Browsing by Subject "Nuclear Pore"
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Item The Role of NS1-BP in Influenza Virus Replication(2012-08-13) Tsai, Pei-Ling; Fontoura, BeatrizInfluenza A viruses are negative-sense, segmented RNA viruses which cause about 500,000 deaths worldwide per year. Genomic studies have shown that the non-structural protein (NS1) of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 is a multifunctional protein localized in the nucleus and in the cytoplasm. In the cytoplasm, NS1 inhibits host signaling pathways that result in down-regulation of interferon expression and innate immune response. In the nucleus, NS1 represses host gene expression. I have shown that NS1 binds an mRNA complex containing NXF1/TAP, NXT/p15, Rae1, and E1B-AP5, which are key components of the mRNA export machinery. By targeting this complex, NS1 blocks host mRNA export, and cells become highly permissive to viral replication. Another intranuclear pool of NS1 was found to interact with a host protein termed NS1-BP, which has been suggested to play a role in pre-mRNA splicing. However, the functions and mechanisms of NS1-BP involved in influenza life cycle remain to be elucidated. To investigate the function of NS1-BP, I first identified its binding partners by immunoprecipitation followed by mass spectrometry. I found interactions of NS1-BP with viral polymerase complex and host RNA polymerase II indicating that NS1-BP has a role in regulating viral RNA transcription and replication. I further showed that low levels of NS1-BP led to a decrease in viral polymerase activity resulting in inhibition of virus replication. Thus, I identified NS1-BP as a novel pro-viral factor required for proper replication of influenza virus. Since NS1 is a key contributor to the virulence of influenza viruses, discovering the function of NS1 interacting partners has major implications for antiviral therapy.Item Toxic PRn Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Block Nuclear Import and Export(2017-03-02) Shi, Kevin; Nijhawan, Deepak; McKnight, Steven L.; Rizo-Rey, José; Rosen, Michael K.Expansion of the (GGGGCC)n hexanucleotide repeat within the first intron of the C9orf72 gene is the mutation that leads to the most prevalent heritable form of amyotrophic lateral sclerosis (ALS). The expanded repeat is aberrantly transcribed from both sense and anti-sense strands relative to the C9orf72 gene, and both transcripts are translated in an ATG-independent manner to yield five distinct poly-dipeptides. Expression of either the glycine:arginine (GRn) or proline:arginine (PRn) poly-dipeptide in Drosophila leads to neurodegneration of the eye, and when introduced into culturing medium, synthetic forms of both peptides lead to the death of human cells. We recently identified PRn poly-dipeptide interacting partners in vivo using an unbiased proteomics approach, demonstrating that the nuclear pore complex (NPC) as a major binding target. The biological significance of the PRn peptide-NPC interaction became apparent when we observed major defects in both mature mRNA export to the cytoplasm and protein import into the nucleus after cells were treated with PRn. The functional nucleocytoplasmic transport defect caused by PRn peptide was due to binding of the peptide to the central channel of the NPC as visualized by super-resolution microscopy. The NPC central channel is comprised of phenylalanine-glycine (FG) domain nucleoporins, and these proteins are essential for the NPC's active transport and passive size-exclusion permeability barrier functions. Purified FG domains of Nup54 and Nup98, both identified in our proteomics study as PRn targets, polymerized into structurally labile, cross-β sheet fibers under physiological conditions. Several lines of evidence suggested that the polymerized state of FG domains is relevant to intact NPC. First, PRn peptides only bound to polymerized Nup54 and Nup98 and not to the soluble versions of either protein, potentially representing how PRn binds to the FG-rich central channel. Second, the aliphatic alcohol 1,6-hexanediol (HD) selectively disrupted the permeability barrier of the NPC, while 2,5-hexanediol had no effect. Similarly, 1,6-HD can effectively solubilize Nup FG polymers in vitro, while 2,5-HD had no effect on polymer stability. Finally, PRn binding to the NPC abrogated the disruption of the permeability barrier by 1,6-HD. Similarly, PRn also protected the Nup FG polymers from solubilization by 1,6-HD. Our study has elucidated a major mechanism by which the C9orf72 expansion associated PRn poly-dipeptide inhibits the transport of macromolecules in and out of the nucleus, leading to a major disruption of cellular physiology. Our results support a model in which the FG domains of the NPC exist in equilibrium between the polymerized and unpolymerized states. By binding to polymerized FG domains and stabilizing them, the PRn peptide is understood to shift the equilibrium toward the polymerized state, with consequent blockage of nuclear transport. The aliphatic alcohol 1,6-hexanediol has the opposite effect, shifting the equilibrium toward the unstructured state of FG domains and disrupting the permeability barrier. The effects of the PRn peptide and aliphatic alcohols on nuclear pore function represent extreme conditions. More subtle changes in the equilibrium between structured and unstructured FG domains may regulate nuclear pore function in living cells.