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HIV-NC is a multifunctional protein which plays an important role in almost every step of the retroviral life cycle. NC is essential in catalyzing stand transfers of HIV-1 reverse transcription, including the annealing of the transactivation response element (TAR) of the viral genome to the complementary TAR DNA in minus-strong-stop DNA. In this dissertation, the research starts with focus on elucidating the reaction mechanism of NC-facilitated TAR DNA/RNA annealing using single molecule spectroscopy (SMS) approaches. The results indicate that nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the partially open “Y” form associated with multiple NC molecules. This encounter complex leads to annealing through the 3’/5’ termini, namely “zipper” pathway and the annealing through the hairpin loop region, namely “kissing” pathway. By employing target oligonucleotides for specific TAR regions, we directly probed kinetic reversibility and the chaperone role of NC. Concentration-dependence of NC chaperoned melting and annealing of TAR hairpins was investigated and the results further support the proposed reaction mechanism. Additionally, we used a single-stranded DNA (ssDNA) as model to study ssDNA conformational change upon NC binding. Here we present observation of NC binding to d(TG)n and d(T)n, including NC effect on flexibility and conformation of these oligonucleotides chains. Our results reveal that the rigidity of ssDNA chain is dramatically reduced through interaction with NC. Meanwhile the results of NC dissociation experiments indicate the interaction of NC/ssDNA is complex and heterogeneous. Finally, we used SMS in vitro to systematically compare and contrast the RNA/protein interactions for the zinc-finger-binding-motif protein (NC) and the arginine-rich-binding-motif (ARM) protein (Tat) encoded by HIV-1. Tat and NC use different RNA binding motifs to recognize and interact with RNA hairpin, giving rise to very different changes in the RNA secondary structure upon protein binding. Competition experiments show that the presence of Tat can effectively inhibit the NC binding-induced local melting of TAR RNA hairpins. These results indicate that Tat specifically binds and stabilizes the TAR RNA hairpin structure, which likely inhibits the local melting of the hairpin induced by NC. / text

Baigiamojo darbo tikslas – ištirti Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezės mielėse Saccharomyces cerevisiae galimybes ir nustatyti ar mielėse susintetintus nukleokapsidės baltymus galima taikyti Paramyxoviridae šeimos virusų infekcijų serologinei diagnostikai. Tikslas pasiektas ištyrus Paramyxoviridae šeimos Sendai, žmogaus parainfluenza, žmogaus respiracinio sincitinio, Nipah, Hendra ir Menangle virusų nukleokapsidės baltymų sintezę mielių Saccharomyces cerevisiae kamiene 214∆pep4. Nustatyta, kad mielėse susintetinti nukleokapsidės baltymai pasižymi natyviems analogiškų virusų nukleokapsidės baltymams būdingomis savybėmis: yra tirpūs, formuoja virusų nukleokpsidę primenančias daleles, kurios nespecifiškai įjungia ribonukleorūgštis, pasižymi antigeninėmis savybėmis, būdingomis natyviems analogiškų virusų nukleokapsidės baltymams. Taip pat mielėse susintetinti nukleokapsidės baltymai išgryninti juos centrifuguojant per tankų sacharozės tirpalo sluoksnį ir CsCl tankio gradiente. Įsitikinta, kad mielėse Saccharomyces cerevisiae susintetinti Paramyxoviridae šeimos virusų nukleokapsidės baltymai reaguoja su atitinkamais šios šeimos virusais užsikrėtusių žmonių ir gyvūnų kraujo serumo antikūnais, todėl tinka šių virusų infekcijų serologinės diagnostikos sistemų kūrimui. Naudojant mielėse susintetintus nukleokapsidės baltymus buvo gauti specifiniai monokloniniai antikūnai reaguojantys su natyviais virusų nukleokapsidės baltymais. Įvertinos gautų monokloninių... [toliau žr. visą tekstą] / The aims of this study was to investigate synthesis of SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV nucleocapside (N) proteins in yeast Saccharomyces cerevisiae, to determine properties of recombinant N proteins and evaluate the feasibility to use them in diagnostics. In this study it was demonstrated, that yeast S.cerevisiae is an excellent host for a high-level production of proteins SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV N proteins as virus nukleokapsid-like particles (vNLP). The yeast-expressed hPIV1, hPIV3, hRSV, NiV, HeV and MenV vNLPs represent useful tools for the development of new virus detection systems and demonstrate the effectiveness of yeast as a host for generation of recombinant proteins organized in complex structures like human virus NLPs.

The aims of this study was to investigate synthesis of SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV nucleocapside (N) proteins in yeast Saccharomyces cerevisiae, to determine properties of recombinant N proteins and evaluate the feasibility to use them in diagnostics. In this study it was demonstrated, that yeast S.cerevisiae is an excellent host for a high-level production of proteins SeV, hPIV1, hPIV3, hRSV, NiV, HeV and MenV N proteins as virus nukleokapsid-like particles (vNLP). The yeast-expressed hPIV1, hPIV3, hRSV, NiV, HeV and MenV vNLPs represent useful tools for the development of new virus detection systems and demonstrate the effectiveness of yeast as a host for generation of recombinant proteins organized in complex structures like human virus NLPs. / Baigiamojo darbo tikslas – ištirti Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezės mielėse Saccharomyces cerevisiae galimybes ir nustatyti ar mielėse susintetintus nukleokapsidės baltymus galima taikyti Paramyxoviridae šeimos virusų infekcijų serologinei diagnostikai. Tikslas pasiektas ištyrus Paramyxoviridae šeimos Sendai, žmogaus parainfluenza, žmogaus respiracinio sincitinio, Nipah, Hendra ir Menangle virusų nukleokapsidės baltymų sintezę mielių Saccharomyces cerevisiae kamiene 214∆pep4. Nustatyta, kad mielėse susintetinti nukleokapsidės baltymai pasižymi natyviems analogiškų virusų nukleokapsidės baltymams būdingomis savybėmis: yra tirpūs, formuoja virusų nukleokpsidę primenančias daleles, kurios nespecifiškai įjungia ribonukleorūgštis, pasižymi antigeninėmis savybėmis, būdingomis natyviems analogiškų virusų nukleokapsidės baltymams. Taip pat mielėse susintetinti nukleokapsidės baltymai išgryninti juos centrifuguojant per tankų sacharozės tirpalo sluoksnį ir CsCl tankio gradiente. Įsitikinta, kad mielėse Saccharomyces cerevisiae susintetinti Paramyxoviridae šeimos virusų nukleokapsidės baltymai reaguoja su atitinkamais šios šeimos virusais užsikrėtusių žmonių ir gyvūnų kraujo serumo antikūnais, todėl tinka šių virusų infekcijų serologinės diagnostikos sistemų kūrimui. Naudojant mielėse susintetintus nukleokapsidės baltymus buvo gauti specifiniai monokloniniai antikūnai reaguojantys su natyviais virusų nukleokapsidės baltymais. Įvertinos gautų monokloninių... [toliau žr. visą tekstą]

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 μg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Hantaviruses are found practically all over the world and cause hemorrhagic fevers in man. Each year about 150,000 people are hospitalized in these zoonotic infections which can be of two types: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), depending on the infecting virus. Hantavirus infections are emerging infectious diseases. That is, the number of reported cases of hantaviral disease is increasing, new hantaviruses are discovered continually, and already known hantaviruses are expected to spread to new areas. Therefore, knowledge and monitoring of these viruses are imperative from a public health perspective. In this thesis, the characterization of a local human Puumala (PUUV) virus isolate is described. Genetic and serological relationships to other hantaviruses are investigated and the viral protein interactions, critical for genome packaging and assembly, are studied. We found that the nucleotide and amino acid sequences of the local PUUV strains are significantly different from the PUUV prototype strain Sotkamo, a difference that indicates that there might be a risk of misdiagnosing PUUV infected patients when using reagents derived from the prototype strain. These data contributed to the introduction of locally derived diagnostic tools to the Laboratory of Clinical Virology at the Umeå University hospital, which is the reference centre for hantaviral diseases in Sweden. Furthermore, when studying the underlying mechanisms of genome packaging, we identified several regions and amino acids absolutely required for nucleocapsid protein interactions. Also, a region that appeared to regulate this interaction was discovered. Finally, the serological immune responses in DNA-vaccinated mice and PUUV infected patients were investigated. We found that the cross-reactive antibody response in vaccinated mice and in infected individuals was unique and independent of homologous titres. Furthermore, four immunodominant epitopes with specific cross-reactive characteristics were identified. Our findings have highlighted the complexity of the serological immune responses to hantavirus infections, and they emphasize the importance of customizing the diagnostic tools and performing clinical analyses on locally derived strains. In conclusion, we believe that these results are valuable in the development of new serological, genetic, and epidemiological tools.

Although wild birds are natural hosts of avian influenza viruses (AIVs), these viruses can be highly contagious to poultry and a zoonotic threat to humans. The propensity of AIV for genetic variation through genetic shift and drift allows virus to evade vaccine mediated humoral immunity. An alternative approach to current vaccine development is induction of CD8+ T cells which responds to more conserved epitopes than humoral immunity and targets a broader spectrum of viruses. Since the memory CD8+ T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte responses induced by intramuscular inoculation of chickens with AIV HA and NP expressing cDNA plasmids or a non-replicating human adenovirus vector were identified through ex vivo stimulation with virus infected, major histocompatibility complex (MHC) matched antigen presenting cells (APCs). The IFN? production by activated lymphocytes was evaluated by macrophage production of nitric oxide and ELISA. MHC-I restricted memory T lymphocyte responses were determined at 10 days and 3, 5, 7 and 9 weeks post-inoculation (p.i). The use of non-professional APCs and APC driven proliferation of cells with CD8+ phenotype correlated with the activation of CD8+ T lymphocytes. The responses specific to nucleocapsid protein (NP) were consistently greater than those to the hemagglutinin (HA) at 5 weeks when the CD8+ T cell responses were maximum. By 8 to 9 weeks p.i., responses to either protein were undetectable. The T lymphocytes also responded to stimulation with a heterologous H7N2 AIV infected APCs. Administration of booster dose induced secondary effector cell mediated immune responses which had greater magnitudes than primary effector responses at 10 days p.i. Flow cytometric analysis (FACS) of the T lymphocytes demonstrated that memory CD8+ T lymphocytes of chickens can be distinguished from naive lymphocytes by their higher expression of CD44 and CD45 surface antigens. CD45 expression of memory lymphocytes further increases upon ex vivo stimulation with APCs expressing AIV. This is the first characterization of avian memory responses following both primary and secondary expression of any individual viral protein.

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-22Bitstream added on 2014-06-13T18:56:05Z : No. of bitstreams: 1 fernandes_cc_me_jabo.pdf: 1349174 bytes, checksum: b0d752648346a29041d0fbd5f330fbf6 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Anticorpos monoclonais se constituem na base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por “phage display” contra a estirpe vacinal (H120) do VBI, foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01, IBVPR05, isoladas de surtos a campo no Brasil e SE-17, isolada nos Estados Unidos. Após três ciclos de “panning”, foi identificado pelo ELISA um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que três desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzido em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus e no estudo de evolução de variantes desse vírus. / Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of outbreaks of infectious bronchitis, because these antibodies are homogeneous, highly specific and fully characterized, allowing the improvement of detection of immunological techniques and antigenic characterization of avian infectious bronchitis virus strains (IBV). We used a phage display library prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments reacting with heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning a set of 15 scFv antibodies was expressed in phages and exhibited crossreaction in ELISA with these three viral strains. Western-blotting analysis showed that three of this clone set were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41 strain of IBV. In conclusion, the recombinant fragments of monoclonal antibodies expressed by phage-display technique have a great potential for future use in immunodiagnostic techniques and study the evolution of variant strains of this virus.

La transcriptase inverse (RT) est un hétéro-dimère p66/p51 avec des activités ADN polymérase et ribonucléase H qui jouent un rôle critique dans le cycle viral du VIH-1. La RT convertit l’ARN génomique viral simple brin en ADN proviral double brin dans le cytoplasme de la cellule infectée. L’efficacité de la RT est augmentée par la protéine de la nucléocapside (NC) grâce à son activité chaperonne vis-à-vis des acides nucléiques et/ou une coopération entre les deux protéines. Dans ce travail, nous avons étudié par la technique de smFRET (single molecule Fluorescence Resonance Energy Transfer) les effets de la NC sur l’interaction entre la RT et un substrat d’ADN au niveau de deux sites de pause de la synthèse de l’ADN(+). Nous avons d’abord réalisé et validé le montage de smFRET. Lors de la validation du montage avec des fluorophores Cy3 encapsulés dans des vésicules lipidiques, nous avons mis en évidence deux mécanismes différents entrainant le photoblanchiment du Cy3. Ensuite, après avoir déterminé les propriétés de liaison à l’équilibre de la RT et la NC sur différents substrats amorce/matrice à l’aide de mesures d’ensemble en solution, nous avons confirmé par smFRET que la RT adopte plusieurs conformations sur son substrat d’ADN, incluant celle qui conduit à la polymérisation de l’ADN. En présence de la NC, nous n’avons observé qu’une réorganisation modérée des différentes conformations du complexe RT/substrat. Par contre, une réorganisation beaucoup plus importante est induite par la NC en présence du dNTP, avec une très forte exaltation des conformations compétentes pour la polymérisation. Nous avons également montré que la NC augmente l’efficacité de synthèse de l’ADN au niveau de sites de pause en diminuant ou en augmentant le temps de dissociation du complexe RT/substrat/dNTP selon le type de site de pause. L’ensemble de ces données permet de mieux comprendre les mécanismes polyvalents par lesquels NC facilite l’activité de la RT. / The reverse transcriptase (RT) is a p66/p51 hetero-dimer with DNA polymerase and ribonuclease H activities which plays a critical role in the viral cycle of HIV-1. RT converts the viral genomic RNA to proviral DNA in the cytoplasm of infected cells. The efficiency of RT is increased by the nucleocapsid protein (NC) through its nucleic acids chaperone properties and/or via direct interaction with RT. In the present work, we investigated the effects of NC on the interaction between RT and its DNA substrate attwo pause sites during the synthesis of (+)DNA by using the smFRET (single molecule Fluorescence Resonance Energy Transfer) technique. In a first step, we implemented and validated the smFRET set-up. Within the validation step, using Cy3 fluorophores encapsulated, in lipid vesicles, we monitored the photobleaching of Cy3 dyes and found out that it was governed by two parallel mechanisms. In a second step, we determined the affinity of RT and NC to different primer/template substrates by using steady-state fluorescence. Then, we confirmed by smFRET that RT adopts different conformations on its DNA substrate, including the one that leads to DNA polymerization. In the presence of NC, we observed only a moderate reorganization of the different conformations of RT/substrate complex. However, NC was found to induce a more important reorganization in the presence of dNTP, with a very strong promotion of the polymerization-competent conformations. We also showed that NC increases the efficiency of DNA synthesis at pause sites by either decreasing or increasing the dissociation time of the RT/substrate/dNTP complex, depending on the type of pause site. Together, these data allow us to further elucidate the mechanisms by which NC facilitates the RT.

La protéine NCp7 du VIH-1 est une cible thérapeutique de choix car, en plus d’être conservée, elle intervient lors de nombreuses étapes du cycle rétroviral. A ce jour, peu de données existent concernant l’interaction possible de NCp7 avec les membranes lipidiques. Or il a récemment été montré que, lors de l’assemblage des virions, le précurseur Gag adopte une conformation repliée qui permettrait à son domaine NC d’interagir avec la membrane plasmique. Dans le but de tester cette hypothèse nous avons utilisé NCp7 sous sa forme mature. Nos résultats indiquent que NCp7, libre ou liée à des acides nucléiques, se lie aux membranes lipidiques chargées négativement, possède la capacité de recruter les lipides chargés négativement de manière à optimiser sa liaison sur la membrane et peut également déstabiliser ces membranes. Finalement, en utilisant un système modèle, nous avons mis au point les conditions de travail pour la poursuite de cette étude à l’aide de la microscopie super-résolutive. / The NCp7 protein is an interesting antiviral target since it is conserved and plays a numerous key roles in the HIV-1 replication cycle. Interestingly, while only few data are currently available on the possible interaction of NCp7 with lipid membranes, it has been recently shown that during assembly, the Gag precursor can adopt a bent conformation where the NC domain may interact with the plasma membrane. In order to check this hypothesis we used the mature NCp7. Our data indicated that the NCp7 protein, free or bound with nucleic acids, binds to negatively charged lipid membrane with high affinity, can recruit negatively charged lipids to optimize its binding to lipid membranes and has the ability of to destabilize negatively charged lipid membranes at high concentrations. Finally, by using a receptor/ligand model system we established the working conditions to investigate Gag/membrane interactions by high resolution microscopy.