NUCLEAR IMPORT OF INFLUENZA-VIRUS RNA CAN BE MEDIATED BY VIRAL NUCLEOPROTEIN AND TRANSPORT FACTORS REQUIRED FOR PROTEIN IMPORT

We have fluorescently labeled one of the eight genomic segments of influenza virus RNA and a recombinant influenza viral protein, the nucleoprotein (NP), to investigate the requirement for their uptake into nuclei of digitonin-permeabilized cells. We found that the influenza viral NP behaves like a nuclear localization sequence (NLS) containing protein. Thus, at 0 degrees C it docks at the nuclear envelope only in the presence of the heterodimeric karyopherin (either karyopherin alpha 1 beta or karyopherin alpha 2 beta), and docking is competitively inhibited by an unlabeled NLS containing substrate. Like other NLS-containing proteins, at 20 degrees C NP is imported into the nucleus after further addition of the GTPase Ran and of p10, In contrast, the fluorescently labeled, 890-nucleotide-long viral RNA segment does not dock to the nuclear envelope or enter the nucleus either in the presence of exogenous cytosol or of karyopherin heterodimer, Ran, and p10, However, in the presence of NP the RNA is able to dock and enter the nucleus with transport requirements indistinguishable from those for docking and entry of NP. These data indicate that uptake of the influenza virus RNA segment is not via a signal in the RNA but via an NLS of a viral protein such as NP.