<p>Adenovirus infected cells provide a good model system for studying nuclear organization during RNA production and transport. This thesis is focused on the dynamic organization of splicing factors during the late phase of Adenovirus infection in HeLa cells, the nuclear localization of viral RNA, and the pathway used for viral RNA transport to the cytoplasm.</p><p>Splicing factors are relocalized from interchromatin granule clusters to sites of transcription in Adenovirus infected cells at intermediate times of infection. Later, splicing factors and viral RNA accumulate posttranscriptionally in interchromatin granule clusters. The release of the splicing factors from transcription sites was energy dependent or preceded by energy requiring mechanisms. Our data indicated that phosphorylation events inhibited by staurosporine, and 3' cleavage of the transcript are two possible mechanisms involved prior to the release of the RNP complex from transcription sites.</p><p>A viral protein derived from orf6 of early region 4, 34K, is important for the nuclear stability and transport of late viral mRNA derived from the major late transcription unit. A viral mutant lacking this region is defective for posttranscriptional accumulation of viral mRNA in interchromatin granule clusters, and for the accumulation of viral RNA in the cytoplasm. These results suggest that posttranscriptional accumulation of viral RNA in interchromatin granule clusters may contribute to the maturation of the RNP complex or sorting of RNAs and proteins, to prepare the final RNP complex for transport to the cytoplasm.</p><p>A previous model suggested that adenoviral late mRNA is transported to the cytoplasm by utilizing the CRM-1 pathway. This pathway can be blocked by the drug leptomycin B. The data presented in paper IV suggests that this model might not be applicable, since leptomycin B did not inhibit adenoviral late gene expression.</p>

<p>In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP).</p><p>Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression.</p><p>Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de-repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4-fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both <i>in vivo</i> and <i>in vitro</i> and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression.</p><p>In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.</p>

<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>

<p>The human tumour suppressor protein p53 is an effective inhibitor of cell growth, by inducing cell cycle arrest and apoptosis. However, p53-induced cell growth inhibition can be detrimental for virus multiplication. Therefore, viruses encode for proteins, which can interfere with the functions of the p53 protein. Human adenoviruses encode for a transcription repressor protein named E1B-55K, which inhibits the activity of the p53 protein during a lytic adenovirus infection.</p><p>In this thesis, we have studied the biochemical characteristics of the E1B-55K protein and how the E1B-55K protein interferes with the function of p53 as a transcription factor.</p><p>Our data show that the E1B-55K protein interacts with the Sin3 co-repressor complex in adenovirus transformed and in adenovirus infected cells. Furthermore, the E1B-55K protein recruites a histone deacetylase activity, indicating that the E1B-55K protein is associated with a functional chromatin modifying complex. We also show that in addition to repressing p53-activated transcription, E1B-55K could also relieve p53-mediated repression of the survivin and Map4 promoters.</p><p>Previous results have shown that E1B-55K inhibits p53 as a transcriptional activator of the p21/CDKN1A promoter. Here we show that the E1B-55K protein prevents p53 from inducing histone H3 and H4 acetylation on p21/CDKN1A promoter, which coincided with the inhibition of p21/CDKN1A protein expression. Notably, the Sin3 complex was detected in the vicinity of the p53 binding site on the p21/CDKN1A promoter, suggesting that the E1B-55K protein blocked p53-mediated histone acetylation by recruitment of a histone deacetylase activity. Inhibition of p21/CDKN1A protein expression might be the reason, why the E1B-55K protein alleviates p53-dependent transcriptional repression of the survivin promoter. </p><p>Finally, we show that oligomerisation of the E1B-55K protein is important for the defined subcellular localization of the protein and for its function as a repressor of p53-activated transcription.</p>

<p>We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration.</p><p>The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins.</p><p>In conclusion, this work presents a new <i>in vivo </i>model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.</p>

We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration. The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins. In conclusion, this work presents a new in vivo model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.

Adenovirus infected cells provide a good model system for studying nuclear organization during RNA production and transport. This thesis is focused on the dynamic organization of splicing factors during the late phase of Adenovirus infection in HeLa cells, the nuclear localization of viral RNA, and the pathway used for viral RNA transport to the cytoplasm. Splicing factors are relocalized from interchromatin granule clusters to sites of transcription in Adenovirus infected cells at intermediate times of infection. Later, splicing factors and viral RNA accumulate posttranscriptionally in interchromatin granule clusters. The release of the splicing factors from transcription sites was energy dependent or preceded by energy requiring mechanisms. Our data indicated that phosphorylation events inhibited by staurosporine, and 3' cleavage of the transcript are two possible mechanisms involved prior to the release of the RNP complex from transcription sites. A viral protein derived from orf6 of early region 4, 34K, is important for the nuclear stability and transport of late viral mRNA derived from the major late transcription unit. A viral mutant lacking this region is defective for posttranscriptional accumulation of viral mRNA in interchromatin granule clusters, and for the accumulation of viral RNA in the cytoplasm. These results suggest that posttranscriptional accumulation of viral RNA in interchromatin granule clusters may contribute to the maturation of the RNP complex or sorting of RNAs and proteins, to prepare the final RNP complex for transport to the cytoplasm. A previous model suggested that adenoviral late mRNA is transported to the cytoplasm by utilizing the CRM-1 pathway. This pathway can be blocked by the drug leptomycin B. The data presented in paper IV suggests that this model might not be applicable, since leptomycin B did not inhibit adenoviral late gene expression.

In this study, adenovirus E1A has been used to target and analyse the transcriptional function of the cellular C-terminal Binding Protein (CtBP). Transcription is a complex biochemical process that represents a major regulatory step in gene expression. Formation of condensed chromatin by histone deacetylation and inhibition of efficient assembly of the transcription machinery are hallmarks of transcriptional repression. During a virus infection, an extensive modulation of the host cell gene expression in favour of viral gene expression can be observed. For example, the transcription regulatory E1A protein from adenovirus has been proven to be a valuable research-tool in characterising cellular proteins controlling eukaryotic gene expression. Expression of a CtBP-binding peptide, encoded by the second exon of E1A, de-repressed transcription from a broad range of promoters, suggesting that CtBP functioned as a repressor of transcription. Artificial promoter recruitment of CtBP, by using different Gal4-fusion proteins, confirmed that CtBP functioned as a repressor. Repression of transcription by Gal4E1A-recruited CtBP was efficiently prevented by a CtBP binding competent E1A peptide, indicating that E1A relieved CtBP mediated repression by displacing CtBP from the promoter. Furthermore, Gal4CtBP repressed both basal and activated transcription in a distance dependent manner, suggesting that CtBP might repress transcription by interfering with the assembly of the basal transcription machinery. Interestingly, CtBP was found to interact with histone deacetylase-1 (HDAC-1) both in vivo and in vitro and endogenous CtBP could also recruit histone deacetylase activity. This might indicate that histone deacetylation was involved in CtBP mediated repression. However, Gal4CtBP mediated repression was insensitive to inhibition of histone deacetylase activity, suggesting an alternative function of HDAC-binding in CtBP mediated repression. In conclusion, this work demonstrates that CtBP can act as a general repressor of activated and basal transcription. Furthermore, although CtBP was shown to recruit histone deacetylase activity the relevance of this binding remains unclear.

Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing. We show that ICP27 inhibits pre-mRNA splicing in vitro in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. In order to learn more about how splicing is organized in the cell nucleus in vivo, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.

The human tumour suppressor protein p53 is an effective inhibitor of cell growth, by inducing cell cycle arrest and apoptosis. However, p53-induced cell growth inhibition can be detrimental for virus multiplication. Therefore, viruses encode for proteins, which can interfere with the functions of the p53 protein. Human adenoviruses encode for a transcription repressor protein named E1B-55K, which inhibits the activity of the p53 protein during a lytic adenovirus infection. In this thesis, we have studied the biochemical characteristics of the E1B-55K protein and how the E1B-55K protein interferes with the function of p53 as a transcription factor. Our data show that the E1B-55K protein interacts with the Sin3 co-repressor complex in adenovirus transformed and in adenovirus infected cells. Furthermore, the E1B-55K protein recruites a histone deacetylase activity, indicating that the E1B-55K protein is associated with a functional chromatin modifying complex. We also show that in addition to repressing p53-activated transcription, E1B-55K could also relieve p53-mediated repression of the survivin and Map4 promoters. Previous results have shown that E1B-55K inhibits p53 as a transcriptional activator of the p21/CDKN1A promoter. Here we show that the E1B-55K protein prevents p53 from inducing histone H3 and H4 acetylation on p21/CDKN1A promoter, which coincided with the inhibition of p21/CDKN1A protein expression. Notably, the Sin3 complex was detected in the vicinity of the p53 binding site on the p21/CDKN1A promoter, suggesting that the E1B-55K protein blocked p53-mediated histone acetylation by recruitment of a histone deacetylase activity. Inhibition of p21/CDKN1A protein expression might be the reason, why the E1B-55K protein alleviates p53-dependent transcriptional repression of the survivin promoter. Finally, we show that oligomerisation of the E1B-55K protein is important for the defined subcellular localization of the protein and for its function as a repressor of p53-activated transcription.