Microinjection of Xenopus laevis oocytes as a system for studying nuclear transport of viruses

Au, Shelly; Cohen, Sarah; Panté, Nelly

doi:10.1016/j.ymeth.2010.02.001

Cited by 17 publications

(20 citation statements)

References 28 publications

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“…Adherent LA9 mouse fibroblast cells (30) and HeLa cells stably expressing a fusion of green fluorescent protein to lamina-associated polypeptide 2␤ (GFP-LAP2␤) (courtesy of U. Kutay, ETH Zurich) were maintained at 5% CO 2 and 37°C in complete Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillinstreptomycin. Wild-type MVM was purified as previously described (2). MVM with an H42R mutation was a gift from P. Tattersall (Yale University School of Medicine).…”

Section: Methodsmentioning

confidence: 99%

“…For the phospholipase A2 (PLA2) inhibition experiment, MVM was incubated with 10 M manoalide (Alexis Biochemicals) for 1 h at room temperature with agitation prior to microinjection. Where indicated, a 50 mM concentration of the caspase inhibitor benzyloxycarbonylVal-Ala-Asp-fluoromethyl ketone (zVAD-fmk), zDEVD-fmk, or zVEID-fmk (Tocris Biosciences) was included in the MVM solution to be injected, resulting in an intracellular inhibitor concentration of approximately 200 M. Oocytes were then incubated at room temperature for 2 h, followed by fixation and preparation for thin-section electron microscopy (EM) as previously described (2,5,39). Quantification of the proportion of ONM damaged was performed using ImageJ software (National Institutes of Health).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were fixed with 4% paraformaldehyde for 1 h and harvested by scraping. The resulting cell pellets were embedded for immunogold EM as previously described (2). For immunogold labeling, grids containing EM sections were blocked (2% BSA, three times for 10 min each), labeled with primary antibody for intact MVM capsids (1:5, monoclonal; provided by P. Tattersall) and secondary antibody (1:50, goat anti-mouse conjugated to 10-nm gold; Ted Pella).…”

Section: Methodsmentioning

confidence: 99%

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Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

Cohen

Marr

Garcin

et al. 2011

J Virol

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Parvoviruses are small, nonenveloped, single-stranded DNA viruses which replicate in the nucleus of the host cell. We have previously found that early during infection the parvovirus minute virus of mice (MVM) causes small, transient disruptions of the nuclear envelope (NE). We have now investigated the mechanism used by MVM to disrupt the NE. Here we show that the viral phospholipase A2, the only known enzymatic domain on the parvovirus capsid, is not involved in causing NE disruption. Instead, the virus utilizes host cell caspases, which are proteases involved in causing NE breakdown during apoptosis, to facilitate these nuclear membrane disruptions. Studies with pharmacological inhibitors indicate that caspase-3 in particular is involved. A caspase-3 inhibitor prevents nuclear lamin cleavage and NE disruption in MVM-infected mouse fibroblast cells and reduces nuclear entry of MVM capsids and viral gene expression. Caspase-3 is, however, not activated above basal levels in MVM-infected cells, and other aspects of apoptosis are not triggered during early MVM infection. Instead, basally active caspase-3 is relocalized to the nuclei of infected cells. We propose that NE disruption involving caspases plays a role in (i) parvovirus entry into the nucleus and (ii) alteration of the compartmentalization of host proteins in a way that is favorable for the virus.In order to replicate successfully, viruses must overcome various barriers in the cell. For viruses that replicate in the cell nucleus, the nuclear envelope (NE) is one such barrier. The NE consists of an inner nuclear membrane (INM) and an outer nuclear membrane (ONM). These membranes are supported by an underlying protein meshwork called the nuclear lamina, composed of the intermediate filament proteins nuclear lamins, which is associated with the nuclear face of the NE. Embedded in the NE are the nuclear pore complexes (NPCs), which are large protein complexes that mediate active transport of molecules up to 39 nm in diameter into and out of the nucleus (40). Because the sizes and structures of viruses vary enormously, viruses have developed surprisingly diverse strategies for delivering their genome and accessory proteins into the nuclei of infected cells (21,26,60,61). Aside from some retroviruses, which are thought to enter the nucleus while the NE is disassembled during mitosis (19), most of these strategies involve partial disassembly of the virion and nuclear transport through the NPC using the cellular nuclear import machinery (i.e., nuclear localization signals, importins, GTP, and Ran) (55). The viral component entering the nucleus may be an intact capsid (e.g., hepatitis B virus capsid, which crosses the NPC intact [40,42]), a naked viral genome (e.g., for herpes simplex virus type 1 which ejects its DNA from its NPC-docked capsid into the nucleus, leaving empty capsids at the NPC [51]), or a viral genome in association with viral proteins (e.g., influenza virus ribonucleoprotein complexes [11]).In general, more is known about the nuclear entry of ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

Cohen

Marr

Garcin

et al. 2011

J Virol

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“…AcMNPV nucleocapsids and VP39-3×mCherry nucleocapsids were obtained by de-enveloping the virus by treatment with 1% NP40 for 1 h at 30°C as described previously (Au et al, 2010;Au and Pante, 2012).…”

Section: Virus Nucleocapsid and Cy3-nls-bsamentioning

confidence: 99%

A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid

Zhou

et al. 2016

Journal of Cell Science

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The transport of macromolecules into the nucleus is mediated by soluble cellular receptors of the importin β superfamily and requires the Ran-GTPase cycle. Several studies have provided evidence that there are exceptions to this canonical nuclear import pathway. Here, we report a new unconventional nuclear import mechanism exploited by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). We found that AcMNPV nucleocapsids entered the nucleus of digitonin-permeabilized cells in the absence of exogenous cytosol or under conditions that blocked the Ran-GTPase cycle. AcMNPV contains a protein that activates the Arp2/3 complex and induces actin polymerization at one end of the rod-shaped nucleocapsid. We show that inhibitors of Arp2/3 blocked nuclear import of nucleocapsids in semi-permeabilized cells. Nuclear import of nucleocapsids was also reconstituted in purified nuclei supplemented with G-actin and Arp2/3 under actin polymerization conditions. Thus, we propose that actin polymerization drives not only migration of baculovirus through the cytoplasm but also pushes the nucleocapsid through the nuclear pore complex to enter the cell nucleus. Our findings point to a very distinct role of actin-based motility during the baculovirus infection cycle.

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“…Subsequent studies using microinjection of the parvovirus minute virus of mice (MVM) into Xenopus oocytes, a popular technique to study the transport of cargo across the NPC [32], found small (100-300 nm) disruptions of the NE, and virions near these breaks and in the perinuclear space adjacent to the breaks in the outer nuclear membrane [33]. These findings were further validated in MVM-infected fibroblast cells [34,35].…”

Section: Introductionmentioning

confidence: 94%

Old foes, new understandings: nuclear entry of small non-enveloped DNA viruses

Fay

Panté

2015

Current Opinion in Virology

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Microinjection of Xenopus laevis oocytes as a system for studying nuclear transport of viruses

Cited by 17 publications

References 28 publications

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

Nuclear Envelope Disruption Involving Host Caspases Plays a Role in the Parvovirus Replication Cycle

A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid

Old foes, new understandings: nuclear entry of small non-enveloped DNA viruses

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