UBC Theses and Dissertations
Nuclear import of purified influenza : a viral ribonucleoproteins Sun, Ying-Hua Betty
The influenza A viral ribonucleoprotein (vRNP) is a macromolecular complex composed of nucleoproteins (NPs), heterotrimetric polymerase complexes (PA, PB1, PB2) and a single stranded viral RNA. Numerous nuclear localization signals (NLSs) have been identified on the vRNP proteins, including two on NP: a classical bipartite NLS (NPcNLS) and a classical nonconventional NLS (NPnNLS). However, the NLS responsible for vRNP nuclear import is not known. Previous studies have demonstrated that NP supports the nuclear import of in vitro assembled vRNA-NP complexes, but, whether NP also supports the nuclear import of authentic vRNPs is not known. Authentic influenza A vRNPs are assembled in the host nucleus and is complete with all four types of vRNP proteins. Unlike in vitro assembled vRNA-NP complexes, authentic vRNPs can, in theory, enter the nucleus using the NLS from any one of the four types of vRNP proteins. I have studied the nuclear import mechanism of authentic vRNPs in mammalian cells in order to determine the functional NLS responsible for vRNP nuclear import. My hypothesis is that authentic vRNPs enter the nucleus using a NP NLS. To test my hypothesis, I have purified authentic vRNPs from influenza A virus. The vRNPs had good structural integrity when visualized by high resolution electron microscopy. Using immunogold electron microscopy, I have shown that both NPnNLS and NPcNLS were exposed on the outer surface of authentic vRNPs; however, more exposed NPnNLS were found in comparison to NPcNLS. Both NLSs were functionally characterized using peptide competition and antibody inhibition experiments in digitonin-perrneabilized cells and microinjected live cells. Only the microinjection studies provided consistent and convincing results. The digitonin-permeabilized cells were incompatible with purified vRNPs, as vRNPs bound strongly to nonspecific sites in the cytoplasm of the digitonin-permeabilized cells. Results from the microinjection studies showed that nuclear import of authentic vRNPs could be inhibited by interfering with the NPnNLS nuclear import pathway, by either introducing excessive NPnNLS peptides or NPnNLS specific antibodies. By contrast, interfering with the NPcNLS nuclear import pathway using similar methods did not inhibited the nuclear import of vRNPs. In these studies, the vRNPs were visualized by indirect immunofluorescence using a monoclonal antibody against NP, because direct labeling of vRNPs to the cy3 fluorochmme abolished the vRNP nuclear import function. The results presented in this thesis demonstrated that while two NP NLSs are exposed on the surfaces of authentic vRNPs, the NPnNLS is the functional NLS responsible for vRNP nuclear import. These results show, for the first time that authentic vRNPs, purified from the influenza A virus, enter the nucleus of mammalian cells using the NPnNLS nuclear import pathway. This makes influenza A vRNP the second example, after the splicesomal uridine-rich small nuclear RNP (U snRNP), that an RNA-protein complex enters the nucleus by one of its protein NLS. Using the new results presented in this thesis, I have proposed a model for the nuclear import mechanism of influenza A vRNPs. These results open the possibility of using NPnNLS as a novel target for anti-viral therapeutics.
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