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Articles by Shelly Au in JoVE
JoVE General
Mikroinjektion av Xenopus laevis oocyter
Department of Zoology, University of British Columbia - UBC
Här visar vi cytoplasmiska mikroinjektion av
Other articles by Shelly Au on PubMed
Microinjection of Xenopus Laevis Oocytes As a System for Studying Nuclear Transport of Viruses
Microinjection of Xenopus laevis oocytes is an excellent system for studying nuclear transport because of the large size of the oocyte and its high nuclear pore complex (NPC) density. In addition, the fact that Xenopus oocytes are not permissive for most mammalian viruses makes this system especially useful for studying nuclear transport of viruses in the absence of the confounding factor of virus replication. In this article, we briefly discuss the contribution of microinjection of Xenopus oocytes to the field of nuclear transport. We then describe the protocols we have developed using microinjection of Xenopus oocytes to study nuclear transport of viral capsids, and summarize variations of the technique that can be used to address many different questions about the nuclear transport of viruses.
How Viruses Access the Nucleus
Many viruses depend on nuclear proteins for replication. Therefore, their viral genome must enter the nucleus of the host cell. In this review we briefly summarize the principles of nucleocytoplasmic transport, and then describe the diverse strategies used by viruses to deliver their genomes into the host nucleus. Some of the emerging mechanisms include: (1) nuclear entry during mitosis, when the nuclear envelope is disassembled, (2) viral genome release in the cytoplasm followed by entry of the genome through the nuclear pore complex (NPC), (3) capsid docking at the cytoplasmic side of the NPC, followed by genome release, (4) nuclear entry of intact capsids through the NPC, followed by genome release, and (5) nuclear entry via virus-induced disruption of the nuclear envelope. Which mechanism a particular virus uses depends on the size and structure of the virus, as well as the cellular cues used by the virus to trigger capsid disassembly and genome release. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.
Nuclear Transport of Baculovirus: Revealing the Nuclear Pore Complex Passage
Baculoviruses are one of the largest viruses that replicate in the nucleus of their host cells. During an infection the capsid, containing the DNA viral genome, is released into the cytoplasm and delivers the genome into the nucleus by a mechanism that is largely unknown. Here, we used capsids of the baculovirus Autographa californica multiple nucleopolyhedrovirus in combination with electron microscopy and discovered this capsid crosses the NPC and enters into the nucleus intact, where it releases its genome. To better illustrate the existence of this capsid through the NPC in its native conformation, we reconstructed the nuclear import event using electron tomography. In addition, using different experimental conditions, we were able to visualize the intact capsid interacting with NPC cytoplasmic filaments, as an initial docking site, and midway through the NPC. Our data suggests the NPC central channel undergoes large-scale rearrangements to allow translocation of the intact 250-nm long baculovirus capsid. We discuss our results in the light of the hypothetical models of NPC function.
