Le virus de la vaccine (VACV) est un grand virus à ADN, modèle du genre orthopoxvirus, et partage plus de 97% d'identité de séquence avec le virus de la variole (VARV), un pathogène humain majeur éradiqué en 1977 grâce au programme de vaccination mondial avec le VACV. Celle-ci ayant été stoppée dans les années 80, un pourcentage significatif de la population mondiale est aujourd'hui considérée comme immunologiquement naïf vis à vis du virus de la variole, ce qui fait de lui un agent bioterroriste potentiel. De plus, la vaccination implique un grand nombre de complications, particulièrement graves chez les personnes immunodéprimées ; et les antiviraux disponibles sont peu développés, ce qui souligne le besoin de nouvelles molécules. Le complexe de réplication apparait comme étant une cible privilégiée, de par son importance dans le cycle viral mais aussi par sa localisation cytoplasmique qui le rend plus accessible aux molécules antivirales. Nous nous sommes intéressés à 4 protéines essentielles de ce complexe : l'ADN polymérase E9, le facteur de processivité composé de la protéine A20 et de l'uracile ADN glycosylase D4 et l'hélicase-primase D5. Nous avons pu exprimer ces protéines de manière recombinante, seules ou en complexe ainsi que les caractériser biochimiquement et biophysiquement. Nous avons finalement abouti à une reconstruction strcuturale du complexe A20D4E9 à basse résolution grâce à la technique de SAXS, ce qui nous a permis de proposer le premier modèle structural de la fourche de réplication du virus de la vaccine. / Vaccinia virus (VACV) is a large DNA virus, prototypic virus of the orthopoxvirus genus, and shows over 97% amino acid sequence identity with the variola virus (VARV), a major human pathogene eradicated in 1977 thanks to the universal vaccination program with VACV. As this vaccination was halted in the 1980s, a significant percentage of the world population is now immunologically naïve, which makes the VARV a potent bioterrorist agent. Vaccination against smallpox may result in a variety of complications, particularly in immunologically depressed patients, and the available antiviral therapeutics are rare, which enhance the need of new molecules. The replication complex appears as an ideal target because of its importance in the viral cycle and its cytoplasmic localization, more accessible for the molecules. We have focused our study on 4 essential proteins of this complex: the DNA polymerase E9, the processivity factor composed by the A20 protein and the uracil DNA glycosylase D4 and the helicase-primase D5. We could express these recombinant proteins, alone and in complex, and characterize them biochemically and biophysically. Using the SAXS technic, we finally reached a low resolution model of the A20D4E9 complex which allow us to propose the first structural model of the vaccinia virus replication fork.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Two vaccinia viruses (VACV) genetically and phenotypically divergent were isolated, in a mixed infection, from a horse lesion during an outbreak of vesicular and exanthematous disease in horses in Southern Brazil and termed Pelotas 1 (P1V) and Pelotas 2 (P2V). This thesis describes studies performed to investigate the pathogenesis of P1V and P2V infection in rabbits and guinea pigs, and to analyze the sequence of genes potentially involved in their phenotype. Chapter 1 investigated the dose-dependent susceptibility of rabbits to P1V and P2V after intranasal (IN) inoculation. Groups of weaning rabbits were inoculated with three doses of each VACV isolate (102.5 TCID50, 104.5 TCID50 e 106.5 TCID50/rabbit). The inoculation resulted in severe respiratory distress and death of most inoculated rabbits regardless the viral strain. Clinical signs started three to six days post-inoculation (pi) and culminated in death or euthanasia at days 5 to 10 pi. Viremia was detected in animals of all groups. All rabbits surviving the infection beyond day 9 pi developed neutralizing antibodies. Interstitial pneumonia, necrossupurative bronchopneumonia and diarrhea were observed in animals which died or were euthanized in extremis. These results demonstrate that P1V and P2V are virulent for rabbits and show no apparent differences in phenotype in this species. Chapter 2 describes the investigation of the susceptibility of rabbits to intradermal (ID) inoculation to VACV, in single or mixed infection. All inoculated animals developed skin lesions characterized by hyperemia, papules, vesicles pustules and ulcers. Infectious virus was detected in cutaneous lesions, lungs and intestine of animals that died during acute infection. These results demonstrate that rabbits develop cutaneous disease and systemic infection after P1V and P2V ID inoculation. Apparently, co-infected animals developed lesions more severe than those submitted to single virus infection. In chapter 3, the susceptibility and the potential of transmission of P1V and P2V by guinea pigs were investigated. For that, guinea pigs were inoculated IN with both P1V and P2V (106 TCID50/ml). The guinea pigs did not showed clinical signs but developed viremia, shed virus in secretions and seroconverted to VACV. Nevertheless, the virus was not transmitted to guinea pig sentinels maintained in close contact or when exposed to food and feces contaminated with VACV. In Chapter 4, four genes involved in virus phenotype/virulence (C7L, K2L, N1L e B1R) were submitted to nucleotide sequencing and analysis. A 15 nucleotide (nt) deletion in K2L gene was identified in P2V. The same pattern of nucleotide deletion was also detected in other genogroup 1 Brazilian VACV isolates. Point mutations were identified in K2L, C7L and N1R genes from P2V isolates when compared to P1V and to a standard VACV strain. The molecular analysis of these genes would not allow the establishment of association between the sequences/genotype and phenotype. However, this analysis indicate that the 15 nt deletion in K2L gene may be used as a molecular marker for genogroup 1 Brazilian VACV isolates. In summary, the results obtained in these studies demonstrate: i. P1V and P2V produce systemic and cutaneous disease in rabbits but they do not exhibit evident differences in virulence for rabbits; ii. Guinea pigs are susceptible to mixed P1V an P2V infection but apparently do not effectively transmit the virus; iii. P1V and P2V present some sequence differences in virulence genes and that a 15 nt deletion in K2L gene may be used as a molecular marker to distinguish between VACV genogroups. / Duas amostras de vírus vaccínia (VACV) geneticamente e fenotipicamente distintas foram isoladas de um mesmo animal em um surto de doença vesicular e exantemática em equinos no Rio Grande do Sul, e denominados Pelotas 1 (P1V) e Pelotas 2 (P2V). Esta tese descreve estudos realizados para investigar a patogenia dos isolados P1V e P2V em coelhos e cobaias, e analisar a sequência de genes potencialmente envolvidos no fenótipo desses isolados. O Capítulo 1 relata a investigação da susceptibilidade dose-dependente de coelhos ao P1V e P2V. Os animais foram inoculados pela via intranasal (IN) com três doses (102,5 DICC50, 104,5 DICC50 e 106,5DICC50/coelho) de cada um dos isolados. A inoculação resultou em enfermidade respiratória grave e morte na maioria dos coelhos, independente do isolado utilizado. Os sinais clínicos iniciaram nos dias 3 e 6 pós-inoculação (pi) e culminaram com a morte ou eutanásia dos animais, 5 a 10 dias pi. Viremia foi detectada em coelhos de todos os grupos. Anticorpos neutralizantes foram detectados em todos os animais que sobreviveram além do dia 9 pi. Pneumonia intersticial com broncopneumonia necrossupurativa e conteúdo líquido intestinal foram lesões observadas em animais inoculados com o P1V ou P2V que evoluíram para a morte ou foram motivo para a eutanásia in extremis. Esses resultados demonstram que P1V e P2V são virulentos para coelhos e não apresentam diferenças evidentes de patogenia nessa espécie. No Capítulo 2 foi investigada a susceptibilidade de coelhos após inoculação de VACV pela via intradérmica (ID). Para isso, os coelhos foram inoculados com um dos isolados ou com ambos. Todos os coelhos inoculados apresentaram lesões de pele caracterizadas por hiperemia, pápulas, vesículas, pústulas e úlceras. Excreção viral foi detectada nas lesões cutâneas e também em amostras de pulmão e intestino de animais que morreram durante a fase aguda da infecção. Os resultados desta inoculação demonstraram que coelhos desenvolvem doença cutânea e sistêmica após a inoculação ID de P1V e P2V. Algumas evidências indicam que os coelhos co-infectados desenvolveram lesões mais severas do que na infecção simples. No Capítulo 3, investigou-se a susceptibilidade e o potencial de transmissibilidade dos isolados P1V e P2V por cobaias. Para isso, cobaias foram inoculadas pela via intranasal (IN) com uma mistura dos isolados P1V e P2V (106 DICC50/ml). As cobaias não apresentaram sinais clínicos, porém excretaram o vírus nas secreções nasais, desenvolveram viremia e soroconverteram para VACV. Apesar disso, o vírus não foi transmitido a sentinelas por contato direto, indireto (aerossóis) ou por água e alimentos contaminados com fezes deliberadamente infectadas com o vírus. No Capítulo 4, quatro genes (C7L, K2L, N1L e B1R) envolvidos no fenótipo do VACV foram amplificados por PCR, sequenciados e submetidos à análise molecular. Uma deleção de 15 nucleotídeos (nt) no gene K2L foi identificada no P2V. Essa mesma deleção também foi identificada em isolados brasileiros do VACV pertencentes ao genogrupo 1. Mutações pontuais foram identificadas nos genes K2L, C7L e N1L no P2V comparando-se com o P1V e cepas de referência do VACV. A análise molecular desses genes não permite associar essas deleções/mutações presentes no P2V com o fenótipo, mas sugere que a deleção de 15 nt no gene K2L possa ser utilizado como marcador molecular de isolados de VACV do genogrupo 1. Em resumo, os resultados obtidos nesses experimentos demonstram que: i. P1V e P2V produzem doença sistêmica e cutânea em coelhos, mas não diferem fenotipicamente nessas espécies; ii. cobaias são susceptíveis à infecção mista pelo P1V e P2V, mas aparentemente não transmitem o vírus com eficiência; iii. P1V e P2V apresentam algumas diferenças em genes de virulência, sendo que a deleção de 15 nt no gene K2L pode ser utilizada como marcador de genogrupos de VACV.

Vaccinia virus (VACV) is the most intensively studied orthopoxvirus and acts as an excellent model to investigate host-pathogen interactions. VACV encodes about 200 proteins, many of which modulate the immune response. This study focusses on two of these: C16 and C4, that share 43.7 % amino acid identity. Given the sequence similarity, we explored whether C16 and C4 have any shared functions, whilst also searching for novel functions. To gain mechanistic insight, we sought to identify binding partners and determine the residues responsible. C16 has two reported functions. Firstly, it inhibits DNA-PK-mediated DNA sensing, and this study found that C4 can perform this function as well. Like C16, C4 associates with the Ku heterodimer to block its binding to DNA leading to reduced production of cytokines and chemokines. For both proteins, the function localised to the C termini and was abrogated by mutating three residues. Secondly, C16 induces a hypoxic response by binding to PHD2. This function was mapped to the N-terminal 156 residues and a full length C16 mutant (D70K,D82K) lost the ability to induce a hypoxic response. In contrast, C4 did not bind PHD2. C4 inhibits NF-κB signalling by an unknown mechanism. Reporter gene assays showed that C16 also suppresses NF-κB activity and, intriguingly, this was carried out by both the N and C termini. C16 acts at or downstream of p65 and the N terminus of C16 associated with p65 independently of PHD2-binding. Conversely, C4 acted upstream of p65, did not display an interaction with p65, and the function was restricted to its C-terminal region. Novel binding partners were identified by a screen utilising tandem mass tagging and mass spectrometry, and selected hits were validated. The C terminus of C16 associated with VACV protein K1, a known NF-κB inhibitor. Additionally, C16 bound to the transcriptional regulator ARID4B. C4 did not interact with these proteins, but the N-terminal region of C4 associated with filamins A and B. The functional consequences of these interactions remain to be determined. In vivo, C4 and C16 share some redundancy in that a double deletion virus exhibits an attenuated virulence phenotype that is not observed by single deletion viruses in the intradermal model of infection. However, non-redundant functions also contribute to virulence in that both single deletion viruses display attenuated virulence compared to a wild-type Western Reserve virus in the intranasal model of infection. Data presented also reveal that C4 inhibits the recruitment of immune cells to the site of infection, as was previously described for C16. Overall, this investigation highlights the complexity of host-pathogen interactions showing that VACV encodes two multifunctional proteins with both shared and unique functions. Moreover, their inhibition of DNA-PK emphasises the importance of this PRR as a DNA sensor in vivo.

IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...