Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.

Made available in DSpace on 2015-04-22T22:14:03Z (GMT). No. of bitstreams: 1 Myuki Alfaia Esashika Crispim.pdf: 2246402 bytes, checksum: d9773c605258310c1244020f1e5cb467 (MD5) Previous issue date: 2007-09-20 / Hepatitis B and D are endemic in the Western Brazilian Amazon region , but few studies have been conducted to investigate the genetic variability of both viruses. The hepatitis B virus (HBV) has a high genetic variability, with eight different genotypes defined (A-H). In present classification hepatitis D virus (HDV) is also supposed to present eight different genotypes (I-VIII). The aim of this study was to describe the genotypes of the virus B and D of the Western Brazilian Amazon region. We selected 190 samples of chronic carriers with HBV and 50 of them presented double infection with HDV. The serum samples of HBV were submitted to the genotyping through Polymerase Chain Reaction (PCR), with type-specific primers . In the reactive samples for HDV RNA by RT-PCR was used the genotyping by Restriction Fragment Lenght Polymorphism (RFLP). The genotype A of HBV was detected as the most frequent, in 91 participants (56,5%), following by genotype F, in 41 (25,5%), and genotype D, in 29 (18,0%). In the HDV genotyping, we found only the genotype III. This study showed that the genotypes A, D and F of VHB and the genotype III of HDV represented the predominant genotypes in the Western Brazilian Amazon. / As hepatites B e D são endêmicas na Amazônia Ocidental Brasileira, mas poucos estudos têm buscado investigar a variabilidade genética de ambos os vírus. O vírus da Hepatite B (VHB) tem uma alta variabilidade genética, sendo definidos oito genótipos distintos (A-H). O vírus da hepatite D (VHD), na atual classificação, também é sugerido apresentar oito genótipos (I-VIII). O presente estudo teve o objetivo de descrever os genótipos do vírus B e D na Amazônia Ocidental Brasileira. Selecionamos 190 amostras de portadores crônicos do vírus da hepatite B, sendo que, destas, 50 apresentavam infecção dupla com o VHD. As amostras de soro do VHB foram submetidas a genotipagem por meio da Reação em Cadeia da Polimerase (PCR), com iniciadores tipo específicos. Em amostras reativas para o VHD RNA por RT-PCR foi realizada a genotipagem por Análise do Polimorfismo do Tamanho de Fragmentos de Restrição (RFLP). Foi detectado o genótipo A como o mais freqüente em 91 participantes (56,5%), seguido pelo F em 41(25,5%), e o D em 29 (18,0%). Na genotipagem para o VHD encontramos somente o genótipo III. Este estudo mostrou que os genótipos A, D e F do VHB e o genótipo III do VHD, representam os genótipos predominantes na Amazônia Ocidental Brasileira.

La surinfection par HDV/ HBV est la forme la plus grave d'hépatite virale chronique et affecte entre 15-20 millions de patients au niveau mondial. HDV n'est pas susceptible aux traitements anti-HBV et le taux de réponse à l'IFNα est <25%. Malgré une progression plus rapide de la maladie hépatique, la majorité des patients présente une suppression de la réplication du HBV. Les détails des interactions entre HDV, HBV et le système immunitaire inné des cellules infectées restent inconnus. Les objectifs de ces travaux de thèse ont été: i) l'étude de l'infection par HDV et son interaction avec la réponse innée cellulaire; ii) l'identification de nouvelles stratégies thérapeutiques anti-HDV; iii) l'exploration de l'interaction entre HDV et HBV. L'approche expérimentale a été basée sur l'infection de cellules dHepaRG, capables d´entretenir des cycles réplicatifs complets de HBV et HDV et ayant une réponse immunitaire innée physiologique. Nous avons observé que: i) l'infection par HDV est associée à un réplication forte dans un nombre limité de cellules, et à une induction de l'expression des ISGs; ii) le traitement des cellules infectées par HDV avec de l'IFNα ne conduit pas à une induction accrue des ISGs et a une faible activité antivirale. Quelques agonistes de PRR, notamment activant la voie NF-kB, induisent une forte diminution de la réplication de HDV; iii) malgré le faible nombre de cellules infectées, HDV et ses protéines induisent une diminution de la réplication de HBV. Ces travaux ouvrent des perspectives importantes concernant la caractérisation de la pathogénèse de l'hépatite delta et l'identification de nouvelles stratégies thérapeutiques immuno modulatrices / HDV/HBV superinfection is the most aggressive form of chronic viral hepatitis and is estimated to affect 15-20 million patients worldwide. HDV is not susceptible to available direct anti-HBV drugs and sustained response to IFNα therapy occurs in less than 1/4 of patients. Despite the faster progression of liver disease, most HDV/ HBV infected patients present a suppression of HBV replication. The details of the interactions between HDV, HBV and the host cell innate immune response remain largely unexplored and research efforts have been limited by the lack of infection models. The aims of this thesis work were: i) to study HDV infection and the interplay with the host innate immune response; ii) to identify novel therapeutic strategies for the inhibition of HDV; iii) to further explore HDV/ HBV interference. The experimental strategy was based on infection of dHepaRG cells, which are known to be permissive to both HBV and HDV full replicative cycles and to present physiological innate immune responses. We observed that: i) HDV infection is associated with a strong, yet transient replication, a potent induction of the expression of ISGs; ii) IFN-α treatment of HDVinfected cells does not induce a further increase of ISG expression and has a modest antiviral activity. Conversely, some PRR agonists, in particular those inducing the NFkB pathway, induce a strong decline in HDV replication; iii) despite the low number of coinfected cells, HDV as well as its encoded proteins exert a repressive effect on HBV replication. Our work opens an array of perspectives on the pathogenesis of hepatitis delta and the identification of novel immune modulatory therapeutic strategies