Un obstacle important dans l’éradication du virus de l’immunodéfience humaine (VIH-1) est l’établissement de réservoirs viraux où le virus reste à l’état latent ainsi que l’absence de vaccin efficace. Bien que les molécules antivirales actuelles permettent d’augmenter l’espérance de vie des personnes vivant avec le VIH-1 (PLWH) ainsi que de diminuer la réplication virale chez cellesci, elles ne contribuent pas à l’élimination de ces réservoirs. La hausse de résistance envers ces molécules inhibitrices nécessite le développement constant de nouvelles molécules. L’une d’entre elles, temsavir (BMS-626529), est un nouvel inhibiteur d’attachement approuvé par la FDA depuis 2020. Sa cible, la glycoprotéine d’enveloppe (Env), est le seul antigène viral présent à la surface des cellules infectées et des virions, représentant donc la cible idéale des anticorps. L’Env mature se trouve sous forme d’hétérodimère (gp120 et gp41) suite au clivage de son précurseur gp160. Temsavir lie sous la boucle β20-β21 de la gp120 et prévient donc, par compétition, l’interaction entre l’Env et le récepteur CD4 de l’hôte. En plus de son rôle en tant qu’inhibiteur d’attachement, temsavir permet de stabiliser le trimère dans sa conformation dite «fermée». Un ancien analogue de temsavir, BMS-806, a montré réduire l’addition de glycans ainsi que de diminuer le clivage du précurseur gp160. Nos études démontrent que temsavir possède également un impact sur ces mécanismes impliqués dans la maturation et la flexibilité de l’Env de plusieurs souches du VIH-1. De ce fait, nous avons investigué l’effet de cette altération sur la conformation des différentes Env. Nos observations montrent que l’effet de temsavir sur le clivage protéolytique est associé à une diminution de la reconnaissance de l’Env par des anticorps ciblant différentes régions de celle-ci. Cette modification de la reconnaissance de l’Env est également associée à l’efficacité de la réponse cytotoxique cellulaire dépendante des anticorps (ADCC) à éliminer les cellules infectées. Les résultats présentés dans ce mémoire, notamment l’effet de temsavir sur la conformation de l’Env, devrait être considéré lors du développement d’immunothérapies ciblant le réservoir viral. / An important obstacle in the eradication of the human immunodeficiency virus (HIV-1) is the establishment of viral reservoirs where the virus remains in a latent state and the absence of a potent vaccine. Although current antiretroviral molecules increase the life expectancy of people living with HIV-1 (PLWH) as well as reduce viral replication, they do not contribute to the elimination of these reservoirs. Also, the increase in drugs resistances towards these inhibitory molecules requires the constant development of new molecules. One of them, temsavir (BMS-626529), is a new attachment inhibitor approved by the FDA since 2020. Its target, the envelope glycoprotein (Env), is the only viral antigen present at the surface of infected cells and virions and thus, is also the main target of antibodies. This mature Env consists of three gp120-gp41 heterodimers after the proteolytic cleavage of its gp160 precursor. Temsavir binds under the β20-β21 loop of gp120 and prevents the interaction between Env and the host CD4 receptor. In addition to its role as an attachment inhibitor, temsavir stabilizes the trimer in its "closed" conformation. A previous analog of temsavir, BMS-806, has been shown to affect the addition of glycans as well as the cleavage of the gp160 precursor. Our studies demonstrate that temsavir also has an impact on these mechanisms involved in the maturation and flexibility of Env of several strains of HIV-1. Therefore, we investigated the effect of this alteration on the conformation of different Env. Our observations showed that the effect of temsavir on proteolytic cleavage is associated with a decrease in Env recognition by antibodies targeting different regions of Env. This modification in Env recognition also appears to be associated with the efficacy of antibody to mediate potent antibody-dependent cellular cytotoxicity (ADCC) against infected-cells. The results presented in this master thesis, should be considered when developing immunotherapies aimed at targeting the viral reservoir in Fostemsavir-treated individuals.

No description available.

Candida parapsilosis is an emerging human opportunistic pathogen causing a wide spectrum of potentially life-threatening infections in immunocompromised hosts. One of the most important virulence factors of Candida spp. is a production of secreted aspartic proteinases (Saps). Presented thesis is mainly focused on the study of secreted aspartic proteinase 1 (Sapp1p) of C. parapsilosis, its processing and secretion under variable conditions and by use of various experimental models. Sapp1p is secreted by C. parapsilosis cells into the extracellular space as a completely processed and fully active enzyme. Experiments studying the C. parapsilosis cell wall (CW) confirmed the prolonged presence of completely processed Sapp1p on the cell surface (CW- Sapp1p). Proteolytic activity assay performed with the intact cells showed that CW-Sapp1p is proteolytically active prior to its release into the extracellular space and is capable of substrate cleavage. Biotinylation experiments with consecutive MS analysis revealed that CW-Sapp1p biotinylation is incomplete but saturable process, leaving partially unlabelled molecules. The accessibility of individual lysine residues in the Sapp1p molecule varied, with exception of four residues that were labelled in all of our experiments performed. The final step of...

Este trabalho teve o objetivo de avaliar o efeito da adição da transglutaminase microbiana (MTGase) na fabricação de pão de forma, através do desenvolvimento de formulação ideal, com combinações de aditivos e enzima, e da avaliação do efeito da enzima nas proteínas, na massa crua, na massa após a fermentação e no produto final. Para comparar a qualidade dos pães produzidos com ou sem enzima, foram testadas três formulações: a básica, livre de aditivos (pão Zero); com a adição de emulsificante e ácido ascórbico (pão Controle); e a preparada com a formulação básica adicionada de enzima (pão MTGase). A avaliação da qualidade dos pães foi feita por meio de medidas físicas e instrumentais. A análise de textura foi realizada pelo método TPA (Texture Profíle Analysis), cujas respostas de firmeza, elasticidade, mastigabilidade e gomosidade podem ser correlacionadas com análises sensoriais. Paralelamente, de amostras de farinha, de massa e de pão foram obtidas as frações protéicas de gliadinas, gluteninas e os resíduos de extração. As gliadinas e as gluteninas foram analisadas por cromatografia líquida de alta eficiência em fase reversa e por eletroforese em gel de poliacrilamida contendo SDS. Os resultados de volume e de firmeza dos diferentes pães apresentaram diferenças significativas a nível de 5%, em que as respostas do pão MTGase foram melhores que as do pão Zero, porém ainda inferiores às do Controle. A melhor formulação foi obtida por meio de um planejamento composto central, com variações nas concentrações de emulsificante, ácido ascórbico e enzima, com os resultados avaliados pela metodologia de superfície de resposta. Exceto para a coesividade, todos os outros parâmetros de volume, dureza (TA), firmeza, elasticidade, mastigabilidade e gomosidade (TPA) apresentaram resultados positivos pela ação da transglutaminase a 0,6%, combinada com 0,2 % de emulsificante e 70 ppm de ácido ascórbico. Os resultados sugerem que a enzima foi capaz de modificar as propriedades químicas das proteínas, o comportamento reológico da massa e as propriedades funcionais do pão, melhorando a força da massa, a textura e o volume dos pães. As análises das frações gliadínicas apresentaram cerca de 3% de Nitrogênio total, em base seca, e as frações glutenínicas apresentaram entre 2 e 5% de Nitrogênio total. Os perfis cromatográficos e eletroforéticos dessas frações sugerem que as gliadinas não foram afetadas pela presença da enzima, que envolveram sobretudo as gluteninas. O conjunto de resultados indica que a aplicação de MTGase em associação com aditivos convencionais pode ser uma alternativa à panificação, embora os mecanismos de sua ação na massa não estejam completamente esclarecidos. / The application of microbial transglutaminase on weak gluten flour used in breadmaking was studied over the process. To verify the enzyme effects, three formulations were tested: Base formulation, characterized by the absence of enzyme and emulsifying agents; Control formulation, comprised by the presence of emulsifying agents and ascorbic acid and MTGase formulation, with the enzyme. Samples of flour, dough and bread were analyzed. The effect of enzyme on bread quality was estimated by parameters of Texture Analysis, Texture Profile Analysis and specific volume. The protein contents from those samples were determined by the total nitrogen in glutenin and gliadin fractions, that were also analyzed by RP-HPLC (reversed phase high performance liquid chromatography) and by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Although the MTGase bread did not reach the same quality parameters as those achieved by the Control samples, it showed as an alternative formulation to reduce the quantity of emulsifying agents and ascorbic acid as compared to the Control. The results indicate that the enzyme modified chemical and functional properties of glutenin fraction, improving dough strength and bread volume. Results of total nitrogen content, and electrophoretic and chromatographic profiles of the protein fractions suggest that while glutenin proteins were modified by enzyme, gliadin proteins were not affected.

Neste trabalho visamos otimizar o isolamento da BnP1, uma SVMP presente no veneno de B. neuwiedi, e isolar disintegrinas desse veneno, ambas originadas de precursores tipo P-II, analisando as seqüências de cDNA e a ação sobre plaquetas. A purificação da BnP1 foi por exclusão molecular e troca iônica; duas novas disintegrinas, D2 e D4, foram isoladas por cromatografia em fase reversa; e outra disintegrina, MS, foi caracterizada a partir do SDS-PAGE. As proteínas foram parcialmente seqüenciadas por espectrometria de massas. Diferentes cDNAs foram clonados a partir da glândula de veneno de B. neuwiedi e os precursores da BnP1 e MS se localizaram em um cluster que inclui SVMPs classe P-I e P-II. O precursor de D4 correspondeu a uma SVMP tipicamente P-II. A BnP1, ao contrário das disintegrinas, não inibiu a agregação plaquetária induzida por diferentes agonistas. Este trabalho indica ainda que a biossíntese de SVMPs da classe P-II pode envolver processamento de mRNA, um novo mecanismo de geração de diversidade das SVMPs. / The aim of this study was to optimize the isolation of BnP1, a SVMP from B. neuwiedi venom, and isolate new disintegrins from this venom, both originated from P-II class precursors, and analyze the sequence of their precursors and their action on platelets. BnP1 was isolated by size exclusion and anion-exchange chromatography; the disintegrins D2 and D4 by reverse-phase chromatography and MS was isolated from SDS-PAGE. These proteins were partially sequenced by mass spectrometry. Several SVMPs cDNAs were cloned from B. neuwiedi venom gland, and BnP1 and MS precursors localized in a cluster enclosing P-I and P-II SVMPs. MS cDNA was tipical of P-II class of SVMPs. BnP1, unlike the disintegrins, was not able to inhibit platelet aggregation induced by different agonists. This work suggested that the biosynthesis of class P-II SVMPs include mRNA processing as a further step to generate venom diversity.

Les colicines sont des toxines antibactériennes d’Escherichia coli qui sont relâchées par les cellules productrices (colicinogènes) dans le milieu extracellulaire en réponse à des conditions de stress environnementaux. Les colicines D et E3 sont des RNases qui clivent respectivement les tRNAArg et le 16S RNA ribosomique. Les deux colicines parvenues au cytoplasme de la cellule cible provoquent ainsi la mort par inactivation de la machinerie de biosynthèse des protéines. L’import de ces deux colicines nécessite d’abord le détournement de deux systèmes cellulaires différents (FepA/TonB ou BtuB/Tol) de leur fonction physiologique, permettant leur translocation à travers la membrane externe. L’idée que par la suite la translocation à travers la membrane interne nécessite au préalable une étape de processing des colicines nucléases est ancienne, mais elle n’a jamais été démontrée formellement. Nos travaux ont permis de montrer qu’une coupure endoprotéolytique des deux colicines constitue une étape de « processing » essentielle de leur action toxique. Nous avons détecté la présence du domaine C-terminal catalytique des deux colicines dans le cytoplasme des cellules cibles préalablement exposées à la toxine. Les mêmes fragments processés (PF) ont été identifiés dans les cellules sensibles et dans les cellules immunes contre ces colicines, qui sont protégées par une protéine d’immunité spécifique, formant un complexe neutre avec le domaine catalytique. Nous avons démontré que la protéase essentielle de la membrane interne, FtsH, est nécessaire au processing des deux colicines pendant leur import. Nous avons montré aussi que la signal-peptidase LepB, une autre enzyme essentielle de la membrane interne, interagit directement avec le domaine central de la colicine D in vitro et ainsi elle est un facteur protéique spécifiquement nécessaire au processing de la colicine D. Cependant ce n’est pas l’activité catalytique de LepB qui est impliquée dans la toxicité de la colicine D, mais elle jouerait un rôle structural. LepB ainsi faciliterait probablement l’association de la colicine D avec la membrane interne en vue de la reconnaissance de la toxine par FtsH. Nous avons aussi montré que la protéase OmpT de la membrane externe est responsable d’une coupure endoprotéolytique alternative, qui refléte probablement son rôle bien connu dans le système de défense des bactéries contre les peptides anti-microbiens. Même si cette coupure in vitro permet de libérer le domaine catalytique des colicines D et E3, il est établit maintenant que la protéase OmpT n’est pas impliquée dans le processing des colicines durant leur import dans le cytoplasme. / Colicins are antibacterial toxins of Escherichia coli that are released into the extracellular medium in response to environmental stress conditions. Colicin D is an RNase that cleaves the anticodon loop of all four isoaccepting tRNAArg. Colicin E3 cleaves 16 S ribosomal RNA. Both colicins provoke cell death by inactivating the protein biosynthetic machinery. Colicin producer cells are protected against both endogenous and exogenous toxin molecules by the constitutive expression of a cognate immunity protein, which forms a tight heterodimer complex with the nuclease domain of the colicin. The import of both colicins first requires the “hijack” of some distinct functions of the target cell (namely the BtuB/Tol and FepA/TonB systems, respectively), this allowing their translocation across the outer membrane. It has long been suggested that the import of nuclease colicins requires protein processing during the translocation across the inner membrane; however it had never been formally demonstrated. Our work shows that the two different RNase colicins E3 and D undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms (PF) were identified in both colicin-sensitive cells and in cells immune to colicins, because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm

Le VIH-1 bourgeonne sous forme immature et doit subir l’étape de maturation afin d’acquérir son caractère infectieux. La maturation protéolytique du précurseur Pr55Gag induit le réarrangement morphologique de la particule alors que le dimère d’ARNg acquiert une compaction optimale. Ces réarrangements conformationnels restent encore inconnus et sont facilités par l’activité chaperonne de la protéine NCp7. Notre but a été de déterminer les différentes étapes menant à l’obtention d’un dimère d’ARNg mature. Nous avons donc étudié la structure des 550 premiers nucléotides du génome par cartographie chimique, à la fois 1. in vitro en présence des protéines Pr55Gag, GagΔp6, intermédiaires contenant le domaine NC et NCp7 et 2. in viro par l’approche hSHAPE-Seq que nous avons développé. Les particules matures et bloquées aux différentes étapes de maturation de Pr55Gag ont été analysées ainsi que des particules matures et totalement immatures traitées avec l’éjecteur de zinc AT-2. Ce traitement permet d’identifier les sites de protection de Pr55Gag et NCp7 ainsi que leur activité déstabilisatrice. / The HIV-1 particle buds from the infected cell as an immature particle and has to undergo a maturation process to become infectious. Proteolytic processing of Pr55Gag triggers morphological rearrangements of the particle whereas the gRNA dimer becomes more stable. Genomic rearrangements remain poorly understood and are facilitated by the RNA chaperone activity of the NCp7 protein. Our goal was to determining the different steps leading to the formation of the mature dimeric gRNA. To this end, the structure of the first 550 nucleotides of the HIV-1 genome was assessed by chemical probing 1. in vitro with Pr55Gag, GagΔp6, NC-containing intermediates and NCp7 proteins and 2. in viro with the hSHAPE-Seq approach we developed. Wild type and mutant viruses mimicking the sequential processing of Pr55Gag were analysed, as well as immature PR- and mature particles treated with the AT-2 zinc ejector, in order to identify the Pr55Gag and NCp7 binding sites and their gRNA destabilising activity.

Abstract Angiopoietins 1 and 2 (Ang1 and Ang2) are the ligands of the Angiopoietin/Tie signalling system, which is a binary pathway offering mechanisms for healthy vessels to reach and maintain their quiescence but also to rapidly respond to activating stimuli leading to a remodelling of endothelium. The latter is linked to disease settings such as inflammation and cancer where endothelial cell (EC) integrity is compromised and is often related to an increase in Ang2 expression. This study focused on the mechanisms enabling Ang1 to mediate both EC stability and migration and molecular and cellular determinants for ligand-specific functions of Ang2 and its isoform Ang2443. The findings revealed that Ang1 induces differential signalling depending on whether it anchors and activates Tie2 in cell-cell junctions in quiescent ECs, or in cell-matrix contacts in mobile ECs, thus leading to cellular phenotypes characteristic of resting and mobile ECs, respectively. In the second part of the thesis Ang2-Tie2 specific cell-extracellular matrix (ECM) contact sites were studied. Formation of Ang2/Tie2 EC-ECM contact sites was dependent on the collagen I and IV matrices, low Ang2 oligomerization state, α2β1-integrins, and intact microtubules. In the third part of the thesis the comparison of Ang2 mRNA splice variant Ang2443 with full length Ang2 (Ang2FL) revealed both redundant and ligand form–specific effects, expression of Ang2443443 increased the amount of monomeric ligand forms due to proteolytic processing and promoted transendothelial migration of cancer cells in vitro. On the other hand, both Ang2443 and Ang2FL were stored in endothelial Weibel-Palade bodies (WPBs), similarly induced Ang2-specific Tie2 cellular redistribution, and were mostly comparable in developmental angio- and lymphangiogenesis. / Tiivistelmä Angiopoietiinit 1 ja 2 (Ang1 ja Ang2) ovat Ang/Tie signalointireitin kasvutekijöitä. Ang1 kasvutekijää tarvitaan sydämen ja verisuoniston sikiöaikaiseen kehittymiseen, se vähentää Tie2 reseptorin kautta verisuonten läpäisevyyttä, mutta edistää myös yksittäisten endoteelisolujen liikkumista. Saman Tie2 signalointireitin toisen kasvutekijän Ang2:n ilmeneminen johtaa verisuonten läpäisevyyden kasvuun tulehduksessa, uusien verisuonten muodostumiseen syöpäkasvaimissa ja syöpäsolujen leviämiseen elimistössä. Väitöskirjatutkimuksessa selvitettiin niitä solutason mekanismeja, joilla Ang1 kykenee välittämään sekä endoteelisolujen tiiviyttä että liikkumista. Lisäksi tutkittiin niitä molekyyli- ja solutason mekanismeja, joilla Ang2 ja sen isomuoto Ang2443 välittävät kasvutekijäspesifisiä vaikutuksiaan. Väitöskirjassa osoitettiin että Tie2 reseptori paikantuu verisuonten endoteelisoluissa Ang1 sitoutumisen seurauksena joko solu-soluliitoksiin, tai yksittäisissä endoteelisoluissa solu-soluväliaine rajapinnalle. Tie2:n siirtyminen solu-soluliitoksiin aktivoi soluissa signalointireittejä, jotka ovat tyypillisiä normaaleille tiiviille verisuonille ja solu-soluväliaineliitoksissa liikkuville endoteelisoluille tyypillisiä piirteitä. Väitöskirjatyön toisessa osassa tutkittiin Ang2:lle ominaisia vaikutuksia ja Ang2-Tie2 kompleksin paikantumista erityisiin solu-soluväliaineliitoksiin. Tämä oli riippuvaista Ang2:n oligomerisaatiosta, kollageenisoluväliaineesta, α2β1-integriinistä ja normaalista mikrotubulusverkostosta. Väitöskirjatyön kolmannessa osassa osoitettiin että Ang2443 isomuodolla on sekä yhteisiä että isomuotospesifisiä piirteitä verrattuna kokopitkään Ang2:een (Ang2FL). Liukoinen Ang2443, mutta ei Ang2FL, esiintyi yleisesti monomeerisenä ligandimuotona proteiinin multimerisaatio-osan pilkkomisen seurauksena. Ang2443 lisäsi myös syöpäsolujen liikkumista endoteelisolujen läpi. Toisaalta sekä Ang2443 että Ang2FL varastoitiin endoteelisoluissa Weibel-Palade varastokappaleisin, ne välittivät samanlaista Tie2 reseptorin paikantumista endoteelisoluissa ja toimivat pääsääntöisesti samanlaisina kasvutekijöinä veri- ja imusuonten kehityksen aikana hiiressä.

In the present thesis, two positive sense single-stranded RNA viruses have been used as models to understand the structure and function of viral-encoded proteins. One of them, Pepper Vein Banding Virus (PVBV; genus Potyvirus; family Potyviridae) is a flexuous, rod-shaped virus that encodes for a polyprotein of size ~340 kDa. The polyprotein undergoes proteolytic processing by viral-encoded proteases, of which Nuclear Inclusion-a Protease (NIa-Pro) is the major protease. It is a serine-like cysteine protease which cleaves between a Q/A or Q/S, present in the context of the heptapeptide recognition sequence. The temporal regulation of intermediates and mature proteins released by NIa-Pro cleavage is crucial for a successful infection. In the present study, histidine-tagged NIa-Pro, Viral Protein genome-linked (VPg), and the cleavage site mutant (E191A) VPg-Pro were over-expressed in E. coli and purified. The protease activity of NIa-Pro was monitored using an HPLC-based protease assay developed using a peptide substrate. NIa-Pro protease activity was found to get modulated upon interaction with VPg and upon undergoing phosphorylation. Both these events have been found to involve the face of NIa-Pro which contains the solvent-exposed Trp143. Mutational studies and molecular dynamics analyses provide evidence that this residue is buried upon interaction of NIa-Pro with VPg, and any perturbation of its orientation influences the active site Cys151 via an extensive interaction network. This interaction was found to enhance the velocity of NIa-Pro protease activity, especially if the two domains were present in trans (VPg+Pro). In addition, the main-chain –NH2 group of Trp143 was found to be hydrogen-bonded to the side chain –OH group of Ser129, the residue which was identified to undergo phosphorylation by host plant kinases. Interestingly, when the two domains were present in cis (E191A VPg-Pro), no phosphorylation was observed. Mutations of Ser129 (to phosphorylation-mimic Asp or phosphorylation-deficient Ala residues) which affected this H-bond were found to disturb Trp143 and Cys151 orientation, which drastically reduced the protease activity of NIa-Pro. Within the polyprotein, VPg is present at the N-terminus of NIa-Pro and the cleavage site between them is suboptimal (E/A). In the present study, VPg-Pro was shown to be covalently linked to the genomic RNA present in the virions. Interestingly, during purification, VPg could only be purified from the soluble when it was expressed at the N-terminus of NIa-Pro. A series of bioinformatics and biophysical analysis of VPg showed that PVBV VPg, like other potyviral VPgs, exists as a molten-globule. Moreover, while VPg was shown to harbour the Walker motifs, it was found to exhibit an ATPase activity only when it was present with the NIa-Pro (especially in cis). Lys47 and Asp88:Glu89 were found crucial for optimal activity. Over all the results demonstrated that there is a reciprocal modulation of structure and function of the VPg and NIa-Pro domains. These results can explain the possible significance of an impeded cleavage rate between the two domains of VPg-Pro during PVBV infection. The precursor, VPg-Pro, could offer the advantage of evading the inhibitory phosphorylation of NIa-Pro by the host, as well as drive certain viral processes by virtue of its ATPase activity. And subsequent cleavage of the domains and their trans interaction could offer a higher turnover rate which might assist sufficient CP production required for viral morphogenesis. Another virus, Tobacco Streak Virus (TSV) that belongs to the Ilarvirus genus of the Bromoviridae family is a spherical virus which forms pleiomorphic icosahedral virus particles. It has a tripartite genome and each RNA is encapsidated individually. In the present thesis, TSV was used as a model to understand the properties of its structural protein-the coat protein (CP), with the aim of deciphering TSV assembly process. Thus, the CP gene from TSV RNA 3 was cloned and over-expressed in E. coli. The coat protein thus expressed formed virus-like particles (VLPs), which could be disassembled into dimers using high CaCl2 concentrations. Reassembly of VLPs was possible from dimers even in the absence of any nucleic acid. Mutational analysis of the N-terminal disordered domain showed that 26 amino acid residues from the amino-terminus could be crucial for capsid heterogeneity while, zinc-binding domain was essential for assembly. Overall, the present study shows that the flexible W-C loop of PVBV NIa-Pro, the disordered N-terminal region of PVBV VPg and the disordered N-terminal region of TSV CP harbour residues crucial for regulation of protein function. Such regulatory elements would ultimately allow viruses to maintain a smaller protein number, and thus a smaller genome size.

Neste trabalho visamos otimizar o isolamento da BnP1, uma SVMP presente no veneno de B. neuwiedi, e isolar disintegrinas desse veneno, ambas originadas de precursores tipo P-II, analisando as seqüências de cDNA e a ação sobre plaquetas. A purificação da BnP1 foi por exclusão molecular e troca iônica; duas novas disintegrinas, D2 e D4, foram isoladas por cromatografia em fase reversa; e outra disintegrina, MS, foi caracterizada a partir do SDS-PAGE. As proteínas foram parcialmente seqüenciadas por espectrometria de massas. Diferentes cDNAs foram clonados a partir da glândula de veneno de B. neuwiedi e os precursores da BnP1 e MS se localizaram em um cluster que inclui SVMPs classe P-I e P-II. O precursor de D4 correspondeu a uma SVMP tipicamente P-II. A BnP1, ao contrário das disintegrinas, não inibiu a agregação plaquetária induzida por diferentes agonistas. Este trabalho indica ainda que a biossíntese de SVMPs da classe P-II pode envolver processamento de mRNA, um novo mecanismo de geração de diversidade das SVMPs. / The aim of this study was to optimize the isolation of BnP1, a SVMP from B. neuwiedi venom, and isolate new disintegrins from this venom, both originated from P-II class precursors, and analyze the sequence of their precursors and their action on platelets. BnP1 was isolated by size exclusion and anion-exchange chromatography; the disintegrins D2 and D4 by reverse-phase chromatography and MS was isolated from SDS-PAGE. These proteins were partially sequenced by mass spectrometry. Several SVMPs cDNAs were cloned from B. neuwiedi venom gland, and BnP1 and MS precursors localized in a cluster enclosing P-I and P-II SVMPs. MS cDNA was tipical of P-II class of SVMPs. BnP1, unlike the disintegrins, was not able to inhibit platelet aggregation induced by different agonists. This work suggested that the biosynthesis of class P-II SVMPs include mRNA processing as a further step to generate venom diversity.