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.