Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-11Bitstream added on 2014-06-13T19:42:38Z : No. of bitstreams: 1 takeda_aas_dr_botib_parcial.pdf: 198000 bytes, checksum: dde840aa4960be4dc4599e18c0694b14 (MD5) Bitstreams deleted on 2014-12-19T18:32:42Z: takeda_aas_dr_botib_parcial.pdf,Bitstream added on 2014-12-19T18:33:33Z : No. of bitstreams: 1 000636567.pdf: 1314615 bytes, checksum: 216d1e404d6340f6787609cd55ed585c (MD5) / A Importina-α (ImpA) participa da via clássica de importação nuclear reconhecendo seqüências de localização nuclear (NLS) presentes em proteínas que apresentam atividades no núcleo. Almejando mais informações a respeito do mecanismo de reconhecimento para importação nuclear, nesse trabalho foram efetuados experimentos de expressão, purificação e cristalografia de raios-X da ImpA de Mus musculus com o peptídeo NLS da proteína de reparo de DNA Ku70 e peptídeos TMNLS, T52NLS, frutos de bibliotecas de peptídeos NLS específicos para isoformas da ImpA de Mus musculus e Homo sapiens, respectivamente. O peptídeo TMNLS, conforme esperado, ligou-se à ImpA de maneira similar ao NLS do antígeno T da SV40. Já o peptídeo não clássico T52NLS, obtido de uma biblioteca de peptídeos NLS para a isoforma 5 de Homo sapiens, acomodou-se ao sítio principal da ImpA de maneira satisfatória, indicando que essa seqüência também apresenta afinidade à isoforma de Mus musculus. Complementar à esse resultado, foi constatada a ligação do T52NLS ao sítio secundário da ImpA e, pela primeira vez, foi observada a ligação de um peptídeo monopartido de maneira alternativa, paralela à ImpA, ao contrário da posição anti-paralela, convencional. Surpreendentemente, resultados do complexo ImpA-NLS de Ku70 indicaramno como monopartido, e ligando-se a ImpA de maneira similar à versão fosforilada do peptídeo NLS do antígeno T da SV40. Posições específicas nos sítios de ligação foram confirmadas como essenciais, bem como resíduos da ImpA conservados nessas regiões, indicando a importância das interações intermoleculares nesses sítios. Adicionalmente foram obtidas informações relevantes, como a importância de resíduos de prolina nos NLSs e a não obrigatoriedade de regiões altamente básicas para o reconhecimento de um NLS pela ImpA / The Importin-α (ImpA) plays a role in the classic nuclear import pathway, recognizing proteins that contain nuclear localization sequences (NLS), which have activities in the nucleus. Aiming additional information about the mechanism of nuclear import recognition, in this work, the expression, purification and X-ray crystallography experiments were performed with ImpA from Mus musculus and NLS peptides from the DNA repair protein Ku70 and the peptides TMNLS and T52NLS obtained from NLS peptide libraries specific for ImpA isoforms of Mus musculus e Homo sapiens, respectively. The peptide TMNLS, as expected, bound to the ImpA in a similar way to SV40 antigen T NLS. However, the non classical peptide T52NLS, obtained from a NLS peptide library for isoform 5 of Homo sapiens, which accomodation into the major site of ImpA was acceptable, showed that this sequence has affinity to the Mus musculus isoform. There was also the binding of T52NLS in the minor site of ImpA and for the first time the binding of a monopartite peptide in an alternative way was observed. It was parallel to the ImpA in spite of the conventional nonparallel position. Surprisingly the results of the ImpA- Ku70NLS complex indicated the peptide as monopartite and bounded to the ImpA similarly to the phosphorilated version of the SV40 antigen T NLS. Specific positions in the binding sites were confirmed as essentials, and conserved residues of ImpA in these regions were highlighted, indicating the importance of intermolecular interactions in these sites. Additional information was obtained like the importance of proline residues in NLS sequences and the non-mandatory highly basic regions for a NLS recognizing by ImpA. Keywords: Importin-!, nuclear import, NLS, X-ray crystallography, Ku70, TMNLS, T52NLS

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. High risk HPV types, including HPV 16, can cause cervical carcinomas upon infecting squamous basal epithelial cells. The HPV E2 protein is a multifunctional protein that regulates viral DNA replication and expression of a large number of cellular and viral genes, including the E6 and E7 viral oncogenes. Previous research in the Moroianu lab has identified a novel alpha-helical nuclear localization signal (NLS) in the C-terminal domain of HPV 16 E2 protein (75). Here, we focused on continuing the dissection of the HPV 16 E2 NLS and on identification of the nuclear import mechanism used by this protein. We identified several residues in the C-terminal domain of HPV 16 E2 (327KHK329) and within the NLS (K299, C300) that enhance the function of the NLS. Additionally, we determined that dimerization of the C-terminal domain plays an important role in the nuclear import of HPV 16 E2 as a mutation that disrupted it led to a significant decrease in the nuclear localization of the protein. We discovered that importin 11 karyopherin is a nuclear import receptor for HPV 16 E2. Our data suggest a nuclear import mechanism for HPV 16 E2 whereby UbcM2/UBE2E3 E2-type ubiquitin-conjugating enzyme acts as an adapter to bind HPV 16 E2 to importin 11 karyopherin for its nuclear import. This is a previously undescribed nuclear import mechanism which may have implications for the control of HPV 16 E2 functions. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Orientador: Marcos Roberto de Mattos Fontes / Banca: Mario Sergio Palma / Banca: Maria Celia Bertolini / Banca: Ivan De Godoy Maia / Banca: Ney Lemke / Resumo: A Importina-α (ImpA) participa da via clássica de importação nuclear reconhecendo seqüências de localização nuclear (NLS) presentes em proteínas que apresentam atividades no núcleo. Almejando mais informações a respeito do mecanismo de reconhecimento para importação nuclear, nesse trabalho foram efetuados experimentos de expressão, purificação e cristalografia de raios-X da ImpA de Mus musculus com o peptídeo NLS da proteína de reparo de DNA Ku70 e peptídeos TMNLS, T52NLS, frutos de bibliotecas de peptídeos NLS específicos para isoformas da ImpA de Mus musculus e Homo sapiens, respectivamente. O peptídeo TMNLS, conforme esperado, ligou-se à ImpA de maneira similar ao NLS do antígeno T da SV40. Já o peptídeo não clássico T52NLS, obtido de uma biblioteca de peptídeos NLS para a isoforma 5 de Homo sapiens, acomodou-se ao sítio principal da ImpA de maneira satisfatória, indicando que essa seqüência também apresenta afinidade à isoforma de Mus musculus. Complementar à esse resultado, foi constatada a ligação do T52NLS ao sítio secundário da ImpA e, pela primeira vez, foi observada a ligação de um peptídeo monopartido de maneira alternativa, paralela à ImpA, ao contrário da posição anti-paralela, convencional. Surpreendentemente, resultados do complexo ImpA-NLS de Ku70 indicaramno como monopartido, e ligando-se a ImpA de maneira similar à versão fosforilada do peptídeo NLS do antígeno T da SV40. Posições específicas nos sítios de ligação foram confirmadas como essenciais, bem como resíduos da ImpA conservados nessas regiões, indicando a importância das interações intermoleculares nesses sítios. Adicionalmente foram obtidas informações relevantes, como a importância de resíduos de prolina nos NLSs e a não obrigatoriedade de regiões altamente básicas para o reconhecimento de um NLS pela ImpA / Abstract: The Importin-α (ImpA) plays a role in the classic nuclear import pathway, recognizing proteins that contain nuclear localization sequences (NLS), which have activities in the nucleus. Aiming additional information about the mechanism of nuclear import recognition, in this work, the expression, purification and X-ray crystallography experiments were performed with ImpA from Mus musculus and NLS peptides from the DNA repair protein Ku70 and the peptides TMNLS and T52NLS obtained from NLS peptide libraries specific for ImpA isoforms of Mus musculus e Homo sapiens, respectively. The peptide TMNLS, as expected, bound to the ImpA in a similar way to SV40 antigen T NLS. However, the non classical peptide T52NLS, obtained from a NLS peptide library for isoform 5 of Homo sapiens, which accomodation into the major site of ImpA was acceptable, showed that this sequence has affinity to the Mus musculus isoform. There was also the binding of T52NLS in the minor site of ImpA and for the first time the binding of a monopartite peptide in an alternative way was observed. It was parallel to the ImpA in spite of the conventional nonparallel position. Surprisingly the results of the ImpA- Ku70NLS complex indicated the peptide as monopartite and bounded to the ImpA similarly to the phosphorilated version of the SV40 antigen T NLS. Specific positions in the binding sites were confirmed as essentials, and conserved residues of ImpA in these regions were highlighted, indicating the importance of intermolecular interactions in these sites. Additional information was obtained like the importance of proline residues in NLS sequences and the non-mandatory highly basic regions for a NLS recognizing by ImpA. Keywords: Importin-!, nuclear import, NLS, X-ray crystallography, Ku70, TMNLS, T52NLS / Doutor

Thesis advisor: Welkin E. Johnson / For lentiviruses such as HIV-1, the viral capsid protein (CA) plays a crucial role in replication by facilitating active transport across nuclear pore complexes (NPCs). Nucleoporin Nup358/RanBP2 – a large, multidomain protein that comprises the main component of cytoplasmic NPC filaments – was previously identified as a potential cofactor for HIV-1 nuclear entry, and its C-terminal cyclophilin-like domain (Nup358Cyp) is able to interact with the CA of both HIV-1 and HIV-2. The importance of this interaction to viral replication is unclear though as certain cell-culture experiments suggest CA interaction with Nup358Cyp is dispensable for viral replication, and the CA of several other lentiviruses like SIVmac do not appear to interact with the Nup358Cyp domain. However, we have found that CA interaction with Nup358 is widely conserved among primate lentiviruses and is maintained by natural selection. The exception, SIVmac, likely reflects an evolutionary trade-off allowing escape from rhesus macaque TRIM5Cyp. Together, our observations are strong evidence that the interaction between viral CA and the Nup358Cyp domain must be biologically relevant in vivo. Specifically, by comparing interactions between multiple SIVsm/HIV-2 lineage CAs and several primate orthologs of Nup358, we identified interspecies differences in the Nup358Cyp domain that affect the CA interaction, but only when assayed in conjunction with the preceding Ran-binding domain 4 (Nup358R4). We next found that selection preserves the interaction during cross-species transmission, resulting in adaptation to differences between the Nup358Cyp homologs of the reservoir and spillover hosts. For example, SIVsm CA does not interact with human Nup358R4-Cyp, while HIV-2 CA interacts with both the human and sooty mangabey orthologs. We confirmed these distinct interaction phenotypes in an extended set of SIVsm/HIV-2 CAs, and mapped the difference to a single position – residue 3173 – in the Nup358Cyp domain. The differing ability to interact with human Nup358R4-Cyp is due to residue 85 in the CA 4-5 loops; most SIVsm strains encode a glutamine at position 85, whereas most HIV-2 strains encode an isoleucine. Reciprocal swaps reverse the interaction phenotypes, such that the SIVsm Q85I CA mutant strongly interacts with human Nup358R4-Cyp, while HIV-2 I85Q CA mutant does not. This difference also correlates with differences in single- and multi-cycle infectivity on human cell lines and levels of nuclear import in HeLa cells. Together, these results indicate that HIV-2 adapted to human Nup358 during emergence in humans. We also examined the ability of our CA panel to interact with Cyclophilin A. While all HIV-2 CA interact with CypA, the ability to interact varied among the other SIVsm CA tested, and was absent for SIVpbj. Thus, conservation of CA interaction with Nup358Cyp does not correlate to the ability to interact with CypA, and is not simply a consequence of maintaining the CA-CypA interaction. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Junona Moroianu / In this study we investigated the hydrophobic interactions between HPV11 E7 and the FG regions of Nup62N through transfection assays with EGFP-11E7 fusion plasmids in HeLa cells and binding assays with GST-Nup62N immobilized on Glutathione-Sepharose beads. We found that EGFP-11cE7 binds to Nup62N. This suggests a possible mechanism for the nuclear import of HPV11 E7 through direct hydrophobic interactions between its carboxy-terminus and the FG region of Nup62. The interaction between HPV11 E7 and CRM1 nuclear export receptor was also examined using similar methods. Binding between these proteins suggest that nuclear export of 11E7 is mediated by CRM1 binding to its leucine-rich nuclear export signal (NES). / Thesis (BS) — Boston College, 2013. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.

Thesis advisor: Junona Moroianu / Some non melanoma skin cancers (NMSC) have been associated with human papillomavirus (HPV) pathogenesis, like epidermodysplasia verruciformis (EV) and squamous cell carcinoma (SCC). EV is a genetically inherited skin disease that develops when the individuals are infected with cutaneous HPV types belonging to the β-genus, especially types 5 and 8. Transgenic mouse lineages expressing all early genes of cutaneous HPV8 develop papillomas, dysplasias and SCC after UV irradiation and this correlates with enhanced HPV8 oncogenes expression. We have previously discovered that the nuclear localization of mucosal HPV16 E7 and HPV11 E7 proteins is mediated by their zinc-binding domain via a Ran-dependent pathway and independent of nuclear import receptors and that a patch of hydrophobic residues within the zinc-binding domain of HPV16 E7 and HPV11 E7 proteins is responsible for their nuclear import via hydrophobic interactions with FG nucleoporins. Here we investigated the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein using confocal microscopy to analyze the intracellular localization of EGFP-8E7, its subdomains and its mutants after transient transfections. We also investigated the nuclear import ability of GST-8E7, its subdomains and mutants using in vitro nuclear import assays in digitonin-permeabilized HeLa cells. In addition, we performed isolation assays to study the direct interaction between HPV8 E7 and two FG nucleoporins, Nup62 and Nup153 or the nuclear export receptor, CRM1. We found that the nuclear import of cutaneous HPV8 E7 is mediated by a nuclear localization signal (NLS) located within its zinc-binding domain. Furthermore, we determined that the hydrophobic residues within the 65LRLFV69 patch are responsible for the nuclear import and nuclear localization of HPV8 E7 via direct hydrophobic interactions with FG nucleoporins, Nup62 and Nup153, whereas the positively charged arginine 66 plays no significant role in the function of the NLS. In addition, we examined the nuclear export mechanism of cutaneous HPV8 E7 protein and showed that it has a leucine-rich nuclear export signal (NES) in its C-terminal domain that is recognized by the CRM1 nuclear export receptor. These studies are essential for understanding the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) have been estimated to be the most common sexually transmitted infection in the United States. In addition to condyloma accuminata, infection of the squamous basal epithelium by high-risk HPVs, notably type 16 (HPV16), has been shown to be the primary etiological agent in the majority of cervical carcinomas. The E7 major transforming protein of HPV16, along with E6, has been linked to tumorigenesis and malignancy. While the E7 protein itself possesses no enzymatic activity, its ability to bind a number of nuclear and cytoplasmic targets subverts a variety of cellular regulatory complexes and facilitates viral replication. Previous studies in the Moroianu Lab have shown the HPV16 E7 oncoprotein to translocate across the nuclear pore complex (NPC) in a facilitated manner dependent on a non-canonical, c-terminal, nuclear localization signal (cNLS) for import, and a consensus leucine-rich nuclear export sequence (NES) for export (28). While the leucine-rich NES has been characterized, a full examination of the cNLS has yet to be performed. Here we present evidence that the karyopherin independent nuclear import mediated by the cNLS of 16E7 is dependent on its c-terminal Zn binding domain. Furthermore, we demonstrate that nuclear import is mediated by the direct interaction of a small patch of hydrophobic residues, 65LRLCV69, with the FG domain of the central FG-nucleoporin Nup62. In addition, we examined a potential regulatory mechanism of 16E7 nucleocytoplasmic translocation. Previous work has shown that a serine conserved in the high-risk HPVs at position 71 is phosphorylated by an unknown kinase. Here we present evidence that while phosphorylation of S71 is not required for either 16E7 nuclear localization or nuclear export in HeLa cells, mimicking phosphorylation of the S71 residue results in a statistically significant shift in the distribution of localization phenotypes of the resultant cell population toward a larger percentage exhibiting more nuclear localization. These data suggest that nucleocytoplasmic transport of 16E7 is, at least in part, a regulated process. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis advisor: Junona Moroianu / The E7 protein of low risk HPV11 has been shown to interact with multiple proteins, including pRb, in both the cytoplasm and the nucleus. High risk HPV16 E7 and low risk HPV11 E7 share a novel nuclear import pathway independent of karyopherins but dependent on the GTPase Ran (Angeline, et al., 2003; Knapp, et al., 2009; Piccioli, et al., 2010). We continued to analyze the nucleocytoplasmic transport of HPV11 E7 in vivo through transfection assays in HeLa cells with EGFP-HPV11 E7 wild type and mutant fusion constructs. We found that nuclear localization of HPV11 E7 is mediated by a nuclear localization signal located in the C-terminal domain which contains a unique zinc-binding domain. Mutations of cysteine residues that interfered with zinc-binding clearly disrupted the nuclear localization of the EGFP-11cE7 and EGFP-11E7 mutants. These data suggest that the integrity of the zinc-binding domain is essential for the nuclear localization of HPV11 E7. In addition, we discovered that HPV11 E7 has a leucine-rich C-terminal nuclear export signal (NES) (76IRQLQDLLL84) mediating the nuclear export of HPV11 E7 in a CRM1-dependent manner. / Thesis (BS) — Boston College, 2011. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology Honors Program. / Discipline: Biology.

Bovine adenovirus (BAdV)-3 is a non-enveloped icosahedral DNA virus, which replicates in the nucleus of infected cells, and is being developed as a vector for vaccination for humans and animals. The genome of BAdV-3 is organized into early, intermediate and late genes and it has thirty three predicted open reading frames (Reddy et al., 1998). The late region of BAdV-3 is divided into seven families (L1-L7) (Reddy et al., 1998). One of the proteins expressed in the L-6 region encodes a protein called pVIII, which is a minor capsid protein connecting the core with the inner surface of the capsid. The objective of the current study was to characterize pVIII protein of BAdV-3 and to examine its role in the life cycle of BAdV-3. Anti-pVIII serum detected a protein of 24 kDa at 12-48 hr post infection and an additional protein of 8 kDa at 24-48 hr post infection. While a 24 kDa protein is detected in empty capsids, only the C-terminal cleaved protein of 8 kDa is detected in the mature virion suggesting that amino acids 147-216 of conserved C- terminus of BAdV-3 pVIII are incorporated in mature virions. The pVIII protein predominantly localizes to the nucleus of BAdV-3 infected cells utilizing the classical importin α /β dependent nuclear import pathway. Analysis of mutant pVIII demonstrated that amino acids 57-72 of the conserved N-terminus bind to importin α-3 with high affinity and are required for the nuclear localization. Detection of hexon associated with both, precursor (24 kDa) and cleaved (8 kDa) form of pVIII suggests that the C-terminus of pVIII interacts with Hexon. Based on yeast II hybrid screening assay, we identified the cellular protein DDX3 as an interacting protein partner of pVIII. Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport (Yedavalli et al., 2004) and induction of interferon production (Schroder et al., 2008; Wang et al., 2009). Here, we provide evidence regarding the involvement of DDX3 in cap dependent cellular mRNA translation and show that targeting of DDX3 by the adenovirus pVIII protein abolishes cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacts with DDX3 in transfected and bovine adenovirus (BAdV-3) infected cells. pVIII inhibited capped mRNA translation in-vitro and in-vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in the cap binding complex. The total amount of eIFs appeared similar in uninfected or BAdV-3 infected cells. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E or PABP. These data indicate that interaction of pVIII with DDX3 depletes eIF3, eIF4E and PABP from the cap-binding complex. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA by excluding functional cap-binding complex from the capped cellular mRNA. BAdV-3 infection of DDX3 positive cells significantly inhibits cellular protein synthesis at late times post-infection. Interestingly, knockdown of DDX3 resulted in significant reduction in virus yield and expression of BAdV-3 late proteins at late times post-infection. Our results suggest that selective translation of BAdV-3 late mRNAs observed at late time post-infection of DDX3 positive cells is abrogated in DDX3 knock down cells. Moreover, the reduction in the extent of protein synthesis is evidenced by less functional 80S and polysomes in pVIII expressing plasmid transfected cells. Alternatively, DDX3 and pVIII binds to BAdV-3 tripartite leader (TPL) and the translation of mRNAs containing TPL at their 5’ ends is enhanced in the presence of pVIII and DDX3 proteins. From this observation, we concluded that pVIII and DDX-3 might promote the translation of late viral mRNAs by interacting with TPL.

Influenza A virus (IAV) replication and transcription occur in the host cell nucleus; a feature which means both the viral genome (vRNA) and mRNA must be exported from the nucleus to the cytoplasm. The mechanism by which vRNA nuclear export is achieved has been well characterised, but how viral mRNAs are exported is poorly understood. The cellular NXF1-dependent mRNA export pathway has been shown to be involved in the export of some viral mRNAs, but how they are recruited to this pathway is unknown. Prior work from our laboratory showed that segment 7 mRNA was inefficiently exported to the cytoplasm in a sub-viral ‘minireplicon’ system, providing the first indication that there were viral requirements for IAV mRNA nuclear export. Further addition of individual viral polypeptides was tested and the effect on segment 7 mRNA export was analysed by fluorescent in situ hybridization (FISH) and confocal microscopy. This identified the NS1 protein as the viral factor required for efficient segment 7 nuclear export. Mutational studies on NS1 were carried out to unveil the mechanistic role of this protein in viral mRNA nuclear export, by plasmid transfection as well as in the context of recombinant viruses. These approaches indicated that both functional domains of NS1 were necessary to preserve the mRNA export function. Furthermore, these mutant proteins were used to examine the association between NS1 and the NXF1-dependent pathway in the context of mRNA nuclear export. Protein-protein and protein-RNA binding assays indicated that interactions between NXF1 and NS1, and NXF1 and segment 7 mRNA were necessary, but not sufficient to promote segment 7 viral mRNA export. Lastly, the role of NS1 protein in the nuclear export of viral mRNAs from other genome segments was studied. The intracellular localisation of most viral mRNAs was not affected by the absence of NS1 or the presence of an export-incompetent NS1 mutant protein. However, segment 4 mRNA exhibited a similar phenotype to segment 7 mRNA in showing a dependence on NS1 for efficient nuclear export. Overall, the results presented in this dissertation suggest that NS1 acts as an adaptor protein between the viral RNA synthesis machinery and cellular export pathway. This provides deeper insights for the characterization of a recently identified function of the IAV NS1 protein, of being required for the efficient nuclear export of mRNA from “late” kinetic class viral genes.