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. Author manuscript; available in PMC: 2015 May 1.
Published in final edited form as: Virology. 2014 Apr 25;0:342–352. doi: 10.1016/j.virol.2014.04.003

Parvovirus particles and movement in the cellular cytoplasm and effects of the cytoskeleton

Sangbom Michael Lyi a,b, Min Jie Alvin Tan a,c, Colin R Parrish a,*
PMCID: PMC4232186  NIHMSID: NIHMS640350  PMID: 24889253

Abstract

Cell infection by parvoviruses requires that capsids be delivered from outside the cell to the cytoplasm, followed by genome trafficking to the nucleus. Here we microinject capsids into cells that lack receptors and followed their movements within the cell over time. In general the capsids remained close to the positions where they were injected, and most particles did not move to the vicinity of or enter the nucleus. When 70 kDa-dextran was injected along with the capsids that did not enter the nucleus in significant amounts. Capsids conjugated to peptides containing the SV40 large T antigen nuclear localization signal remained in the cytoplasm, although bovine serum albumen conjugated to the same peptide entered the nucleus rapidly. No effects of disruption of microfilaments, intermediate filaments, or microtubules on the distribution of the capsids were observed. These results suggest that movement of intact capsids within cells is primarily associated with passive processes.

INTRODUCTION

Cell infection by viruses that replicate in the nucleus involves viral components being delivered into the cytoplasm and then transfer of the genome to the nucleus, generally along with viral proteins or capsid components (Greber and Fornerod, 2005; Marsh and Helenius, 2006). The processing or transport of infecting capsids or nucleocapsids within the cytoplasm, and the transport of the genome to the vicinity of or into the nucleus can be complex as the cytoplasm prevents the free diffusion of virus-sized particles (Lukacs et al., 2000; Seksek et al., 1997). For adenoviruses, herpesviruses, and at least some retroviruses, viral proteins and structures are actively transported within the cytoplasm to the vicinity of the nucleus (Lagache et al., 2009b), while for other viruses, including papillomaviruses and polyomaviruses endosomal mechanisms are used to transport the capsids to the endoplasmic reticulum or other compartments (Engel et al., 2011; Gruenberg, 2009; Sapp and Bienkowska-Haba, 2009).

The capsids of parvoviruses or adeno-associated viruses (AAVs) bind receptors on the cell surface, enter the cells by receptor-mediated endocytosis, and then traffic within endosomes to the microtubular organizing center (MTOC) (Ding et al., 2005; Harbison et al., 2008; Harbison et al., 2009; Vendeville et al., 2009). Release from endosomes appears to be quite slow and requires the activity of a phospholipase A2 in the unique region of the viral protein 1 (VP1), and many parvoviral capsids are retained within endosomes for up to several hrs (Farr et al., 2005; Zadori et al., 2001). Expression of PLA2 in cells can alter the cellular morphology (Deng et al., 2013). Because of the slow release of the capsids, in studies of viral entry it can be difficult to know whether caspids being detected are within the cytoplasm or endosomes.

The roles of the different cytoskeleton elements in viral infection appear to be complex. In some studies infection has been shown to depend on the presence of an intact microtubular cytoskeleton, and capsids of autonomous parvoviruses (canine parvovirus (CPV) and porcine parvovirus), and at least some adeno-associated viruses (AAVs) have been suggested to be trafficked within the cytoplasm in association with the molecular motor dynein (Kelkar et al., 2006; Kelkar et al., 2004; Suikkanen et al., 2003a). Addition of peptides to AAV type-2 capsids that were predicted to bind dynein light chain (LC8) also enhanced retrograde transport in axons (Xu et al., 2005). However, other studies have suggested that an intact cytoskeleton is less important for cell infection (Hirosue et al., 2007), and it is unclear whether cytoplasmic trafficking of parvovirus capsids is an active trafficking mechanism, occurs by diffusion, or involves some combination of those processes. A role of intermediate filaments and vimentin in infection by the MVM parvovirus has been reported in localization of virions around the nucleus, and the filaments became rearranged in cells that have taken up virions from the cell surface and in many infected cells (Fay and Pante, 2013). After cells are infected there may be extensive changes in the cellular architecture that result from virus replication and expression of the viral NS1 protein (Nuesch et al., 2005).

When free in the cytoplasm parvovirus capsids may become conjugated to ubiquitin, and in some cases the capsid proteins are degraded by proteosomal systems (Boisvert et al., 2010; Ros and Kempf, 2004; Yan et al., 2002). However the effects reported vary for different viruses, and while proteosomal inhibitors such as MG138 enhance transduction by AAV type-2 or type-5 (Ding et al., 2003; Yan et al., 2004), they inhibit infection by autonomous parvoviruses (Ros and Kempf, 2004), and it may be difficult to distinguish direct and indirect effects of the drugs. AAV2 capsids may also be modified by ubiquitin addition to surface exposed tyrosines (Tyr), and mutating one or more of the several Tyr on the capsid surface can enhance transduction due to alterations that capsid modification (Zhong et al., 2008a; Zhong et al., 2008b).

The processes of nuclear entry and exit of parvovirus capsids are still not understood in detail, and may vary between viruses and perhaps cell types. When capsids of autonomous parvoviruses or AAV2 enter cells by receptor-mediated endosomal processes only a low proportion are seen to enter the nucleus by microscopy (Bantel-Schaal et al., 2002; Harbison et al., 2009; Seisenberger et al., 2001). Purified CPV capsids microinjected into the cytoplasm of cells and detected after cell fixation remained in the cytoplasm for more than 2 hrs (Vihinen-Ranta et al., 2002; Vihinen-Ranta et al., 2000). Several AAV serotypes may infect cells more rapidly, and higher proportions were recovered in the nuclear-associated fractions a few hrs after uptake from the cell surface (Sonntag et al., 2006; Zhong et al., 2008b). Both endosomal release and nuclear transport have been associated with the release or exposure of the N-termini of some or all of the 5 or 6 VP1s in each capsid, which contain both the phospholipase A2 enzyme activity and sequences made up of basic amino caids, similar to classic nuclear localization sequences (NLS) (Sonntag et al., 2006; Vihinen-Ranta et al., 2002). The VP1 unique regions are usually sequestered within the capsid but become exposed in the endosome (Farr and Tattersall, 2004; Suikkanen et al., 2003b), and some would be on the outside of capsids that enter the cytoplasm (Vihinen-Ranta et al., 2002).

After replication parvovirus capsids assemble in the nucleus, and in many cases appear to be retained there. However, capsids may also be trafficked to the cytoplasm or even out of the cell. The export of intact newly-produced minute virus of mice (MVM) capsids out of the nucleus occurred efficiently for some virus-cell combinations, and was regulated by the phosphorylation of Ser and Thr within the N-terminal sequence of VP2 exposed on the outside of the newly produced full capsids (Maroto et al., 2004). The process of nuclear export and cytoplasmic and extra-cellular transport of the capsids was also associated with remodeling of the actin cytoskeleton by the enzyme gelsolin, which is modified by the viral NS1 protein (Bar et al., 2008).

Capsids in the cytoplasm are reported to directly alter the structure of the nuclear envelope. When MVM capsids were injected into the cytoplasm of Xenopus oocytes, they changed the integrity and morphology of the nuclear envelope as seen by electron microscopy, with damage particularly to the outer nuclear membrane (Au et al., 2010; Cohen and Pante, 2005). When purified capsids were added to digitonin-permeabilized fibroblasts the nuclear envelope morphology changed to show ruffles and patches, as detected by staining for Lamin A/C (Cohen et al., 2006). The nuclear breakdown was reported to involve enzymes involved in nuclear changes that occur during mitosis, including protein kinase C which acted on cdk-2, and which was acted on by caspase-3 (Porwal et al., 2013).

Here we further examine the intracellular trafficking of parvoviral capsids by examining the distribution of capsids within the cytoplasm of live cells after microinjection. Fluorescently labeled capsids generally remained close to the location where they were injected, with little movement even over periods of hours, and similar effects were seen with unlabeled capsids. That localization was not significantly altered by changes in the structures of the microfilaments or microtubules, or by the presence of intermediate filaments.

RESULTS

Here we examined the locations and movement of fluorescently labeled virus capsids after injection into live cells, and compared the results to those seen for unlabeled capsids in cells that were fixed after various times of incubation. Capsids were labeled with Alexa488 or Alexa594 and were in the form of single particles as described previously (Harbison et al., 2009). Initial studies showed no obvious differences in the distribution or movement of full or empty capsids (results not shown), and most studies were conducted with full particles, which represent the infectious virions.

We initially examined the distribution of capsids in feline NLFK cells which express feline transferrin receptor type-1 (TfR) and which bind and endocytose the virus, as well as within TRVb cells, which are derived from CHO cells and which lack the TfR and which do not bind viruses. The latter cells are efficiently infected when expressing the feline TfR from a plasmid (Goodman et al., 2010; Parker et al., 2001), indicating that the cytoplasmic and nuclear transport processes required for infection are functional. When capsids were released from the microinjection needle near NLFK cells some were subsequently found on the surface and filopodia of the cells, indicating that they bound receptors and were endocytosed (Fig. 1A), so that using those cells would show both injected and endocytosed capsids. However, receptor-negative TRVb cells showed no evidence of surface bound virus under the same conditions (Fig. 1B), and those were therefore used for the remainder of these studies.

Figure 1. Distribution of Alexa594-labeled full CPV capsids or 20 nm Cy5-labeled polystyrene microspheres after injection into the cytoplasm of cells, with images collected after incubation at 37°C as indicated. In each field some cells are injected, while others were left as non-injected controls. Feline NLFK cells express functional receptors, while TRVb cells are receptor deficient.

Figure 1

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A) CPV capsids injected into NLFK cells, B) CPV capsids in TRVb cells, C) 20nm Cy5 microbeads in TRVb cells.

When fluorescently labeled CPV capsids were injected into the cytoplasm of live NLFK or TRVb cells they remained near the position of injection for between 2 mins and 1 hr, with little movement being observed (Figs. 1A and 1B). Twenty nm diameter Cy5-labeled polystyrene nanospheres injected into TRVb cells also showed little movement within the cytoplasm (Fig. 1C).

Previous studies have indicated that parvovirus capsids in the cytoplasm can alter the nuclear morphology and nuclear envelope integrity (Au et al., 2010; Cohen et al., 2006; Cohen and Pante, 2005). Cells expressing Lamin A/C-GFP were injected with Alexa594-labeled full capsids (Fig. 2A) or with un-labeled capsids (Figs. 3A–B), and examined using widefield and confocal microscopy. The capsids remained near where they were injected (Fig. 2A) with only a small proportion becoming localized adjacent to the nuclear envelope (Figs. 2A, 3A–B). Some of the virus-injected cells showed subtle changes in the distribution of Lamin A/C that were not seen in the non-injected cells or in cells injected with labeled BSA (Fig. 2B). When Alexa488-70kDa dextran was injected into the cytoplasm it mostly remained in that location after 1.5 to 2 hrs with limited movement into the nucleus (Fig. 4A). When injected along with capsids the 70 kDa dextran entered the nucleus at similar rates as in the absence of capsids (Fig. 4B).

Figure 2.

Figure 2

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Distribution of microinjected (A) Alexa594-full CPV capsids (red) or (B) Alexa594-BSA (red) in TRVb cells expressing Lamin A/C-GFP (green), after various times of incubation at 37°C. The phase images of the same cells are also shown. The cells injected are enclosed by a white outline.

Figure 3. Distribution of unlabeled CPV capsids within cells after injection at different concentrations, with different nuclear stains. Cells were fixed after 1 hr incubation at 37°C, and capsids were detected with anti-capsid antibody 8.

Figure 3

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The distribution of capsids injected at concentrations of (A) 5mg/ml or (B) 50 μg/ml in cells expressing Lamin A/C.

Figure 4.

Figure 4

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Effect of co-injected capsids on the distribution of fluorescent dextran in the cell. The dextran was injected alone (A) or along with capsids (B), and the cells then incubated for 80 or 90 mins at 37°C.

Capsids were conjugated to NLS peptides, with an average of 5 or 28 peptides attached to each capsid in two different conjugated preparations. Peptide conjugated capsids were injected into cells and incubated at 37°C, and then the capsids detected using antibody staining after fixing the cells. Most of the capsids remained in the cytoplasm, while a few became localized within the nucleus (Figs. 5A–B). As a control we injected BSA conjugated to the same NLS peptide, and that entered the nucleus very quickly, mostly in less than 30 mins (Fig. 5C).

Figure 5. The effect of addition of NLS-peptides to CPV capsids, compared to BSA conjugated to the same peptide. TRVb cells were injected with NLS-conjugated capsids, then the cells fixed 60 mins after injection and capsids detected with an antibody, while the nuclei were stained with DAPI.

Figure 5

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A and B) The distribution of CPV capsids conjugated with an average of 28 (A) or 5 (B) fluorescene-NLS peptides per capsid, after injection into cells expressing Lamin A/C GFP and incubation for 60 mins.

C) BSA conjugated to fluorescene-NLS peptide and injected into TRVb cells, then incubated at 37°C. Images were collected at the indicated times after injection.

Labeled capsids injected into cells expressing GFP-actin did not show any specific association of particles with the actin filaments, and after incubating the injected cells for 1 hr and then adding cytochalasin only minor changes in the distribution of the particles were seen (Fig. 6). In cells expressing YFP-tubulin some particles appeared to be aligned with the microtubules, but addition of nocodazole to the cells after incubating for 1 hr did not result in significant changes in the capsid distribution (Fig. 7A). When the microtubules were stabilized with Paclitaxel before injection of virus, particles became distributed in similar patterns to those seen in untreated cells (Fig. 7B). We examined wildtype and vimentin −ve SW13 cells for the distribution of capsids after injection (Fig. 8A and B), and saw similar distributions of the particles, and the injected particles also did not significantly alter the distribution of the vimentin in the cells (Fig. 8C).

Figure 6.

Figure 6

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A) The distribution of Alexa594-CPV full capsids (red) injected into cells expressing Actin-GFP (green), and incubated at 37°C for various times. Cytochalasin (Cyto) was added after 60 mins incubation, and the cells incubated for a further 30 mins. Images on the right of the 5, 30 and 60 min times show a higher magnification view of the capsid distribution on the cytoplasm.

B) Tracking the movement of individual particles in the absence or presence of cytochalasin.

C) Plots showing the measured differences in the movement of the capsids in the presence or absence of cytochalasin.

Figure 7. Distribution of microinjected Alexa594-CPV full capsids (red) followed in live cells expressing YFP-tubulin (green), and in some cases subjected to various treatments.

Figure 7

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A) Injected cells incubated at 37°C for up to one hr, as indicated. Nocodazole was added to the cells after 60 mins incubation, and then the cells incubated for a further 30 mins.

B) Capsids in cells that had been pretreated with Paclitaxel (taxol) for 2 hrs at 37°C.

Figure 8. Distribution of CPV capsids microinjected into SW13 cells that either express vimentin (vimetin +ve) or that lack vimentin expression (vimentin -ve), at various times after injection.

Figure 8

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A) Distribution of labeled capsids in wildtype cells.

B) Distribution of labeled capsids in SW13 cells that lacking vimentin.

C) The co-distribution of vimentin and capsids in SW13 cells. Vimentin = green, capids = red.

DISCUSSION

Here we examine the localization of labeled capsids after microinjection to cells, and followed their distribution during subsequent incubations. While microinjection is an artificial means of introducing virions into the cytoplasm or nucleus, these studies provide additional information about this step of the capsid-cell interaction that is difficult to observe when capsids are taken up through receptor-mediated processes due to the difficulty of distinguishing between capsids within the various endosomes and those that were free in the cytoplasm. In most cases the capsids remained close to the site of injection for 1 to 2 hrs at 37°C, while as expected labeled BSA or dextran became distributed throughout the cytoplasm within a few mins. The relative lack of capsid movement is likely related to their size and to the tight packing of the cytoplasm, and we showed that 20 nm diameter Cy5-labeled polystyrene nanospheres also showed little movement after injection under the same conditions. Previous studies of nanoparticle movement in the cytoplasm of cells have showed a strong dependence on the diameter of the particle, with those of 25 nm diameter or greater showing limited movement, being restricted by the physical properties of the cytoplasm (Boulo et al., 2007; Lagache et al., 2009a; Luby-Phelps, 2000; Novak et al., 2009; Seksek et al., 1997; Wirtz, 2009). The 25mn diameter parvovirus particle is close to the size where diffusion was observed in previous studies – reported to be below 40 nm – although in this study we also saw limited movement of 20 nm diameter nanoparticles (Fig. 1C).

To examine the possibility that the capsids are confined or transported by cytoskeletal components we examined their association with actin, tubulin, or vimentin. There was no clear association with either the microtubules or microfilaments, and nocodazol, paclitaxel, or cytochalasin D treatment did not substantially alter the distribution of the injected capsids. These results contrast with studies which have suggested that CPV capsids are actively transported by cytoplasmic dynein (Kelkar et al., 2006; Suikkanen et al., 2003a), but are consistent with others that show no direct role of microtubules in the transduction by AAV capsids (Hirosue et al., 2007). Here localization of the capsids remained close to the positions where they were deposited by the injection process, and there was little movement during incubation at 37°C. The capsid distribution seen differs from those seen in studies where cells were fixed at various times after virus was added to the cells. During normal receptor-mediated entry processes capsids are rapidly carried within endosomes to the vicinity of the microtubule organizing center (Aniento et al., 1993; Hirokawa, 1998), and so active long-distance transport of free capsids in the cytoplasm is not necessarily required. In previous studies the actin cytoskeleton influenced the release from the cell of newly produced MVM particles, but the effect was associated with alterations of vesicular transport and not to direct effects on the virions (Bar et al., 2008).

MVM capsids injected into Xenopus ooctyes influenced the nuclear envelope morphology as seen by electron microscopy, and effects were also seen in digitonin-permeabilized fibroblasts incubated with virions, by activating enzymes involved in nuclear envelope breakdown (Au et al., 2010; Cohen et al., 2006; Cohen and Pante, 2005; Porwal et al., 2013). Here we show that capsids microinjected into live cells do alter the nuclear morphology, as seen in the distribution of Lamin A/C, but the virus in the cytoplasm did not appear to enhance the nuclear entry of 70 kDa dextran, and the virions did not obviously enter the nucleus in greatly increased amounts. These results suggest that capsids do not simply permeabilize the nucleus under the conditions of this study, and the role any direct virus-nuclear interaction on infection remains unclear.

Three different sequences or structures have been reported to influence nuclear trafficking of parvoviral capsid proteins or capsids. VP2 alone can enter the nucleus in an unassembled or oligomeric form (Yuan and Parrish, 2001), perhaps by using a structure that is formed when the protein trimerizes (Lombardo et al., 2000). The N-terminal sequence of the VP2 protein and phosphorylated Ser and Thr residues of the MVM control nuclear export of full capsids (Maroto et al., 2004). Sequences in the N-terminal domain of VP1 that include a NLS-like motif can become exposed to the outside of the capsid during endocytosis (Cotmore et al., 1999; Cotmore et al., 2010; Riolobos et al., 2006; Vihinen-Ranta et al., 2002). Here we saw only low amounts of labeled virus entering the nucleus, even when conjugated to an 5 or 28 copies of an NLS peptide. The nuclear pore has a diameter for free diffusion of ~9 nm, but has been reported to allow passage of particles of 30–40 nm in diameter (Labokha and Fassati, 2013; Pante and Kann, 2002; Solmaz et al., 2013), suggesting that the 25 nm diameter capsids could be trafficked into the nucleus by active processes. Alternatively, if the NLS is engaged by α/β-importin complex this may alter the capsid distribution. Infection clearly involves a series of coordinated steps, including the release of the VP1 N-terminus and the 3′-end of the viral DNA. The nuclear entry processes may therefore show similarities to those reported for other viral capsids, including adenovirus, hepatitis B virus, and herpesviruses, which dock onto or close to the nuclear pore in a partially disassembled form, allowing release of the genome for transport into the nucleus, sometimes along with accessory proteins (Krautwald et al., 2009; Pasdeloup et al., 2009; Rabe et al., 2009; Schmitz et al., 2010; Trotman et al., 2001).

These results provide new information about the localization and movement of parvovirus capsids in the cytoplasm of host cells, and their interactions with the nucleus over periods of hours. The capsids injected here do not recapitulate some of the conditions encountered during normal cell entry, such as the low pH exposure of receptor-bound capsids, but do show some processes that determine the virus distribution within the cytoplasm. The limited movement of the capsids observed here is likely not a specific barrier to cell infection, as after endosomal transport the incoming particles end up very close to the nucleus (Bantel-Schaal et al., 2009; Harbison et al., 2009; Liu et al., 2013; Mani et al., 2006; Suikkanen et al., 2002), from which position they should readily reach the nuclear pore. This would therefore differ from the infection pathways required for viruses such as herpesvirus and HIV which enter through the plasma membrane and hence need to traverse the entire cytoplasm. The data provides a foundation for testing variables of capsid processing during cytoplasmic and nuclear transport in future studies.

MATERIALS AND METHODS

Cells and viruses

Feline NLFK cells were grown in a 1:1 mixture of McCoy’s 5A and Liebovitz L15 media with 5% fetal bovine serum. CHO-derived cells lacking transferrin receptor 1 (TRVb cells) (McGraw et al., 1987) were grown in Ham’s F12 medium containing 5% fetal bovine serum. In some cases cells were transfected with plasmids expressing green fluorescent protein (GFP)-actin, yellow fluorescent protein (YFP)-tubulin, or GFP-lamin-A/C. Transfected cells were grown for 2 or 3 passages before use in these studies, or positive cells were selected by growth in the presence of 400 μg/ml of G418. SW13 cells that were vimentin +ve or −ve were used to study the role of intermediate filaments (Hedberg and Chen, 1986; Ho et al., 1998).

Viruses were derived from the infectious plasmid clone of CPV type-2 (CPV-d) (Parrish, 1991). Plasmids were transfected into NLFK cells and recovered viruses were titrated using TCID50 assays (Parker and Parrish, 1997). Virus capsids were concentrated by polyethylene glycol precipitation followed by sucrose gradient centrifugation, then dialyzed against either PBS or 20 mM Tris-HCl (pH 7.5) and stored at 4°C (Agbandje et al., 1993; Nelson et al., 2008).

Labeled proteins, capsids, and nanospheres

Bovine serum albumen (BSA) that was conjugated to both rhodamine and to a large T-antigen nuclear localization sequence (NLS) peptide was obtained from Sigma (St Louis, MO). Purified BSA or capsids were conjugated with Alexa488 or Alexa594 (Invitrogen, San Diego, CA) using methods recommended by the supplier. Briefly, full or empty CPV capsids at 1 mg/ml in 0.1 M bicarbonate buffer (pH 8.3) were conjugated with Alexa dye for 1 h at 22°C, and then 150 mM (final) hydroxylamine was added to neutralize the labeling reaction. The labeled proteins were separated from the free dye by passage through a P10 chromatography column (Millipore, Billerica, MA). An NLS peptide that included fluorescene was obtained from New England Peptides (Gardner, MA), and had the sequence Ac-PKKKRKVEDPYGK(FITC)GC-OH. That peptide was conjugated to BSA or to capsids by the hetero-bifunctional reagent, sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) as recommended by the manufacturer (Thermo Scientific, Rockford, IL). Cy5 labeled polystyrene nanospheres (20nm diameter) were purchased from Fluorophorex (Fall River, MA). Vimentine in cells was stained with mouse monoclonal anti-Vimentin Clone V9 antibody (V6630, Sigma), and detected with goat anti-mouse Alexa488.

Cell microinjection

NLFK or TRVb cells in medium lacking phenol red were microinjected with ~6 nl volumes using an Eppendorf Femtojet micromanipulator and InjectMan NI2 microinjector (Eppendorf, Hauppauga, NY), using needles prepared with a P-97 micropipette puller (Sutter Instrument Co., Novato, CA). Samples of capsids, proteins, or nanospheres were prepared in microinjection buffer (48 mM K2HPO4, 4.5 mM KH2PO4, 14 mM NaH2PO4, pH7.2), injected into cells, and then the cells were incubated at 37°C for varying times. Where labeled materials were injected, or where GFP-expression was present, the cells seeded in Falcon 50 mm gridded dishes (MatTek, Ashland, MA) or in Delta T4 culture dishes (Bioptechs, Butler, PA) in a 37°C warming chamber with a stage warmer (World Precision Instruments, Sarasota, Fl). The cells were examined for fluorescence and images collected at different intervals. In other cases the cells were incubated at 37°C after injection, fixed after various times with 4% paraformaldehyde, then the virus detected by staining with Alexa Fluor 594-labeled rabbit or mouse anti-capsid monoclonal antibody 8. In some experiments, the nuclear DNA of the cells was stained with DAPI, or the nuclear pores were stained with antibody Mab414 (Abcam, Cambridge, MA) that recognizes the conserved FXFG repeats in nucleoporins, followed by Alexa Fluor 488-labeled goat anti-mouse IgG. Some cells were treated 30 mins before or at various times after injection with 10 μM cytochalasin D (C8273), 20 μM nocodazole (M1404), or 100 nM Paclitaxel (T7191) (all from Sigma).

Microscopic analysis

Cells were examined using wide field microscopy with a Nikon TE300 microscope and a Hamamatsu OrcaER charge-coupled-device camera or by confocal imaging with a Zeiss LSM510 microscope. Images were prepared using either Simple PCI software (Hamamatsu, Sewickley, Pa), Image J software (US National Institutes of Health, Bethesda, MD), ZEN 2010 (Zeiss, Thornwood, NY) and/or Adobe Photoshop (Adobe, San Jose, CA).

Acknowledgments

Wendy Weichert and Virginia Scarpino provided excellent technical support. Supported by grants AI 28385 and AI 33468 from the National Institutes of Health to C.R.P..

Footnotes

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