A new mechanism for nuclear import by actin-based propulsion used by a baculovirus ... · 2016. 7....

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SHORT REPORT A new mechanism for nuclear import by actin-based propulsion used by a baculovirus nucleocapsid Shelly Au 1 , Wei Wu 1 , Lixin Zhou 1 , David A. Theilmann 2 and Nelly Pante ́ 1, * ABSTRACT 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. KEY WORDS: Actin, AcMNPV, Arp2/3 complex, Baculovirus, Nuclear import INTRODUCTION The main mechanism of nuclear transport is by passage through the nuclear pore complex (NPC). Molecules from 5 to 39 nm (Mohr et al., 2009; Pante and Kann, 2002) cross the NPC by bearing one or more nuclear localization sequences (NLSs). NLSs are recognized by nuclear transport receptors (NTRs) that translocate the cargo through the NPC (Cautain et al., 2015; Wente and Rout, 2010). The small GTPase Ran regulates the binding and dissociation of NTRs and NLS-containing cargos. Numerous studies have provided evidence for deviations to this conventional nuclear import mechanism (reviewed by Wagstaff and Jans, 2009). For example, nuclear import of cyclin-B1Cdc2 occurs independently of Ran but requires importin-β (Takizawa et al., 1999) and some nuclear import pathways are importin-β independent, for which the cargo acts as an NTR (Kumeta et al., 2012). Viruses and subviral particles use both conventional and unconventional pathways to enter the nucleus (Fay and Pante, 2015a,b). The cytoskeleton enhances this process by accelerating viral transport through the cytoplasm toward the nucleus (Wagstaff and Jans, 2009). Although most viruses use microtubule-mediated transport (Dodding and Way, 2011), baculoviruses use actin-based motility. Autographa californica nucleopolyhedrovirus (AcMNPV), the most-studied baculovirus, releases its nucleocapsid into the cytoplasm during cell entry (Rohrmann, 2013). The nucleocapsid is subsequently propelled towards the nucleus by inducing actin polymerization at one of its ends through VP78/83 (a single protein encoded by the baculovirus VP78/83 gene), a viral WiskottAldrich syndrome protein (WASP)-like protein (Lanier and Volkman, 1998; Machesky et al., 2001; Mueller et al., 2014; Ohkawa et al., 2010). VP78/83 is a nucleation-promoting factor that activates actin polymerization by the host Arp2/3 complex (Goley et al., 2006). The rod-shaped nucleocapsid (30×250300 nm) then enters the nucleus by crossing the NPC lengthwise (Au and Pante, 2012; Au et al., 2013). Whether baculoviruses use a conventional or an unconventional nuclear import pathway remains to be determined. In any case, proteins of the AcMNPV nucleocapsid do not contain any obvious functional NLS that could mediate the nuclear import of the nucleocapsid. Here, we show that nuclear import of AcMNPV nucleocapsids occurs independently of the Ran-GTPase cycle, but depends on Arp2/3-mediated F-actin nucleation. We further demonstrate the involvement of actin polymerization in the nuclear import of AcMNPV by reconstituting nuclear import of nucleocapsids in purified nuclei supplemented with G-actin and Arp2/3 under permissive conditions for actin polymerization. Our data suggest that baculovirus-induced actin polymerization is involved in the mechanism of nuclear import of AcMNPV nucleocapsids. RESULTS AND DISCUSSION AcMNPV nucleocapsid enters the nucleus of semi- permeabilized cells in the absence of exogenous cytosol In order to study nuclear import of AcMNPV nucleocapsids, we used the well-established nuclear import assay with digitonin- permeabilized cells (Adam et al., 1990; Cassany and Gerace, 2009). In this system, nuclear import is assayed by incubating the digitonin-permeabilized cells with a fluorescently labeled cargo and either exogenous cytosol or purified recombinant nuclear transport factors and an energy-regenerating system. Control experiments with a 70-kDa dextran labeled with Texas Red, which does not diffuse through the NPC, demonstrated that digitonin did not disrupt the nuclear envelope (data not shown). Control experiments with a Cy3-conjugated NLS-tagged bovine serum albumin (Cy3NLS-BSA) showed that this cargo was efficiently imported into the nucleus in the presence of exogenous cytosol (rabbit reticulocyte lysate; RRL) and an energy-regenerating system, whereas in the absence of RRL and the energy-regenerating system, nuclear import was inhibited as expected (Fig. 1A). Strikingly, when the import assay was performed with purified AcMNPV nucleocapsids, nuclear import of the nucleocapsid Received 2 May 2016; Accepted 3 June 2016 1 Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada. 2 Summerland Research and Development Centre, AAFC, Summerland, British Columbia, Canada. *Author for correspondence ( [email protected]) N.P., 0000-0002-0926-9378 2905 © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2905-2911 doi:10.1242/jcs.191668 Journal of Cell Science

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SHORT REPORT

A new mechanism for nuclear import by actin-based propulsionused by a baculovirus nucleocapsidShelly Au1, Wei Wu1, Lixin Zhou1, David A. Theilmann2 and Nelly Pante1,*

ABSTRACTThe transport of macromolecules into the nucleus is mediated bysoluble cellular receptors of the importin β superfamily and requires theRan-GTPase cycle. Several studies have provided evidence that thereare exceptions to this canonical nuclear import pathway. Here, wereport a new unconventional nuclear import mechanism exploited bythe baculovirus Autographa californica multiple nucleopolyhedrovirus(AcMNPV). We found that AcMNPV nucleocapsids entered thenucleus of digitonin-permeabilized cells in the absence of exogenouscytosol or under conditions that blocked the Ran-GTPase cycle.AcMNPV contains a protein that activates the Arp2/3 complex andinduces actin polymerization at one end of the rod-shapednucleocapsid. We show that inhibitors of Arp2/3 blocked nuclearimport of nucleocapsids in semi-permeabilized cells. Nuclear import ofnucleocapsids was also reconstituted in purified nuclei supplementedwith G-actin and Arp2/3 under actin polymerization conditions. Thus,we propose that actin polymerization drives not only migration ofbaculovirus through the cytoplasm but also pushes the nucleocapsidthrough the nuclear pore complex to enter the cell nucleus. Ourfindings point to a very distinct role of actin-based motility during thebaculovirus infection cycle.

KEY WORDS: Actin, AcMNPV, Arp2/3 complex, Baculovirus,Nuclear import

INTRODUCTIONThe main mechanism of nuclear transport is by passage through thenuclear pore complex (NPC). Molecules from 5 to 39 nm (Mohret al., 2009; Pante and Kann, 2002) cross the NPC by bearing one ormore nuclear localization sequences (NLSs). NLSs are recognizedby nuclear transport receptors (NTRs) that translocate the cargothrough the NPC (Cautain et al., 2015; Wente and Rout, 2010). Thesmall GTPase Ran regulates the binding and dissociation of NTRsand NLS-containing cargos. Numerous studies have providedevidence for deviations to this conventional nuclear importmechanism (reviewed by Wagstaff and Jans, 2009). For example,nuclear import of cyclin-B1–Cdc2 occurs independently of Ran butrequires importin-β (Takizawa et al., 1999) and some nuclear importpathways are importin-β independent, for which the cargo acts as anNTR (Kumeta et al., 2012).Viruses and subviral particles use both conventional and

unconventional pathways to enter the nucleus (Fay and Pante,2015a,b). The cytoskeleton enhances this process by accelerating

viral transport through the cytoplasm toward the nucleus (Wagstaffand Jans, 2009). Although most viruses use microtubule-mediatedtransport (Dodding and Way, 2011), baculoviruses use actin-basedmotility. Autographa californica nucleopolyhedrovirus (AcMNPV),the most-studied baculovirus, releases its nucleocapsid into thecytoplasm during cell entry (Rohrmann, 2013). The nucleocapsid issubsequently propelled towards the nucleus by inducing actinpolymerization at one of its ends through VP78/83 (a single proteinencoded by the baculovirus VP78/83 gene), a viral Wiskott–Aldrichsyndrome protein (WASP)-like protein (Lanier and Volkman, 1998;Machesky et al., 2001; Mueller et al., 2014; Ohkawa et al., 2010).VP78/83 is a nucleation-promoting factor that activates actinpolymerization by the host Arp2/3 complex (Goley et al., 2006).The rod-shaped nucleocapsid (30×250–300 nm) then enters thenucleus by crossing the NPC lengthwise (Au and Pante, 2012; Auet al., 2013). Whether baculoviruses use a conventional or anunconventional nuclear import pathway remains to be determined. Inany case, proteins of the AcMNPV nucleocapsid do not contain anyobvious functional NLS that could mediate the nuclear import of thenucleocapsid.

Here, we show that nuclear import of AcMNPV nucleocapsidsoccurs independently of the Ran-GTPase cycle, but depends onArp2/3-mediated F-actin nucleation. We further demonstrate theinvolvement of actin polymerization in the nuclear import ofAcMNPV by reconstituting nuclear import of nucleocapsids inpurified nuclei supplemented with G-actin and Arp2/3 underpermissive conditions for actin polymerization. Our data suggestthat baculovirus-induced actin polymerization is involved in themechanism of nuclear import of AcMNPV nucleocapsids.

RESULTS AND DISCUSSIONAcMNPV nucleocapsid enters the nucleus of semi-permeabilized cells in the absence of exogenous cytosolIn order to study nuclear import of AcMNPV nucleocapsids, weused the well-established nuclear import assay with digitonin-permeabilized cells (Adam et al., 1990; Cassany and Gerace, 2009).In this system, nuclear import is assayed by incubating thedigitonin-permeabilized cells with a fluorescently labeled cargoand either exogenous cytosol or purified recombinant nucleartransport factors and an energy-regenerating system. Controlexperiments with a 70-kDa dextran labeled with Texas Red,which does not diffuse through the NPC, demonstrated thatdigitonin did not disrupt the nuclear envelope (data not shown).Control experiments with a Cy3-conjugated NLS-tagged bovineserum albumin (Cy3–NLS-BSA) showed that this cargo wasefficiently imported into the nucleus in the presence of exogenouscytosol (rabbit reticulocyte lysate; RRL) and an energy-regeneratingsystem, whereas in the absence of RRL and the energy-regeneratingsystem, nuclear import was inhibited as expected (Fig. 1A).Strikingly, when the import assay was performed with purifiedAcMNPV nucleocapsids, nuclear import of the nucleocapsidReceived 2 May 2016; Accepted 3 June 2016

1Department of Zoology, University of British Columbia, Vancouver, BritishColumbia, Canada. 2Summerland Research and Development Centre, AAFC,Summerland, British Columbia, Canada.

*Author for correspondence ([email protected])

N.P., 0000-0002-0926-9378

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occurred under both permissive (RRL with the energy-regeneratingsystem) and non-permissive (without RRL and the energy-regenerating system) nuclear import conditions with comparablenuclear import efficiency (Fig. 1A–C; Movies 1,2).

Nuclear import of AcMNPV nucleocapsids in digitonin-permeabilized cells occurs through the NPC and isindependent of RanSome viruses enter the nucleus through nuclear envelope disruptions,instead of the NPC (Cohen et al., 2006; Cohen and Pante, 2005).Thus, to determine whether AcMNPV nucleocapsids enter thenucleus through the NPC or through the nuclear envelope, weblocked transport through the NPCs with wheat germ agglutinin(WGA), which blocks the NPC by binding to nucleoporins (Nups),

and found that nucleocapsids remained in the cytoplasm (Fig. 1D).Thus, themode of nuclear entry of AcMNPVnucleocapsids observedin Fig. 1A was not the result of nuclear envelope disruptions by thevirus. This is in agreement with Ohkawa et al. (2010) who found thatnuclear import of nucleocapsids was inhibited in cells microinjectedwith WGA before AcMNPV infection and in cells that have theirNPCs blocked with a dominant-negative form of importin-β, whichdoes not release endogenous import cargo from the NPC, andtherefore clogs the NPC and inhibits nuclear transport (Gorlich et al.,1996; Kutay et al., 1997).

In contrast to the results with WGA, nuclear import ofnucleocapsids was not inhibited by importazole (Fig. 2), whichdisrupts the interaction of importin-β and Ran-GTP (Soderholmet al., 2011). This result indicates that the nucleocapsid either does

Fig. 1. Nuclear import of baculovirusnucleocapsids in semi-permeabilized cells.(A) Digitonin-permeabilized HeLa cells wereincubated with Cy3–NLS-BSA or AcMNPVnucleocapsids in the presence or absence ofexogenous cytosol (+RRL and –RRL, respectively)with or without an energy-regenerating system(+E and –E, respectively). Arrowheadspoint to nucleocapsids inside the nucleus.(B,C) Quantification of the number of cells withfluorescent cargo in the nucleus (B) andnucleocapsid number per nucleus (C) (mean±s.e.m.;n=200). (D) Digitonin-permeabilized HeLa cells pre-treated with WGAwere incubated with Cy3–NLS-BSA or AcMNPV nucleocapsid and the import assaywas performed in the presence of RRL and anenergy-regenerating system. Arrowheads point tonucleocapsids. In A and D, nucleocapsid wasimmunolabeledwith antibodyagainst VP39 (red) andDAPI (pseudocolored in gray) was used to observethe nucleus. Scale bars: 10 µm.

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not use importin-β or uses importin-β but the binding of Ran-GTPto importin-β is not required for its nuclear import. Consistent withthe latter, nuclear import of the nucleocapsid was not inhibited inthe presence of GTPγS (Fig. 2), a non-hydrolyzable GTP analogthat inhibits Ran-dependent nuclear import (Melchior et al., 1993).Another virus that uses importin-β but has its nuclear importinhibited by GTPγS is hepatitis B virus (Rabe et al., 2003).Collectively, these results indicate that nuclear import ofAcMNPV nucleocapsids occurs through the NPC and isindependent of Ran.

It is possible that the nucleocapsid has importin-β like properties.However, comparison of all baculovirus proteins from currentlysequenced viral genomes did not identify any protein withhomology to importin-β (data not shown).

Nuclear import of AcMNPV nucleocapsids in digitonin-permeabilized cells is inhibited by conditions that inhibitF-actin polymerizationGiven that the baculovirus nucleocapsid hijacks the host Arp2/3complex during infection to promote actin polymerization at one

Fig. 2. Nuclear import of AcMNPV nucleocapsid is not inhibited by importazole or GTPγS in semi-permeabilized cells. Digitonin-permeabilized HeLacells were incubated with Cy3–NLS-BSA (A) or AcMNPV nucleocapsids (B) under permissive (RRL with an energy-regenerating system; RRL+E) or non-permissive (RRL without an energy-regenerating system; −RRL−E) conditions in the presence or absence of importazole (Imp) or GTPγS. In B, highmagnification images of cells indicated with white boxes are shown in the lower panels. Arrowheads point to nucleocapsids inside the nucleus. DAPI(pseudocolored in gray) was used to observe the nucleus. Scale bars: 10 µm. (C) Quantification of the number of cells with nucleocapsids in the nucleus for allexperimental conditions shown in B (mean±s.e.m.; n=200).

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of its ends, creating a ‘comet tail’ that propels the nucleocapsidtowards the cell nucleus (Mueller et al., 2014; Ohkawa et al.,2010) and digitonin-permeabilized cells contain G-actin, F-actinand Arp2/3 (Fig. S1A–C), we hypothesized that nuclearimport of the nucleocapsid could also be driven by thepropulsive force of actin polymerization involving Arp2/3. Totest our hypothesis, the import assay was performed with semi-permeabilized cells treated with CK666, an Arp2/3 inhibitor(Nolen et al., 2009). We found that nuclear import ofnucleocapsids was significantly reduced in cells incubatedwith CK666 (Fig. 3). These results indicate that F-actinnucleation promoted by Arp2/3 is involved in the mechanismof nuclear import of the nucleocapsid. Given that the Arp2/3activity for nucleating G-actin depends on nucleation-promotingfactors (Firat-Karalar and Welch, 2011) and the only nucleation-promoting factor present in our experiment is the viral proteinVP78/83 on nucleocapsids, we conclude that baculovirus-induced actin polymerization is involved in the mechanism ofnuclear import of AcMNPV. In agreement with this conclusion,nuclear import of the nucleocapsid was inhibited when thedigitonin-permeabilized cells were treated with cytochalasinD, which disrupts F-actin and inhibits actin polymerization(Fig. S1D).

Nuclear import of AcMNPV nucleocapsids can bereconstituted in isolated nuclei supplemented with G-actinand Arp2/3 under actin polymerization conditionsTo further test the involvement of actin polymerization in thenuclear import of nucleocapsids, we assayed for nuclear import ofnucleocapsids in purified nuclei using an actin polymerizationassay. In this assay, F-actin nucleation occurs in the presence of bothan actin nucleation-promoting factor (such as VP78/83) and Arp2/3.Therefore, we incubated purified nuclei with nucleocapsids andperformed the actin polymerization assay in the presence andabsence of Arp2/3. As shown in Fig. 4B, nucleocapsids were foundin the nucleus when the actin polymerization assay was performedin the presence of Arp2/3 under permissive conditions for actinpolymerization (presence of G-actin and actin polymerizationbuffer containing ATP). In contrast, no nucleocapsids were found inthe nucleus when the assay was performed in the absence of actinpolymerization buffer (Fig. 4B). When Arp2/3 was omitted in theassay very few nucleocapsids were found in the nucleus (Fig. 4B).Moreover, experiments with VP39–3×mCherry nucleocapsidsincubated with pyrene–G-actin under conditions that allow for actinpolymerization, in which pyrene-conjugated actin becomes fluorescentupon polymerization, and visualized without fixation yielded actincomet tails attached to VP39–3×mCherry nucleocapsids (Fig. 4D).

Fig. 3. Nuclear import of AcMNPV nucleocapsid is inhibited by an Arp2/3 inhibitor in semi-permeabilized cells. Digitonin-permeabilized HeLa cells wereincubated with nucleocapids under permissive (RRL with an energy-regenerating system; RRL+E) or non-permissive (RRL without an energy-regeneratingsystem; −RRL−E) conditions in the absence of any inhibitors (A) or the presence of the Arp2/3 inhibitor CK666 (B) or the inactive compound CK689 (C).Arrowheads point to nucleocapsids (red). DAPI (pseudocolored in gray) was used to observe the nucleus. Scale bars: 10 µm. (D) Quantification of the number ofcells with nucleocapsids in the nucleus for all experimental conditions shown in A–C (mean±s.e.m.; n=200). ***P<0.01, ****P<0.01 (unpaired Student’s t-test).

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The involvement of actin polymerization in the nuclear importof AcMNPV is supported by live-cell images of High Five cellstransiently expressing EGFP–actin and infected with VP39–3×mCherry virus in which nucleocapsids physically depressingthe nuclear envelope are depicted (Ohkawa et al., 2010).Although in infected cells it is not possible to separate theprocess of nuclear import from the cytoplasmic trafficking of thenucleocapsids, HeLa cells transduced with AcMNPV in thepresence of CK666 had a significant decrease in nuclearaccumulation of nucleocapsids (Fig. S2A,B). However, whenexamined by electron microscopy, although some nucleocapsidswere found attached to the nuclear envelope (Fig. S2C), theywere never found in the nucleus.Based on our results, we propose that AcMNPV uses the

propulsive force of actin polymerization to drive not only themigration of the nucleocapsid through the cytoplasm, but alsothe nuclear import of the nucleocapsid. In this proposed mechanismthe actin comet tail formed at one end of the nucleocapsid pushes thenucleocapsid through the NPC for nuclear import. In agreementwith this model, the nucleocapsid crosses the NPC lengthwise withthe end containing the actin-assembly-inducing VP78/83 proteinaway from the NPC (Au and Pante, 2012). Our model implies thatthe phenylalanine-glycine-rich nucleoporin (FG-Nup) permeability

barrier at the NPC central channel can be breached by the propulsiveforce of actin polymerization.

MATERIALS AND METHODSCells, antibodies and inhibitorsHeLa cells (American Type Culture Collection) were cultured in completeDMEM medium supplemented with 10% fetal bovine serum (FBS).Spodoptera frugiperda clone 9 (Sf9) cells were grown in Sf-900 II serum-free medium (Invitrogen) supplemented with 2% FBS. Antibodies usedwere mouse anti-VP39 (1:500, provided by Robert Kotin, National HeartLung and Blood Institute, NIH), rabbit anti-Nup153 (1:100, Abcam-84872),rabbit anti-Arp2 (1:300, Abcam, ab47654), and mouse anti-fibrillarin(1:1000, Abcam, 38F3) antibodies. Phalloidin–FITC was from Sigma-Aldrich. Secondary antibodies were from Invitrogen. The Arp2/3 inhibitorCK666, its inactive form CK689, and GTPγS were from EMD Millipore.The importin-β inhibitor importazole was from Sigma-Aldrich.

Virus, nucleocapsid and Cy3–NLS-BSARecombinant AcMNPV was propagated in E. coli (DH10B) and amplifiedat a multiplicity of infection (MOI) of 1 in Sf9 cells. Virus was maintainedby infecting Sf9 cells grown in Sf-900-II serum-free medium at MOI=1 andwas harvested 5 days post infection by centrifugation at 1000 g for 10 min at4°C. The viral titre was determined by TCID50 end-point dilution assayusing Sf9 cells (O’Reilly et al., 1992). AcMNPV was purified through acontinuous 15–60% (w/v) sucrose gradient centrifugation.

Fig. 4. Reconstitution of nuclear import of AcMNPV nucleocapsids in isolated nuclei supplemented with G-actin and Arp2/3. Isolated HeLa nucleiincubatedwith (A) 70-kDa dextran conjugated to TexasRed or (B) nucleocapsids andG-actin in the presence of an actin polymerization buffer containing ATP (toprow; +G-actin+ATP), this buffer and Arp2/3 (middle row; +G-actin+ATP+Arp2/3), or in the absence of the actin polymerization buffer (bottom row; +G-actin–ATP).Nuclei were labeled with an antibody against Nup153 (green). Arrowheads point to nucleocapsids (red). (C) Quantification of the number of nucleocapsids in thenucleus per isolated nuclei for all experimental conditions shown in B (mean±s.e.m.; n=150). **P<0.01 (unpaired Student’s t-test). (D) Baculovirus actin comettails formed in vitro by incubating VP39–3×mCherry nucleocapsids with G-actin labeled with pyrene and Arp2/3 under permissive conditions for actinpolymerization. Polymerization of pyrene-labeled G-actin induced by VP78/83 on the nucleocapsid resulted in fluorescent F-actin comet tails (blue). Scale bars:10 µm.

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AcMNPV VP39–3×mCherry virus expressing both wild-type VP39 andVP39 fused to three copies of mCherry was generated as describedpreviously (Biswas et al., 2016). The VP39–mCherry fusion construct wasderived from the WOBCAT vector (Ohkawa et al., 2010).

AcMNPV nucleocapsids and VP39–3×mCherry nucleocapsids wereobtained by de-enveloping the virus by treatment with 1% NP40 for 1 h at30°C as described previously (Au et al., 2010; Au and Pante, 2012).

BSA covalently attached to the NLS of SV40T antigen(CGGGPKKKRKVED) at a ratio of 5:1 (NLS:BSA) was custom made(Sigma-Genosys). NLS-BSA was labelled with Cy3 (Amersham-Biosciences) according to the manufacture’s protocol.

Nuclear import assay with semi-permeabilized cellsAdherent HeLa cells were permeabilized with 20 µg/ml digitonin (Sigma-Aldrich) in transport buffer (20 mM HEPES, pH 7.4, 110 mM potassiumacetate, 1 mM EGTA, 5 mM sodium acetate, 2 mMmagnesium acetate, and2 mM dithiothreitol) for 4 min. Permeabilized cells were washed withtransport buffer and incubated with transport buffer containing 70-kDadextran conjugated to Texas Red (Invitrogen), Cy3-labeled cNLS-BSA, orpurified nucleocapsids for 60 min at 37°C in the presence or absence of 20%rabbit reticulocyte lysate (RRL; Promega), a energy regenerating system(0.4 mM ATP, 0.45 mM GTP, 4.5 mM phosphocreatine and 18 U/mlphosphocreatine kinase; Sigma-Aldrich), complete-Mini EDTA-freeProtease Inhibitor Cocktail (Roche) at 10 μg/ml, and 1.6 mg/ml BSA.Next, cells were washed with transport buffer three times and prepared forimmunofluorescence microscopy.

For inhibitor treatment, permeabilized cells were pre-treated with0.5 mg/ml WGA (0.5 mg/ml, 30 min), importazole (40 µM, 1 h), orCK666 or CK689 (1 mM, 1 h). For some treatments, the drug was alsopresent in the import mixture (importazole at 40 µM, and CK666 or CK680at 1 mM).

Nuclei purificationNuclei from HeLa cells were isolated by osmotic swelling under isotonicconditions. Briefly, pelleted cells were resuspended in Earle’s balance saltsolution (1.8 mM CaCl2, 5.3 mM KCl, 0.8 mM MgSO4, 117 mM NaCl,26 mM NaHCO3, 1 mM NaH2PO4-H2O, 5.6 mM glucose) and centrifugedfor 5 min at 600 g, followed by resuspension in 10 volumes of isotonicbuffer (30 mM Tris-HCl pH 8.3, 100 mM NaCl, 5 mM MgCl2) containing0.5% (v/v) of NP40, incubation on ice for 4 min with gentle mixing, andcentrifugation for 5 min at 600 g. The supernatant was discarded and theresulting pellet containing isolated nuclei was resuspended in isotonicbuffer. Isolated nuclei were adhered to glass coverslips by centrifugation for10 min at 2000 g through a 30% sucrose cushion onto a coverslip.

Nuclear import assay with purified nucleiCoverslips containing isolated nuclei were incubated with AcMNPVnucleocapsids, 40 µM importazole and skeletal muscle G-actin from anactin polymerization kit (Cytoskeleton Inc). Next, actin polymerizationbuffer (final concentration on the coverslip: 50 mM KCl, 2 mM MgCl2,5 mM guanidine carbonate and 1 mM ATP) was added and the actinpolymerization assay was performed according to the manufacturer’sprotocol. After 60 min, cells were washed with PBS and prepared forimmunofluorescence microscopy. For experiments in the presence ofArp2/3 complex, 50 nM of this complex was added to the coverslip prior toadding the actin polymerization buffer.

Visualization of actin tails on VP39–3×mCherry nucleocapsidsmCherry nucleocapsids were mixed with pyrene-conjugated skeletal muscleG-actin from an actin polymerization kit (Cytoskeleton Inc) in a well ofan eight-well glass bottom μ-slide (Ibidi). Next, actin polymerization bufferwas added and samples were incubated at 37°C in the dark for 30 min. Afterthis incubation time, samples were visualized immediately by confocalmicroscopy.

Immunofluorescence microscopyHeLa cells or purified nuclei on glass coverslips assayed as indicated abovewere fixed with 3% paraformaldehyde (PFA) in PBS for 10 min,

permeabilized with 0.2% Triton X-100 in PBS for 5 min, blocked withPBS containing 1% BSA and 10% goat serum for 30 min at 37°C, andlabelled with a primary antibody against VP39 or Nup153 for 1 h at 37°C.Samples were washed three times for 10 min each with PBS, followed byincubation with fluorescently labeled secondary antibodies (1:1000dilution) for 45 min at 37°C. Coverslips were washed three times for10 min each with PBS, and mounted with Prolong Gold antifade reagentcontaining DAPI (Invitrogen).

Confocal microscopy and image analysisAll fluorescence microscopy images were acquired using a FluoviewFV1000 confocal laser-scanning microscope (Olympus). Quantificationwas performed using Prism (GraphPad Prism Software, Inc) and analyzedusing an unpaired Student’s t-test.

AcknowledgementsWe are grateful to Dr Robert Kotin (NIH) for the VP39 antibody and to Drs MatthewWelch and Taro Ohkawa (University of California, Berkeley) for the VP39-3xmCherry construct.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsS.H. and N.P. designed the study and wrote the manuscript. W.W. performed theexperiments with VP39–3×mCherry nucleocapsids, L.Z. performed the westernblotting and immunolabeling of Arp2/3. S.H. performed all other experiments andanalyzed the data. D.A.T. provided viruses and expertise. All authors discussed theresults and commented on the manuscript.

FundingThis work was supported by grants from the Natural Sciences and EngineeringResearch Council of Canada (NSERC) [grant number RGPAS-412254-11 andRGPIN-227926-1].

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.191668.supplemental

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