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 vitro transport system with digitonin-treated infected cells. We first monitored 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 immunofluorescence 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 the presence of LMB. Finally, we performed in vitro RNA synthesis assays using digitonin-treated infected cells and exported fractions. The exported vRNP was RNA synthesis-competent. Analyses using glycerol density gradients showed that a major fraction of M1 and NS2 was not complexed with the exported vRNP. These results suggest that nuclear export of RNA synthesis-competent 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 discussed.