Nucleocytoplasmic transport: the influenza virus NSl protein regulates the transport of spliced NS2 mRNA and its precursor NSl mRNA

Influenza virus unspliced NSl mRNA, like retroviral pre-mRNAs, is efficiently exported from the nucleus and translated in the cytoplasm of infected cells. With human immunodeficiency virus (HIV), the transport of viral pre-mRNAs is facilitated by the viral Rev protein. We tested the possibility that the influenza virus NSl protein, a nuclear protein that is encoded by unspliced NSl mRNA, has the same function as the HIV Rev protein. Surprisingly, using transient transfection assays, we found that rather than facilitating the nucleocytoplasmic transport of unspliced NSl mRNA, the NSl protein inhibited the transport of NS2 mRNA, the spliced mRNA generated from NSl mRNA. The efficient transport of NS2 mRNA from the nucleus to the cytoplasm occurred only when the synthesis of the NSl protein was abrogated by amber mutations. The NSl protein down-regulated the export of NS2 mRNA whether or not it was generated by splicing, indicating that the NSl protein acted directly on transport. Actinomycin D chase experiments verified that the NSl protein acted on the transport and not on the differential stability of NS2 mRNA in the nucleus as compared to the cytoplasm. In addition, the NSl protein inhibited the transport of NSl mRNA itself, which contains all of the sequences in NS2 mRNA, particularly when NSl mRNA was released from the splicing machinery by mutating its 3'-splice site. Our results indicate that the NSl protein-mediated inhibition of transport requires sequences in NS2 mRNA. The transport of the viral PBl protein, nucleocapsid protein, hemagglutinin, membrane protein, and M2 mRNAs was not affected by the NSl protein. When the NS2 mRNA sequence was covalently attached to the PBl mRNA, the transport of the chimeric mRNA was inhibited by the NSl protein. Our results identify a novel function of the influenza virus NSl protein and demonstrate that post-transcriptional control of gene expression can also occur at the level of the nucleocytoplasmic transport of a mature, spliced mRNA.