Alonso-Caplen F V, Nemeroff M E, Qiu Y, Krug R M
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1179.
Genes Dev. 1992 Feb;6(2):255-67. doi: 10.1101/gad.6.2.255.
Influenza virus unspliced NS1 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 NS1 protein, a nuclear protein that is encoded by unspliced NS1 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 NS1 mRNA, the NS1 protein inhibited the transport of NS2 mRNA, the spliced mRNA generated from NS1 mRNA. The efficient transport of NS2 mRNA from the nucleus to the cytoplasm occurred only when the synthesis of the NS1 protein was abrogated by amber mutations. The NS1 protein down-regulated the export of NS2 mRNA whether or not it was generated by splicing, indicating that the NS1 protein acted directly on transport. Actinomycin D chase experiments verified that the NS1 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 NS1 protein inhibited the transport of NS1 mRNA itself, which contains all of the sequences in NS2 mRNA, particularly when NS1 mRNA was released from the splicing machinery by mutating its 3'-splice site. Our results indicate that the NS1 protein-mediated inhibition of transport requires sequences in NS2 mRNA. The transport of the viral PB1 protein, nucleocapsid protein, hemagglutinin, membrane protein, and M2 mRNAs was not affected by the NS1 protein. When the NS2 mRNA sequence was covalently attached to the PB1 mRNA, the transport of the chimeric mRNA was inhibited by the NS1 protein. Our results identify a novel function of the influenza virus NS1 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.
流感病毒未剪接的NS1 mRNA，与逆转录病毒前体mRNA一样，能有效地从细胞核输出并在受感染细胞的细胞质中进行翻译。对于人类免疫缺陷病毒（HIV），病毒Rev蛋白促进病毒前体mRNA的转运。我们测试了流感病毒NS1蛋白（一种由未剪接的NS1 mRNA编码的核蛋白）是否具有与HIV Rev蛋白相同功能的可能性。令人惊讶的是，通过瞬时转染实验，我们发现NS1蛋白非但不促进未剪接的NS1 mRNA的核质转运，反而抑制了NS2 mRNA（由NS1 mRNA产生的剪接后mRNA）的转运。只有当NS1蛋白的合成被琥珀突变消除时，NS2 mRNA才能有效地从细胞核转运到细胞质。无论NS2 mRNA是否通过剪接产生，NS1蛋白都会下调其从细胞核到细胞质的转运，这表明NS1蛋白直接作用于转运过程。放线菌素D追踪实验证实，NS1蛋白作用于转运过程，而非作用于NS2 mRNA在细胞核与细胞质中的差异稳定性。此外，NS1蛋白还抑制其自身mRNA（包含NS2 mRNA中的所有序列）的转运，特别是当通过突变其3'剪接位点使NS1 mRNA从剪接机制中释放出来时。我们的结果表明，NS1蛋白介导的转运抑制作用需要NS2 mRNA中的特定序列。病毒PB1蛋白、核衣壳蛋白、血凝素、膜蛋白和M2 mRNA的转运不受NS1蛋白影响。当NS2 mRNA序列共价连接到PB1 mRNA上时，NS1蛋白会抑制嵌合mRNA的转运。我们的结果确定了流感病毒NS1蛋白的一种新功能，并证明基因表达的转录后调控也可发生在成熟剪接mRNA的核质转运水平。
