Inhibition of nuclear export of ribonucleoprotein complexes of influenza virus by leptomycin B

We have studied nuclear export of influenza virus components using an in vi tro transport system with digitonin-treated infected cells. We first monito red the efficiency of export of the viral ribonucleoprotein (vRNP) complex by analyzing viral components with western blotting. We used leptomycin B ( LMB), an inhibitor of nuclear export signal (NES) - and its receptor, CRM1/ Exportin1 - mediated protein export. LMB efficiently inhibited vRNP export, while it did not affect the subcellular localization and export of matrix protein (M) 1 and nonstructural protein (NS) 2. Second, indirect immunofluo rescence assays also revealed that vRNP export is sensitive to LMB. NS2 in NS2-transfected cells was not accumulated in nuclei in the presence of LMB. while NS2 in infected cells was found slightly accumulated in nuclei in th e presence of LMB. Finally, we performed in vitro RNA synthesis assays usin g digitonin-treated infected cells and exported fractions. The exported vRN P was RNA synthesis-competent. Analyses using glycerol density gradients sh owed that a major fraction of MI and NS2 was not complexed. with the export ed vRNP. These results suggest that nuclear export of RNA synthesis-compete nt vRNP is dependent on a LMB-sensitive pathway and that there would be two types of NS2, i.e. LMB-sensitive and -insensitive NS2. The involvement of viral late proteins in vRNP export during late stages of infection is discu ssed. (C) 2001 Elsevier Science B.V. All rights reserved.