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Influenza A virus (family Orthomyxoviridae) is one of the most important human pathogens, causing annual epidemics with significant worldwide mortality, and sporadic but potentially devastating pandemics. The influenza A viral genome encodes 14 proteins and consists of 8 segments of negative-stranded RNA. During infection, the virus exploits the host cell signaling machinery to ensure efficient replication. The PI3K/Akt and Ras/ERK are two of the signaling cascades that are induced for virus survival. Influenza A virus replicates in the nucleus, hence the newly synthesized RNPs must be exported from the nucleus and exported to the cell membrane. Although the detailed mechanism of vRNP nuclear export is not yet fully elucidated, several studies on this process have begun to emerge. Influenza A virus nucleoprotein nuclear export is CRM1-dependent. Ran-binding protein 3 (RanBP3) is a Ran-interacting protein that is best known for its role as a cofactor of CRM1-mediated cargo nuclear export. In this study, we investigated the role of RanBP3 during the influenza A virus life cycle. We found that RanBP3 was phosphorylated at Ser58 in early and late phases of infection. Knockdown of RanBP3 expression led to a vRNP nuclear retention, suggesting that RanBP3 is involved in vRNP nuclear export. Moreover, we demonstrated that RanBP3’s function during vRNP nuclear export is regulated by phosphorylation at Ser58, and the RanBP3 phosphorylation is modulated by both PI3K/Akt and Ras/ERK/RSK pathways in the late phase of viral infection. In conclusion, this study has shown that RanBP3 is a host factor that has a vital role during the late stage of influenza A virus replication, specifically as a co-factor in CRM1-mediated nuclear export. Identifying this host factor will contribute to the understanding of the mechanism of vRNP transport.

Supplementary Information Video attached / <p> Machado-Joseph disease (MJD), also known as Spinocerebellar ataxia type 3 (SCA3) is one of nine poly glutamine neurodegenerative diseases caused by an expansion of CAG DNA triplets in the genes resulting in an expanded poly glutamine tract in the expressed proteins. These proteins are unrelated in function yet all manifest as specific neurological diseases. The Truant lab and others have previously shown that six of the nine polyglutamine proteins display nucleocytoplasmic shuttling capabilities and that this shuttling is affected by polyglutamine expansion. It is believed that deciphering the mechanism of nucleocytoplasmic transport may be important in understanding the normal function of these proteins, which in turn may lead to a better understanding of the pathogenesis of disease. Studies that looked at the subcellular localization of the MJD/SCA3 protein, ataxin-3, have shown that the normal protein is variably distributed between the nucleus and the cytoplasm, whereas mutant ataxin-3 is localized primarily in the nucleus. Using fluorescent protein technology and fluorescence microscopy, this thesis project attempts to analyze the nucleocytoplasmic shuttling capabilities of ataxin-3 and to evaluate the potential mechanisms that govern its translocation into and out of the nucleus. </p> <p> It was revealed that ataxin-3 is able to shuttle into and out of the nucleus and that the shuttling dynamics are dependent on the length of the poly glutamine tract. As well, two putative, CRMl dependent nuclear export signals and a putative, importin-a/~1 dependent, classical, nuclear localization signal were tested and shown to be nonfunctional as transport signals. It was then discovered that ataxin-3 is marginally leptomycin B (an inhibitor ofCRMl dependent nuclear export) sensitive in NIH3T3 and MCF7 cells, more sensitive to the drug in STHdhQ71Q7 cells and even more so in HEK 293 cells. This suggests that an exogenous factor mediates the nuclear import of ataxin-3 through the CRMl pathway. Subsequently, four known binding partners, hHDACl, hHDAC2, hHDAC6 and hHRAD23b, were tested for their potential ability to shuttle ataxin-3. It was concluded that although hHDAC6 had the greatest effect on ataxin-3 subcellular localization, we believe that it does not mediate its nuclear import or export. Future studies would involve an investigation as to how and why different polyglutamine lengths affect the nucleocytoplasmic shuttling of ataxin-3 and to identify the factor(s) that cause ataxin-3 to be more sensitive to LMB treatments in HEK 293 cells. </p> / Thesis / Master of Science (MSc)

Yes / Tumour suppressor proteins, such as p53, BRCA1, and ABC, play key roles in preventing the development of a malignant phenotype, but those that function as transcriptional regulators need to enter the nucleus in order to function. The export of proteins between the nucleus and cytoplasm is complex. It occurs through nuclear pores and exported proteins need a nuclear export signal (NES) to bind to nuclear exportin proteins, including CRM1 (Chromosomal Region Maintenance protein 1), and the energy for this process is provided by the RanGTP/RanGDP gradient. Due to the loss of DNA repair and cell cycle checkpoints, drug resistance is a major problem in cancer treatment, and often an initially successful treatment will fail due to the development of resistance. An important mechanism underlying resistance is nuclear export, and a number of strategies that can prevent nuclear export may reverse resistance. Examples include inhibitors of CRM1, antibodies to the nuclear export signal, and alteration of nuclear pore structure. Each of these are considered in this review.

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<P> The first histone acetyltransferase to be described was GCN5, from the yeast species Saccharomyces cerevisiae. To date, the GCN5-related N-acetyltransferases (GNATs) comprise one of the largest enzyme superfamilies with over 10,000 identified members in sequenced genomes. This protein is known to acetylate specific lysine residues on the amino-terminal tails of nucleosomal histones, thereby loosening their contact with the tightly packed DNA and facilitating transcription. </p> <p> In this study, I determined that GCN5 is able to shuttle between the nucleus and the cytoplasm using fluorescence recovery after photobleaching (FRAP). Mutational studies revealed that its nuclear import is regulated by a classical bipartite nuclear localization signal (NLS) that is dependent on the transporters importin a and f3. In contrast, we found that GCN5 lacks a CRM1-dependent nuclear export signal (NES), as demonstrated by mutational and leptomycin B (LMB) studies; instead, IKB, a previouslydescribed transcription inhibitor with a CRMl-dependent NES, was found to modulate the export of GCN5 from the nucleus. This was initially discovered while performing the LMB assays, for which IKB served as a positive control, and was subsequently confirmed by mutational studies and protein complementation assays (PCAs). Furthermore, while the PCAs demonstrated a physical interaction between these two proteins in vivo, GST pull-down experiments were employed to confirm their interaction in vitro. </p> <p> Furthermore, this study also revealed that over-expression of GCN5-e YFP in NIH 3T3 cells causes -10% of the transfected cells to exhibit nuclear GCN5-eYFP-associated filaments; these structures were confirmed to be F -actin filaments comprised of f3-actin through co-localization studies with both TRITC-phalloidin and a mRFP-f-actin construct. GCN5's acetyltransferase activity was shown to be responsible for the formation ofthese filaments through mutation of its catalytic residue. Moreover, a protein complementation assay (PCA) demonstrated an in vivo interaction between GCN5 and f-actin, while FRAP analysis of a single filament showed that GCN5-e YFP molecules rapidly and randomly associate with these filaments along their entire length. Together these results suggest that GCN5's acetyltransferase activity is responsible for the structural maintenance of these filaments. Finally, GCN5-eYFP-associated filaments were found to be spatially separate from both lamin A (a nuclear envelope structural protein) and DNA; however, this does not exclude the possibility of an indirect interaction between these cellular constituents, as treatment of a live cell with Hoechst DNA stain, which disrupts the structure of DNA, was shown to disturb the structural integrity of these filaments. </p> / Thesis / Master of Science (MSc)