The distribution and function of the Adenovirus L4-33K protein Akusjärvi Department of Medical Biochemistry and Microbiology, Department of Medicine, Uppsala University, Sweden Introduction The adenoviral genome consists of double-stranded DNA, which encodes for about 40 genes that are transcribed from both strands of the genome. The genes are divided into three classes: Early (red), intermediate (black), and late (yellow). We are studying how two intermediately expressed proteins, L4-22K and L4-33K (2), affect transcription and splicing of the late regions. Our results so far, point to L4-33K being an alternative splicing factor (1) and L4-22K being a transcription factor (unpublished results). Our aim is to structurally and functionally dissect these two proteins, and to investigate their subcellular localization. Fig 1. Organization of the genome of adenovirus 5. Material and methods Immunocytochemistry - Cells were transfected with 100-500 ng L4-33K wt spliced or mutant expressing plasmids. For infections, wt Ad 5 was used at a concentration of 5 FFU/cell. Cells were stained 24h post transfection and 16h post infection, using anti-flag and anti-72K antibodies. Western blot - 293-cells were transfected with 2 ug L4-33K wt plasmid and harvested 24h post transfection, by use of isoB/NP40 buffer or RIPA buffer. Proteins were separated on SDS-PAGE, transferred to an NC membrane and detected using anti-flag antibody. S1 nuclease protection assay - 293-cells were cotransfected with increasing amounts of L4-33K wt and mutant plasmids with L1 reporter plasmid. RNA was harvested 24h post-transfection, and analysed by S1 analysis using a 5’-end labelled L1 probe. The gene expression is temporally regulated at the level of transcription, alternative splicing and polyadenylation. When the replication starts, the transcriptional activity from the major late promoter (MLP) is enhanced and the usage of the late region 3’ splice sites is increased. Results We have previously shown that L4-33K can enhance L1 splicing in vitro and in vivo (1). When examining the effects of different L4-33K mutant proteins in vitro (1) and in vivo (Fig. 2), we observe that they have varying effects on L1 splicing. The SG192 mutant and the 3xSG mutant are reduced in their capacity to activate splicing. The ds mutant has a negligible effect on splicing. When examining the distribution of wt L4-33K in immunocytochemistry, it is very similar to that of a nucleolaminar protein (Fig. 4). Figure 6. Schematic drawing of the primary sequence of L4-33K, highlighting the ds area and the mutated serine residues. From these results we can see that the region between residue 170 and 197 in L4-33K, which is deleted in the ds mutant (Fig. 6), is important. When mutating these residues, which contain the small RS repeats, we can change both the nuclear and the nucleolaminar localization of L4-33K. We are currently characterizing this region further. Figure 4. Subcellular localization of L4-33K in uninfected or infected 293-cells. An examination of the L4-33K mutants by immunocytochemistry shows that the SG192 mutant is more located in the cytoplasm than the wt protein. The 3xSG mutant is almost evenly distributed between the cytoplasm and nucleoplasm, whilst the ds mutant is predominantly located in the cytoplasm. Note that the nucleolaminar localization is lost with the 3xSG and ds mutants. Conclusions Figure 2. The effects of L4-33K mutants on L1 splicing in vivo. L4-33K is localized in the nuclear lamina of cells. The area important for this localization has been found in the C-terminal domain. The small RS repeats therein are crucial for both the splicing enhancer activity and the sub-cellular distribution of the protein. The mutant proteins also localizes differently from wt L4-33K. The SG192 and 3xSG mutants are partially found in the cytoplasm, and the ds mutant is present in almost equal levels in the nucleus and cytoplasm. References Törmänen, H., Backström, E., et al. (2006). "L4-33K, an adenovirus-encoded alternative RNA splicing factor." J Biol Chem 281(48): 36510-7. Larsson, S., et al. (1992). "Control of adenovirus major late gene expression at multiple levels." J Mol Biol 225(2): 287-98. Figure 3. Western blot of different fractions of cells transfected with L4-33K wt spliced and mutant expression plasmids. Figure 5. Subcellular localization of L4-33K mutants in uninfected 293-cells.