| Influenza is an RNA virus which replicates in the nucleus and buds from the plasma membrane. As such, its ribonucleoproteins (RNPs) must undergo bidirectional transport across the nuclear envelope. This thesis examines the transport of incoming influenza viral RNPs into the nucleus during viral entry and the export of newly synthesized viral RNPs out of the nucleus towards the end of infection.;In contrast, I found that export of newly synthesized viral RNPs depended on association with M1. During infection, the export of newly synthesized NP from the nucleus coincided with the onset of M1 synthesis. In cells in which M1 was not expressed, NP remained confined to the nucleus. When the transport of newly synthesized M1 into the nucleus was blocked by cytoplasmically microinjected anti-M1 antibodies, NP remained in the nucleus. By double-label immunocytochemistry, M1 was found associated with newly assembled viral RNPs in the nucleus, at the nuclear pore, and in the cytoplasm. Thus, M1 appeared to bind to the viral RNPs in the nucleus and to escort them through the nuclear pore to their site of budding at the plasma membrane.;Together, the results of this thesis show that M1 modulates the directionality of influenza viral RNP transport into and out of the nucleus.;Upon penetration of the influenza virus nucleocapsid into the host cell cytoplasm, the viral RNA and associated proteins are transported to the nucleus, where viral transcription and replication occur. Using quantitative confocal microscopy, I found that over half of cell-associated nucleoprotein (NP) entered the nucleus with a half time of 10 min after penetration into CHO cells. Microinjection and immunoelectron microscopy experiments indicated that the NP entered the nucleus through the nuclear pore as part of an intact RNP structure and that its transport was an active process. Transport of the incoming RNPs into the nucleus was not dependent on an intact microfilament, microtubule, or intermediate filament network. Subsequent to penetration, the matrix (M1) protein appeared to dissociate from the RNP structure and to enter the nucleus independently of the RNP. I found that 50% of penetrated M1 entered the nucleus with a half time of 25 min after penetration into CHO cells. Nuclear transport of M1 appeared to occur by passive diffusion. While the entry of incoming M1 into the nucleus was not a prerequisite for infection, the separation of M1 from the incoming viral RNPs was. Amantadine, an antiviral agent which induces an early block in influenza infection, was found to block the dissociation event, and to thereby prevent import of incoming RNPs into the nucleus. |
