title: Ultrastructural analysis of the nuclear localization sequences on influenza A ribonucleoprotein complexes.
abstract: The influenza A genome consists of eight single-stranded RNA molecules, each associated with an oligomeric core of the structural protein, nucleoprotein, to form a distinct viral ribonucleoprotein (vRNP) complex. vRNPs are the entities responsible for the transcription and replication of the influenza viral RNAs in the nuclei of host cells. Thus, nuclear targeting and localization of the vRNPs are a critical step in the infection and life cycle of influenza A. We have recently shown that the nuclear import of vRNPs derived from influenza A virions is independently mediated by two nuclear localization sequences (NLSs) on nucleoprotein: NLS1, spanning residues 1-13 at the N terminus, and NLS2, spanning residues 198-216 in the middle of the protein, with NLS1 being the principal mediator. To better understand the structural basis for the differences in the ability of NLS1 and that of NLS2 to mediate nuclear import of influenza vRNPs, we analyzed the levels of surface exposure of these NLSs on vRNPs by both dot blotting and immunogold labeling of vRNPs in their native state. We found that NLS1 is much more accessible to its corresponding antibody compared with NLS2. Electron microscopy of immunogold-labeled vRNPs further showed that 71% of vRNPs were labeled with one to six gold particles located throughout the vRNP for NLS1. In contrast, less than 10% of vRNPs were labeled with an antibody against NLS2, usually with a single gold particle located at one end of the vRNP. In addition, a regular periodicity of repeat was observed with gold particles labeling for NLS1, indicative of a highly regular helical conformation present in the vRNPs. These findings provide the underlying structural basis for the enhanced ability of NLS1 in mediating nuclear import of influenza vRNPs and add to our understanding of the ultrastructural features of vRNP complexes derived from influenza A virions.
authors.affiliation: Department of Zoology, University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4.
authors.forename: Winco W H
authors.initials: WW
authors.lastname: Wu
authors.forename: Lindsay L
authors.initials: LL
authors.lastname: Weaver
authors.forename: Nelly
authors.initials: N
authors.lastname: Panté
mesh_terms: D000595:Amino Acid Sequence; D007150:Immunohistochemistry; D009980:Influenza A virus / Q000737:chemistry*; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012367:RNA, Viral / Q000737:chemistry*; D012261:Ribonucleoproteins / Q000737:chemistry* / Q000648:ultrastructure; D014764:Viral Proteins / Q000737:chemistry*; D014771:Virion / Q000737:chemistry
chemical_list: D019913:Nuclear Localization Signals; D012367:RNA, Viral; D012261:Ribonucleoproteins; D014764:Viral Proteins