<p>Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. </p><p>Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing.</p><p>Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.</p>

<p>Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged <i>in vitro</i> culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon.</p><p>In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.</p>

<p>Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing.</p><p>Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity <i>in vitro</i>.</p><p>Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns.</p><p>Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts.</p><p>Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in <i>E. Coli</i>.</p>

<p>Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. </p><p>Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors.</p><p>In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. </p><p>Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity.</p><p>Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.</p>

<p>Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system.</p><p>The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation.</p><p>Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex.</p><p>Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription.</p><p>In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.</p>

Pre-mRNA splicing and alternative pre-mRNA splicing are key regulatory steps controlling geneexpression in higher eukaryotes. The work in this thesis was focused on a characterization of thesignificance of exonic and intronic splicing enhancer elements for pre-mRNA splicing. Previous studies have shown that removal of introns with weak and regulated splice sitesrequire a splicing enhancer for activity. Here we extended these studies by demonstrating thattwo "strong" constitutively active introns, the adenovirus 52,55K and the Drosophila Ftzintrons, are absolutely dependent on a downstream splicing enhancer for activity in vitro. Two types splicing enhancers were shown to perform redundant functions as activators ofSplicing. Thus, SR protein binding to an exonic splicing enhancer element or U1 snRNP bindingto a downstream 5'splice site independently stimulated upstream intron removal. The datafurther showed that a 5'splice site was more effective and more versatile in activating splicing.Collectively the data suggest that a U1 enhancer is the prototypical enhancer element activatingsplicing of constitutively active introns. Adenovirus IIIa pre-mRNA splicing is enhanced more than 200-fold in infected extracts. Themajor enhancer element responsible for this activation was shown to consist of the IIIa branchsite/polypyrimidne tract region. It functions as a Janus element and blocks splicing in extractsfrom uninfected cells while functioning as a splicing enhancer in the context of infected extracts. Phosphorylated SR proteins are essential for pre-mRNA splicing. Large amount recombinantSR proteins are needed in splicing studies. A novel expression system was developed to expressphosphorylated, soluble and functionally active ASF/SF2 in E. Coli.

Viruses modulate biosynthetic machineries of the host cell for a rapid and efficient virus replication. One important way of modulating protein activity in eukaryotic cells is by reversible phosphorylation. In this thesis we have studied adenovirus and vaccinia virus, two DNA viruses with different replication stategies. Adenovirus replicates and assembles new virions in the nucleus, requiring the host cell transcription and splicing machinieries, whereas vaccinia virus replicates in the cytoplasm, only requiring the cellular translation machinery for its replication. Adenovirus uses alternative RNA splicing to produce its proteins. We have shown that adenovirus takes over the cellular splicing machinery by modulating the activity of the essential cellular SR family of splicing factors. Vaccinia virus, that does not use RNA splicing, was shown to completely inactivate SR proteins as splicing regulatory factors. SR proteins are highly phosphorylated, a modification which is important for their activity as regulators of cellular pre-mRNA splicing. We have found that reversible phosphorylation of SR proteins is one mechanism to regulate alternative RNA splicing. We have demonstrated that adenovirus and vaccinia virus induce SR protein dephosphorylation, which inhibit their activity as splicing repressor and splicing activator proteins. We further showed that the adenovirus E4-ORF4 protein, which binds to the cellular protein phosphatase 2A, induced dephosphorylation of a specific SR protein, ASF/SF2, and that this mechanism was important for regulation of adenovirus alternative RNA splicing. Inhibition of cellular pre-mRNA splicing results in a block in nuclear- to cytoplasmic transport of cellular mRNAs, ensuring free access of viral mRNAs to the translation machinery. We propose that SR protein dephosphorylation may be a general viral mechanism by which mammalian viruses take control over host cell gene expression.

Type 1 diabetes is a multifactorial disease resulting from the selective destruction of insulin-producing β-cells within the pancreatic islets of Langerhans. The mechanisms of β-cell death are not fully understood but cytokines are important mediators of this process. In the present study we found that the combination of IL-1β, TNF-α and IFN-γ induced a nitric oxide-dependent disruption of the mitochondrial membrane potential in rat insulin-producing RINm5F-cells, which seems to be a necessary event for both RINm5F-cell apoptosis and necrosis. The antiapoptotic protein Bcl-2 was able to prevent cellular death in RINm5F cells, most probably by counteracting the mitochondrial permeability transition. These results pointed out the potential of such antiapoptotic genes as gene therapy tools, to allow enhanced resistance against autoimmune destruction of β-cells in type 1 diabetes. For this purpose we used a progesterone-antagonist (RU 486)-inducible gene transfer system to achieve an efficient and controlled Bcl-2 overexpression in primary rat β-cells. However, in our experience, prolonged in vitro culture revealed adenoviral-induced islet cell necrosis, a process that was not prevented by Bcl-2 overexpression. Moreover, we observed that specific adenoviral genotypes correlate with differential induction of necrosis in both human and rat pancreatic islet cells. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell-toxicity, our results showed that they were unable to build up an efficient antiviral response following infection and that their survival was dependent on the exogenous addition of α-interferon. In conclusion, adenoviral techniques for overexpression of antiapoptotic proteins in insulin-producing cells may provide useful tools against β-cell directed autoimmune destruction. However, understanding the specific interactions of the viral gene products with cellular proteins and how they are involved in β-cell death regulation is fundamental for an efficient and safe application of gene therapy approaches to type 1 diabetes.

Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors. In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity. Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.

Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system. The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation. Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex. Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription. In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.