The adenovirus major late transcription unit (MLTU) encodes for most of the mRNAs that are translated into the structural proteins of the virus capsid. Transcription from the MLTU is directed by the major late promoter (MLP), which is highly activated during the late phase of infection. This thesis discusses how the adenovirus-encoded L4-22K protein regulates the MLP at both the level of transcription and pre-mRNA splicing. The study shed new light on the complex regulation of the early to late shift of adenoviral gene expression. Here we show that the L4-22K protein has opposing effects on MLP transcription, functioning both as an activator and a repressor protein. The stimulatory effect mainly depends on the direct interaction of the L4-22K protein with the downstream element (DE element) located approximately 100 nucleotides downstream of the transcription initiation site. In addition to the DE element we also show that the promoter-proximal upstream element (UPE) acts as an L4-22K responsive enhancer element in the MLP. Preliminary data suggests that the activation of MLP transcription via DE and UPE differs mechanistically. The transactivation domain of the L4-22K protein is localized to the conserved carboxy-terminus of the protein. Our results also defined a novel low affinity L4-22K binding site, the R1 region, which functions as a repressor element in MLP transcription. At high concentrations L4-22K binds to R1 and recruits the cellular transcription factor Sp1 to a DNA segment covering the major late first leader 5´ splice site that is embedded in the R1 region. Sp1 binding to R1 results in a suppression of L4-22K-mediated activation of MLP transcription. This self-limiting effect on MLP transcription might have a function to fine-tune the MLTU gene expression. Interestingly, the L4-22K protein binds with the same sequence specificity to both the R1 double-stranded DNA and R1 single-stranded RNA (ssRNA). L4-22K binds to the R1 ssRNA with the same polarity as the MLTU nascent RNA. This binding results in the recruitment of U1 snRNA to the major late first leader 5´ splice site. This enhanced U1 snRNA recruitment leads to a suppression of MLP transcription and simultaneously an increase of major late first intron splicing.

Regulation of adenoviral gene expression is a complex process directed by viral proteins controlling a multitude of different activities at distinct phases of the virus life cycle. This thesis discusses adenoviral regulation of transcription and splicing by two proteins expressed at the late phase: L4-22K and L4-33K. These are closely related with a common N-terminus but unique C-terminal domains. The L4-33K protein is an alternative RNA splicing factor inducing L1-IIIa mRNA splicing, while L4-22K is stimulating transcription from the major late promoter (MLP). The L4-33K protein contains a tiny RS-repeat in its unique C-terminal end that is essential for the splicing enhancer function of the protein. Here we demonstrate that the tiny RS-repeat is required for localization of the protein to the nucleus and viral replication centers. Further, we describe an auto-regulatory loop where L4-33K enhances splicing of its own intron. The preliminary characterization of the responsive RNA-element suggests that it differs from the previously defined L4-33K-responsive element activating L1-IIIa mRNA splicing. L4-22K lacks the ability to enhance L1-IIIa splicing in vivo, and here we show that the protein is defective in L1-IIIa or other late pre-mRNA splicing reactions in vitro. Interestingly, we found a novel function for the L4-22K and L4-33K proteins as regulators of E1A alternative splicing. Both proteins selectively upregulated E1A-10S mRNA accumulation in transfection experiments, by a mechanism independent of the tiny RS-repeat. Although L4-22K is reported to be an MLP transcriptional enhancer protein, here we show that L4-22K also functions as a repressor of MLP transcription. This novel activity depends on the integrity of the major late first leader 5’ splice site. The model suggests that at low concentrations L4-22K activates MLP transcription while at high concentrations L4-22K represses transcription. So far, characterizations of the L4-22K and L4-33K proteins have been limited to human adenoviruses 2 or 5 (HAdV-2/5). We expanded our experiments to include HAdV-3, HAdV-4, HAdV-9, HAdV-11 and HAdV-41. The results demonstrated that the transcription- or splicing-enhancing properties of L4-22K and L4-33K, respectively, are evolutionarily conserved and non-overlapping. Thus, the sequence-based conservation is mirrored by the functions, as expected for functionally important proteins.

Adenoviruses have been extensively studied in the field of gene regulation, since their genes are subjected to a tightly controlled temporal expression during the virus lifetime. The early-to-late shift in adenoviral gene expression distinguishes two completely different programs in gene expression. The adenoviral L4-33K protein, which is the subject of this thesis, was previously implicated to be a key player in the transition from the early to the late phase of infection. Here we show that L4-33K activates late gene expression by functioning as a virus-encoded alternative RNA splicing factor activating splicing of transcripts containing weak 3’ splice sites; a feature common to the viral genes expressed at late times of infection. The splicing enhancer activity of L4-33K was mapped to a tiny arginine/serine (RS) repeat in the carboxyl-terminal domain of the protein. Also, the subcellular distribution to the nucleus with enrichment in the nuclear membrane and subnuclear redistribution to viral replication centers during a lytic infection was observed to depend on this motif. RS repeats are common features for the cellular splicing factors serine/arginine-rich (SR) proteins, which in turn are regulated by reversible phosphorylation. We further show that L4-33K is phosphorylated by two cellular protein kinases, the double-stranded DNA-dependent protein kinase (DNA-PK) and protein kinase A (PKA) in vitro. Interestingly, DNA-PK and PKA have opposite effects on the control of the temporally regulated L1 alternative RNA splicing. DNA-PK functions as an inhibitor of the late specific L1-IIIa pre-mRNA splicing whereas PKA functions as an activator of L1-IIIa pre-mRNA splicing. In summary, this thesis describes L4-33K as an SR protein related viral alternative splicing factor. A tiny RS repeat conveys splicing enhancer activity as well as redistribution of L4-33K to replication centers. Finally, DNA-PK and PKA that phosphorylates L4-33K are suggested to be novel regulatory factors controlling adenovirus alternative splicing.