| Summary: | Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the archetype of the Baculoviridae family, is an enveloped, rod-shaped, double-stranded DNA virus that replicates in the nucleus of its host cells. Baculoviruses have been used extensively as pesticides and in biological systems. Despite their importance, the mechanism by which baculovirus deliver its genome into the nucleus has been the subject of considerable debate. Molecules <39 nm in diameter enter the nucleus through nuclear pore complexes (NPCs) embedded within the nuclear envelope. Because the diameter of AcMNPV capsids (30 x 300 nm) falls below this limit, we hypothesize that AcMNPV capsids enter the nucleus via NPCs. In this thesis, we aim to visualize the mechanism of nuclear import used by the baculovirus AcMNPV capsid, to understand the role of cellular proteins facilitating viral capsid delivery into the nucleus, and to demonstrate the role of cellular actin in mediating nuclear import of the baculovirus capsid. We found for the first time that an intact AcMNPV capsid is able to traverse the NPC, importing the entire capsid into the nucleoplasm. This transport occurs through the NPC central channel, which is able to open up completely to accommodate the AcMNPV capsid. Nuclear transport of these capsids was inhibited by physical blockade of NPCs, as well as low temperature conditions, which inhibits facilitated transport and dynamic structural changes of the NPC. We also showed that nuclear import of AcMNPV capsids occurred independently of importin-β and the Ran cycle, but required activation of actin nucleation. Finally, we demonstrated through targeted knockdown that Nup62 and Nup153 are not essential in mediating nuclear import of AcMNPV capsids. Conversely, these depletion studies showed that Nup358 enhances the nuclear import efficiency of AcMNPV capsids. Our results support a model in which the intact baculovirus capsid, after being released into the cytoplasm during infection, enters the nucleus through the NPC, by a mechanism that does not require importin-β, Ran, and the three FG-Nups tested, but is dependent on actin nucleation. Because baculovirus capsids are among the largest cargos that translocation through the NPC, our results provide exciting new insights into how the NPC functions. === Science, Faculty of === Zoology, Department of === Graduate
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