring Tat encapsidationThe demonstration that Tat is present into viral particles may bring a new point of view on HIV-1 replication cycle, especially for viral reverse-transcription and transcription steps. Our results could initiate the development of Tat encapsidation inhibitors that might have future therapeutic application.

Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel “blocking” (or “competitive”) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 96-117). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 110-133). Also issued on the Internet.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Stuttgart, Univ., Diss., 2004.

München, Techn. Univ., Diss., 2002.

München, Techn. Univ., Diss., 2002.

Le circovirus porcin de type 2 est responsable de la maladie d’amaigrissement du porcelet. Il est différent du circovirus porcin de type 1 qui est non pathogène. Nous disposons de peu de données pouvant expliquer pourquoi le virus PCV2 est pathogène et le virus PCV1 ne l’est pas. De plus les bases moléculaires soutenant la pathogénicité du PCV2 et l’immunodépression induite par le PCV2 sont mal comprises. Dans cette étude nous avons montré que la protéine gC1qR était capable d’interagir de façon différentielle avec les protéines de capside (Cap) de circovirus pathogène PCV2 et non pathogène PCV1. La protéine de capside de PCV1 isolée d’un porcelet issu d’un élevage était incapable d’interagir avec la protéine gC1qR. Il a été également montré que la région de la protéine Cap PCV2 impliquée dans l’interaction avec gC1qR était comprise dans les 59 acides aminés N-terminaux, région riche en arginine. Il a été également mis en évidence que les transcrits de gC1qR étaient sous-régulés in vitro et in vivo après une infection par le virus PCV2 au temps court de l’infection. Une sous-régulation de gC1qR induite par ARN interférence en cellules permissives rénales de porc PK15 n’induisait cependant ni une diminution de la réplication du virus PCV2, ni une diminution de formation de ses particules infectieuses. Ce travail apporte de nouveaux éléments pour comprendre l’adaptation des souches de circovirus porcins à leur hôte ainsi que son interaction avec les protéines de son hôte. / The porcine circovirus type 2 is the causal agent of the post-weaning multisystemic wasting syndrome. It is different from porcine circovirus type 1 which is non-pathogenic. We have little data that could explain why the PCV2 is pathogenic and PCV1 is not. The molecular basis supporting the pathogenicity of PCV2 and the induced immune-depression is misunderstood. It has been shown that the capsid protein (Cap) of the porcine circovirus was able to interact differentially with the capsid protein of the pathogenic circovirus PCV2 and nonpathogenic PCV1. Cap proteins from PCV1 virus isolated from a piglet was unable to interact with gC1qR. It has also been shown that the Cap PCV2 region involved in the interaction with gC1qR was included among the 59 N-terminal amino acids, an arginine-rich region. It was also shown that gC1qR transcripts were down-regulated in vitro and in vivo after infection with PCV2 virus at the beginning of infection. A siRNA-mediated downregulation of gC1qR in the PK15 permissive cells did not induce a modification of the replication of PCV2 virus and neither the production of infectious viral particles. This work provides new evidence for understanding the adaptation of porcine circovirus strains to their host as well as its interaction with its host proteins.

Le norovirus est le virus de la gastroentérite virale aiguë chez les humains et les animaux. Chaque année, il est responsable de la mort de près de 220 000 personnes et un coût de près de 65 milliards d’euros.C’est un petit virus à ARN simple brin de polarité positive. Sa capside icosaédrique est composée de 90 dimères d’une seule protéine structurale (VP1) à deux domaines : le domaine de spicule (P) exposé à l’environnement biologique, et le domaine shell (S) qui est le module d’assemblage de la capside. Un intermédiaire d'assemblage serait le pentamère de dimères, mais expérimentalement c'est un intermédiaire de 10-11 dimères qui a été rapporté.En utilisant des approches computationnelles (modélisation par homologie, recuit simulé, simulation de dynamique moléculaire avec des systèmes tout atomes ou en gros grains) nous cherchons à déterminer les bases moléculaires de l’assemblage de la capside des norovirus.Nos résultats sur la brique d’assemblage de la capside (dimère de VP1) indiquent la présence d’une asymétrie pouvant avoir un rôle dans les premières étapes d’autoassemblage, que nous confirmons expérimentalement par SAXS.Nos résultats sur de plus gros assemblages (pentamère et hexamère de dimère) révèlent également une rupture de symétrie dans le pentamère de dimère, laissant une position privilégiée pour l’insertion d’un dimère supplémentaire et favorisant une croissance anisotropique.Nous complétons et précisons ainsi le modèle d’assemblage initialement publié par notre équipe en 2013. / Noroviruses are the leading cause of acute viral gastroenteritis in humans and animas. Each year, they are responsible for 220 000 deaths and cost close to 65 billion euros.It’s a small single-stranded positive sense RNA virus. The capsid is composed of 90 dimers of a single structural protein (VP1) which consists of two main domains: the protruding domain (P) exposed to the biological environment, and the shell domain (S) which is the assembly module of the capsid .Using computational approaches such as homology modelling, simulated annealing, molecular dynamic simulations in all atoms and coarse grains systems, we are looking to determine the molecular basis of norovirus capsid assembly.Our results on the capsid assembly brick (dimer of VP1) indicate the presence of an asymmetry that can have a role in the first stages of self-assembly, which we confirm experimentally by SAXS.Our results on larger VP1 assemblages (pentamer and hexamer of dimers) also reveal a disruption of symmetry in the pentamer of dimer, leaving a preferred position for the insertion of an additional dimer and promoting anisotropic growth.We complete and specify the assembly model originally published by our team in 2013.