INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA human parvovirus that is under development as a gene therapy vector (1, 22, 29, 63). Recombinant AAV-2 (rAAV) carrying reporter genes has been shown to readily transduce several organs, including muscle, brain, and eye (2, 7, 21, 29, 63). However, the transduction efficiency of rAAV appears to vary greatly among different tissues. In part, the lack of AAV-2 cell surface receptors (56) is thought to contribute to the inefficient infection of different tissue types (16).

The nuclear events influencing rAAV transduction, such as the phosphorylation status of the D-sequence-binding protein and the conversion of single-stranded DNA genomes to circular forms, have been partially elucidated (14, 15, 48, 51-53). However, the mechanisms underlying rAAV uptake and trafficking to the nucleus still remain largely undefined. The identification of several receptors and coreceptors has contributed to the understanding of AAV-2 binding to cells, the first step required for internalization. Heparan sulfate proteoglycan (HSPG), the first identified receptor for AAV-2 (56), appears to function primarily in virus attachment to the cell surface. Efficient AAV-2 infection has also been suggested to require a second coreceptor, such as human fibroblast growth factor receptor 1 (47) or V5 integrins (55). Integrins are molecules involved in cell adhesion and motility (9, 33) and have also been implicated in adenovirus infection. In this context, integrins interact with small intracellular signaling molecules, such as Rho, Rac, and Cdc42 GTPases, and can act through actin fibers to facilitate motility and endocytic pathways (42, 45). Furthermore, integrin clustering has been shown to activate a focal adhesion kinase, known as pp125^FAK, through tyrosine phosphorylation (39). Tyrosine-phosphorylated focal adhesion kinase then recruits phosphatidylinositol-3 kinase (PI3K), resulting in the activation of PI3K pathways (35). PI3Ks are members of a family of lipid kinases composed of a p85 regulatory subunit and a p110 catalytic subunit (28). Activation of this PI3K pathway leads to the generation of phosphoinositol-3,4-biphosphate and phosphatidylinositol-3,4,5-triphosphate (PIP₃). These messengers are involved in vesicular trafficking (43) and the rearrangement of cytoskeletal proteins such as actin (28). Interestingly, activated Rac1 and PI3K pathways are also required for the internalization of adenovirus (35).

Productive transduction by many viruses requires that virions gain access to the nucleus following infection. However, cytoskeletal network proteins such as actin and tubulin prevent free diffusion of large particles in the cytoplasm (54). These microtubules and microfilaments not only serve as barriers but also act as "highways" to facilitate the trafficking of these particles to specific destinations such as the nucleus or lysosomes. For example, nuclear targeting of adenovirus (49) has been shown to require functional microtubules and microfilaments (19, 34, 35, 57). Using Cy3-labeled AAV-2, we investigated the mechanisms of endocytosis and nuclear trafficking for this virus. Our results indicate that endocytosis of AAV-2 occurs through an V5 integrin/Rac1-dependent mechanism and that subsequent trafficking of the virus to the nucleus requires activation of PI3K pathways as well as functional microtubules and microfilaments.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Analysis of purity of rAAV stocks. The tgAAVCF vector, an AAV-2 vector encoding the cystic fibrosis transmembrane conductance regulator (CFTR) transgene, was produced and purified by Targeted Genetics (Seattle, Wash.) using column chromatography. tgAAVCF was free of detectable replication-competent adenovirus and replication-competent AAV (<3 IU/10¹⁰ DNase-resistant particles), bacteria, fungi, mycoplasmas, and endotoxin. To further assess the purity of the virus to be used for coupling to the Cy3 dye, decreasing amounts of tgAAVCF (from 20 to 1 µl) were electrophoresed on a 12% polyacrylamide-Tris-glycine gel (Novex); 50 ng of -galactosidase protein (Sigma) was used as a reference standard. The gel was run for about 1.5 h at 140 V in 1× running buffer (5.8 M Tris, 28.8 M glycine, 0.05% [vol/vol] sodium dodecyl sulfate [SDS]). The gel was removed, washed for 10 min in 7.5% (vol/vol) acetic acid, then stained for 1 h with Sypro-Orange (Molecular Probes) and destained for 10 min in 7.5% acetic acid. The gel was scanned using a Storm Imager and then analyzed using Image Quant Software (Molecular Dynamics). Data analysis was performed to calculate the purity of virus based on the percentage of viral capsid proteins (VP1, VP2, and VP3) to total protein staining.

Cy3 labeling of rAAV and analysis in HeLa cells. Purified rAAV was dialyzed and concentrated in conjugation buffer (0.1 M sodium carbonate [pH 9.3]) using Centricon 30 ultrafilters (Millipore) prior to the labeling reaction. The lyophilized Cy3 dye was also dissolved in conjugation buffer. Briefly, samples of virus (5 × 10¹¹ particles) were incubated for 30 min at room temperature with the N-hydroxysuccinimide (NHS)-ester carbocyanine (Cy3) dye in a reaction volume of 1 ml. The solutions were then transferred to dialysis chambers (10,000-molecular-weight cut-off; Gibco-BRL) and dialyzed for 24 h against two changes of buffer containing 20 mM HEPES (pH 7.5) and 150 mM NaCl. Lastly, the samples were dialyzed overnight in Dulbecco's modified Eagle's medium (DMEM) with no serum and concentrated in a Centricon 30. Dye-to-virus particle (D/P) ratios of the Cy3-labeled virus samples were calculated according to the manufacturer's instructions (Amersham Life Science) and were approximately equal to 1. The dialyzed Cy3-virus solution (Cy3AAV) was used directly for infecting HeLa cells (multiplicity of infection [MOI] of 10,000 DNA particles/cell) on glass slides at 4°C for 60 min (in the absence of serum). Following binding of the labeled virus at 4°C, slides were washed in serum-free medium twice and either fixed immediately for analysis or shifted to 37°C for continued infection in the presence of medium containing 10% serum. It should be noted that low-temperature incubation of HeLa cells resulted in subtle alterations in cell structure, as indicated by rounding of cells and cytoplasmic blebbing. However, these changes were independent of virus application.

Heparin competition assay. HeLa cells were incubated with CellTracker Green 5-chloromethylfluorescein diacetate (Molecular Probes) for 0.5 h at 37°C to permit visualization of subcellular compartments. Cells were then infected with Cy3-labeled rAAV at an MOI of 10,000 DNA particles/cell in the presence of increasing concentrations of heparin (40 µM, 200 µM, and 1 mM; Sigma) at 4°C for 60 min. After the incubation, cells were washed in phosphate-buffered saline (PBS) and fixed in 2% paraformaldehyde.

Immunocytochemistry. Cy3AAV-infected HeLa cells were incubated for 1 h at 4°C to allow virus binding. Cells were fixed in 100% methanol by incubating at 20°C for 10 min. After three washes in PBS, blocking was performed in 20% goat serum for 30 min. Samples were incubated in 1.5% goat serum-PBS containing 25 µg of primary B1 monoclonal anticap antibody (ARP Inc.; catalog no. 03-61058) per ml for 1 h at room temperature, followed by three washes with 1.5% goat serum-PBS. Fluorescein isothiocyanate (FITC)-labeled secondary antibody (anti-mouse immunoglobulin G [IgG]; Boehringer Mannheim Corp.) was added and incubated for another 30 min. After extensive washing in 1.5% goat serum-PBS, the slides were coverslipped with Citifluor mountant.

Preparation of Ad.N17Rac1 and Ad.CMVLacZ and Ad.K44Adynamin stocks. Three recombinant adenovirus vectors expressing either -galactosidase (Ad.CMVLacZ) (20), a dominant negative mutant of Rac1 (Ad.N17Rac1) (32), or a dominant negative mutant of dynamin (Ad.K44Adynamin) (10, 27) were used for functional studies. Recombinant adenoviral stocks were generated as previously described (18) and stored in 10 mM Tris with 20% glycerol at 80°C. The particle titers of adenoviral stocks were determined by A₂₆₀ readings and were typically 10¹³ DNA particles/ml. The functional titers of adenoviral stocks were determined by plaque titering on 293 cells and expression assays for encoded proteins. Typically the particle-to-plaque-forming unit ratio was equal to 25.

Morphologic assays to test involvement of V5 integrin and Rac1 in rAAV endocytosis. Several assays were performed to assess the involvement of V5 integrin and Rac1 in Cy3AAV endocytosis. These studies utilized an adenoviral vector encoding a dominant mutant to Rac1 (Ad.N17Rac1) or blocking antibodies to V5 integrin. Affinity-purified mouse anti-human IgG V5 integrin monoclonal antibody was obtained from Chemicon International (catalog no. MAB1961; Temecula, Calif.). This monoclonal antibody has been previously demonstrated to block vitronectin binding to V5 receptors (62). Affinity-purified goat anti-mouse IgG control antibody was obtained from Boehringer Mannheim Biochemicals (catalog no. 605240; Indianapolis, Ind.). For blocking antibody experiments, HeLa cells were incubated with either anti-human V5 or anti-mouse IgG at a final concentration of 2 µg/ml in the presence of Cy3AAV for 1 h at 4°C. For dominant inhibitor studies, cells were infected with either Ad.N17Rac1 or Ad.CMVLacZ at MOIs of 200 and 1,000 DNA particles/cell, respectively, for 48 h prior to Cy3AAV binding for 1 h at 4°C. Following viral binding at 4°C, samples were washed and either fixed immediately or incubated for an additional 10 to 120 min at 37°C. For blocking antibody experiments, Cy3AAV infections were performed in the continued presence of antibodies (2 µg/ml). The amount of membrane-bound and endocytosed Cy3AAV was determined as described below using computer-aided image analysis.

Wortmannin, nocodazole, and cytochalasin B assays. HeLa cells were treated with wortmannin (0.1 and 1 µM; Sigma), nocodazole (20 and 100 µM; Sigma), or cytochalasin B (5 or 20 µM) for 1 h at 37°C. After the slides were washed in DMEM, Cy3AAV was added to the cells (MOI of 10,000 particles/cell). Cells were then incubated at 4°C for 1 h, followed by washing and continued incubation in the presence of wortmannin, nocodazole, or cytochalasin B at 37°C for 2 h. Control cells were similarly infected with Cy3AAV but did not receive any inhibitor.

Rac1 activation assay. Rac1 activation assays were performed using a modified previously described protocol (23). pGEX-PBD (PBD encodes the p21 binding domain of Pak1, an effector molecule of activated Rac-1) was kindly provided by Richard Cerione (4). Glutathione-S-transferase (GST)-PBD fusion protein was purified from DL21 cells (Amersham Pharmacia Biotech, Piscataway, N.J.) transformed with pGEX-PBD. Bacteria were grown at 37°C to log phase and treated with 1 mM isopropyl-1-thio--D-galactopyranoside (IPTG) for 2 h. The cells were centrifuged, and the cell pellet was resuspended in lysis buffer (20 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM MgCl₂, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg of leupeptin per ml, and 10 µg of aprotinin per ml). Cells were then further lysed by three rounds of sonication (each lasting for 30 s). The lysate was subsequently centrifuged at 10,000 × g for 15 min, and the fusion proteins were isolated from the supernatant using a bulk GST purification kit (obtained from Amersham Pharmacia Biotech, Piscataway, N.J.). The purified protein appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining. Protein concentrations were determined using the Bradford assay. For selective precipitation of GTP-bound Rac1, the GST-PBD fusion protein (50 µg) was first bound to agarose-conjugated anti-GST antibody (20 µg) (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif.; catalog no. sc-138 AC) in 500 µl of lysis buffer at 4°C overnight. Subsequently, samples were centrifuged at 2,500 × g for 5 min and then washed three times with lysis buffer. These PBD-bound agarose beads were used for precipitation of GTP-bound Rac1 from virally infected HeLa cells as described below.

PI3K activation assays. The PI3K activation assay was modified from a previously published protocol by Upstate Biotechnology (Lake Placid, N.Y.). Confluent monolayers of HeLa cells were infected with tgAAVCF virus at an MOI of 5,000 DNA particles/cell and incubated at 37°C for 0, 5, or 15 min. HeLa cells were lysed in a lysis buffer containing 1% NP-40, 10 mM NaF, 2 mM sodium pyrophosphate, 0.4 mM Na₃VO₄, 0.05 M Tris (pH 7.4), and 0.15 M NaCl, with protease inhibitor cocktail (100 µg of pepstatin, 10 µg of leupeptin, and 50 µg of aprotinin per ml plus 0.5 M PMSF). Cells were then further lysed by four rounds of sonication (each lasting for 30 s). Cellular debris were removed by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined using the Bradford assay. The catalytic complex of PI3K was immunoprecipitated using anti-PI3K p85 antibody (Santa Cruz) conjugated to Gamma Bind Plus Sepharose (Amersham-Pharmacia) at 4°C overnight. The beads were washed with PBS three times and incubated with 500 µg of cellular lysate at 4°C overnight. Samples were washed three times with buffer 1 (1% NP-40 and 100 µM Na₃VO₄), three times with buffer 2 (100 mM Tris HCl [pH 7.5], 500 mM LiCl, 100 mM Na₃VO₄), and twice with buffer 3 (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 100 µM Na₃VO₄), and then resuspended in 50 µl of buffer 3. A 10-µl amount of each sample was used for Western blotting against anti-p85 antibody to ensure equivalent levels of immunoprecipitation of the PI3K complex. PI3K assays were performed using sonicated L--phosphatidylinositol (Avanti Lipids, Alabaster, Ala.) as the substrate. L--Phosphatidylinositol was dissolved in 10 mM Tris (pH 7.5) with 1 mM EGTA and added to the cellular extracts in the presence of 14 mM MgCl₂ for 5 min at room temperature. Then 30 µCi of [-³²P]ATP (Amersham-Pharmacia) and 63 µM ATP were mixed with the samples and allowed to react for 5 to 15 min at room temperature. The desired product (PIP₃) was extracted by addition of chloroform-methanol (1:1) along with 160 µM HCl. The samples were dried under nitrogen flow and then resuspended in chloroform-methanol (1:1) prior to analysis by thin-layer chromatography (TLC) using silica gel coated with 10% potassium oxalate. Chloroform-methanol-water-ammonium hydroxide (60:47:11.3:2) was the separation solvent. The production of PIP₃ was quantitated using a phosphoimager and exposed to X-ray film.

Hirt DNA preparations, subcellular fractionation, and Southern blotting for viral DNA. HeLa cells were treated with inhibitors (nocodazole, cytochalasin B, wortmannin, anti-mouse IgG, and integrin blocking antibody as described above) for 1 h at 37°C. For studies evaluating Rac1 involvement, cells were infected with Ad.N17Rac1 or Ad.CMVLacZ at an MOI of 1,000 particles/cell 48 h prior to rAAV infection. Following the above treatments, cells were then washed and incubated with AV.GFP3ori virus (15) at an MOI of 1,000 DNA particles/cell for 1 h at 4°C. Following binding, cells were washed with PBS three times and either harvested directly by scraping or trypsinization or shifted to 37°C for 2 h to promote internalization of virus prior to harvesting. Cell harvesting by trypsinization following by washing in PBS was used to remove extracellular bound virus. Extraction of low-molecular-weight Hirt DNA (viral DNA) and Southern blotting were performed according to protocols described previously (25, 52). In addition to the above methods for determining the extent of internalized and external membrane-bound virus, nuclear and cytoplasmic extracts were prepared to evaluate nuclear accumulation of virus following treatment with nocodazole, cytochalasin B, and wortmannin. Cellular and nuclear fractions were prepared according to a modified procedure of Andrew and Faller (3). Hirt DNA extractions from each of these subcellular fractions were performed as described above to recover viral DNA.

Image analysis. Phase-contrast and Texas red channels were superimposed to mark the nuclear and cell boundaries using the Volume Trace Motif Version 3.1 program (The University of Iowa Image Analysis Facility, Iowa City, Iowa). A histogram equalization was performed on each image to maximize the contrast for boundary visualization. Viral particle distributions in the nucleus and in the cytoplasm were assessed using the tal_program (Randall Frank, Brainvox, Human Neuroanatomy and Neuroimaging Lab, Department of Neurology, University of Iowa). Cy3 pixels (AAV) were visualized as white pixels (255 = absolute value for a white pixel) by setting the threshold to the maximum gray level value of background pixels (tal_threshold). Using tal_stat, the pixel mean for a region was determined. The number of white pixels was calculated from the formula of pixel mean = [(number of white pixels × 255) + (number of black pixels × 0)]/(total pixels). The numbers of regional pixels as membrane bound versus intracellular or cytoplasmic versus nuclear were divided by the number of total pixels in order to determine the regional percentages of Cy3AAV. It must be acknowledged that due to our sampling of nuclear cross-sections, it is possible that we have underestimated the extent of nonnuclear AAV particles. Nevertheless, comparative analysis between samples using this method of evaluation is informative. Three-dimensional (3-D) image reconstruction was used to evaluate whether Cy3-labeled capsid proteins entered the nucleus. In these studies the nucleus and cytoplasm of individual 0.5-µm phase-contrast confocal scans were pseudocolored in blue and green, respectively. The individual confocal layers (both phase-contrast and red channel) were then reconstructed into a 3-D image using the Voxblast program developed by the Image Analysis Core Facility at the University of Iowa.

Transmission electron microscopy. We used 300-mesh copper Formvar-coated grids (90 nm) treated with 0.5% polyvinyl formaldehyde in ethylene dichloride. Samples were placed on coated grids, stained in 1% ammonium molybdate for 30 s, and then examined using a Hitachi H-7000 transmission electron microscope.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Functional characterization of Cy3-labeled rAAV. The bifunctional NHS-ester carbocyanine-Cy3 was conjugated to highly purified rAAV virions as explained in Materials and Methods. To confirm that Cy3 was labeling capsid proteins and not other minor impurities in the virus samples, HeLa cells infected with Cy3-labeled particles were stained with a B1 antibody developed against the three AAV-2 capsid proteins. Results from this study confirmed that all Cy3-labeled particles colocalized with AAV capsid proteins on the surfaces of HeLa cells following binding at 4°C for 1 h (Fig. 1A). These results are consistent with the estimated purity (>99%) of viral stocks used in labeling reactions (Fig. 1B). As seen in Fig. 1B (lanes 2 to 7), only three bands, VP1, VP2, and VP3, were detected on SDS-PAGE. In order to determine if the conjugation reaction had altered the capsid structure of rAAV, both the labeled (Cy3AAV) and unlabeled (AAV) virus samples were examined by transmission electron microscopy (Fig. 1C). No alterations were detected in the capsid structure of rAAV after the labeling reaction. Additionally, to evaluate whether Cy3 labeling decreased the functional properties of rAAV, replication center assays (50) were performed on labeled and mock-labeled virus. The particle-to-infectious unit ratio was approximately 1,000 in both cases and was not altered by the presence of Cy3 (data not shown).

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Cy3 conjugation to AAV capsids. HeLa cells were infected with rAAV labeled with Cy3 as explained in Materials and Methods. (A) Fluorescent colocalization of Cy3AAV (left panel in red) with AAV capsid proteins stained with FITC-labeled anti-cap B1 antibody (center panel in green). The two channels are superimposed in the right panel, demonstrating colocalization of Cy3AAV and capsid proteins (in yellow). SDS-PAGE analysis, following staining with Sypro-Orange, was used to evaluate the purity of the rAAV preparation used for Cy3 labeling (B). Lane 1 contains molecular size markers, and the sizes (in kilodaltons) are given to the left of the gel. Various amounts of rAAV from 20 to 1 µl were loaded onto lanes 2 to 7 in a 12% polyacrylamide-Tris-glycine gel (Novex). The locations of capsid proteins VP-1, -2, and -3 are marked to the right of the gel. Lane 8 contains 50 ng of -galactosidase protein as a reference. (C) Ultrastructure of unlabeled (upper panel) and Cy3-conjugated AAV (lower panel) by transmission electron microscopy. The binding of Cy3-labeled AAV to its receptor on HeLa cells was tested for competition by increasing amounts of heparin (D). Numbers below each panel indicate the doses of heparin used in these assays.

View larger version (95K): [in this window] [in a new window]   FIG. 2.   Time course of AAV translocation to the nucleus. HeLa cells were incubated with Cy3AAV at 4°C for 1 h to promote virus binding. Following binding, cells were either immediately washed and fixed (A) or washed and then shifted to 37°C for 0.5 (B), 1 (C), 2 (D), or 3 (E) h prior to fixation. Cells were then analyzed by confocal fluorescent microscopy. Each image is a stack of three consecutive 0.5-µm layers, with each panel representing confocal phase contrast (top), gray scale Cy3 (middle), and superimposed images (bottom). The nucleus (nu) of each cell is marked for clarity. 3-D reconstructed images were generated from 25 (F and G) or 5 (H and I) consecutive 0.5-µm confocal layers obtained after both phase-contrast and red channel scans from a cell infected for 2 h at 37°C. Cellular architecture was generated from the individual phase-contrast confocal images, with the nucleus pseudocolored in blue and the cytoplasm in green. The same cell is represented in each panel with a different angle of rotation. The percentage of nucleus-associated Cy3AAV particles was determined using morphometric image analysis as explained in Materials and Methods (n = 7 cells analyzed for each time point) and is graphically represented in panel J. Values in panel J are the mean ± standard error of the mean (SEM) percentage of particles associated with the nucleus for each time point.

Endocytosis of rAAV is inhibited by blocking antibodies against integrin V5. It has been reported that efficient AAV-2 infection requires coreceptors such as human fibroblast growth factor receptor 1 (47) or V5 integrins (55). In order to test whether V5 integrins mediate rAAV binding and/or endocytosis in HeLa cells, infections with Cy3AAV were performed in the presence of a blocking anti-human V5 integrin monoclonal antibody or control anti-mouse IgG. As seen in Fig. 3, neither anti-human V5 integrin antibodies (panel E) nor control anti-mouse IgG (panel C) interfered with the binding of Cy3AAV to HeLa cells, since they were indistinguishable from untreated controls (panel A). These findings suggest that V5 integrin is not required for binding of AAV-2 to HeLa cells. However, when the temperature was shifted to 37°C for 45 min to initiate endocytosis, uptake of Cy3AAV was inhibited only in cells treated with anti-human V5 integrin antibody (panel F). The anti-human V5 integrin antibody appeared to prevent endocytosis, leaving virus at the membrane. No effect on Cy3AAV endocytosis was seen in cells treated with anti-mouse IgG (panel D), which were indistinguishable from untreated cells (panel B). These results suggested that V5 integrin is involved in endocytosis of rAAV virus in this HeLa cell model.

View larger version (124K): [in this window] [in a new window]   FIG. 3.   V5 integrin is required for the endocytosis of AAV-2 virus. Cy3AAV was incubated alone (A and B) or in the presence of control anti-mouse IgG antibody (C and D) or anti-human V5 integrin-blocking antibody (E and F) at 4°C for 1 h. Cells were then either washed and fixed immediately (A, C, and E) or washed and incubated for an additional 45 min at 37°C (B, D, and F). Representative confocal photomicrographs are shown for each condition. Each of the panels shows the confocal phase-contrast image (left), gray scale stack of Cy3AAV images (middle), and superimposed images (right). Each confocal image is a stack of six consecutive 0.5-µm layers that intersect the nucleus. The nucleus (nu) of each cell is marked for clarity. Southern blotting of Hirt DNA isolated from rAAV-infected cells was performed as an alternative approach to evaluate rAAV endocytosis in cells treated with anti-mouse IgG or anti-human V5 integrin (G). HeLa cells were infected with unlabeled AV.GFP3ori virus (MOI of 1,000 DNA particles/cell) for 1 h at 4°C, washed, and shifted to 37°C for 2 h. Cells were harvested by either direct scraping to determine viral binding ( trypsin) or by trypsinization to remove extracellular virus (+ trypsin). Hirt DNA was prepared from cells treated with the various conditions, and Southern blots were hybridized with P32-labeled EGFP cDNA probe. Single-stranded rAAV genomes are marked by a 1.6-kb hybridizing band.

Endocytosis of AAV-2 is blocked by expression of a dominant negative mutant form of Rac1 (N17Rac1). Integrins have been demonstrated to associate with small intracellular signaling molecules, such as Rho, Rac, and Cdc42 GTPases (42, 45). The Rac GTP-binding protein has been shown to control mitogenic and oncogenic signals through NADPH-oxidase superoxide production (26, 44). Interestingly, reactive oxygen species have been implicated in augmenting rAAV transduction by an as yet unknown mechanism (53). Given the correlation between reactive oxygen species involvement in rAAV-2 transduction and results demonstrating a role for integrins in rAAV endocytosis, we hypothesized that Rac1 and V5 integrin may interact in the endocytic events controlling AAV-2 infection.

View larger version (70K): [in this window] [in a new window]   FIG. 4.   AAV endocytosis requires Rac1 activation. A dominant inhibitor of Rac1 (N17Rac1) was used to evaluate the involvement of the small GTPase Rac1 in AAV endocytosis. HeLa cells were either uninfected (A to C) or infected with Ad.CMVLacZ (D to F) or Ad.N17Rac1 (G to I) virus at an MOI of 1,000 (D, F, G, and I) or 200 (E and H) particles/cell 48 h prior to incubation with Cy3AAV at 4°C for 1 h. Following incubation with Cy3AAV, cells were either washed and immediately fixed to examine viral binding (A, D, and G) or shifted to at 37°C for 45 min to examine endocytosis (B, C, E, F, H, and I). Representative confocal photomicrographs are given for each condition, representing six 0.5-µm stacked layers that intersect the nucleus. Each of the panels shows the confocal phase contrast image (left), gray scale stack of Cy3AAV images (middle), and superimposed images (right). The nucleus (nu) of each cell is marked for clarity. Panel J represents the mean percentage ± SEM (n = 7) of Cy3AAV particles internalized within a 45-min time period for each condition, as determined by computer-aided image analysis. Southern blotting of Hirt DNA isolated from rAAV-infected cells was performed as an alternative approach to evaluate rAAV endocytosis in Ad.N17Rac1- and Ad.CMVLacZ-infected (1,000 DNA particles/cell) HeLa cells (K). At 48 h following adenoviral infection, HeLa cells were infected with unlabeled AV.GFP3ori virus (MOI of 1,000 DNA particles/cell) for 1 h at 4°C, washed, and either harvested directly or shifted to 37°C for 2 h prior to harvesting. Cells were harvested by either direct scraping to determine viral binding ( trypsin) or by trypsinization to remove extracellular virus (+ trypsin). Hirt DNA was prepared from the various conditions, and Southern blots were hybridized with a 32P-labeled EGFP cDNA probe. Single-stranded rAAV genomes are marked by a 1.6-kb hybridizing band. Rac1 activation assays were performed as described in Materials and Methods. A Western blot detecting GST-PBD-precipitated GTP-bound Rac1 is given in panel L. Cells were infected with unlabeled tgAAVCF virus (MOI of 5,000 DNA particles/cell) or mock infected with vehicle alone for the exposure times indicated above each lane, then 500 µg of HeLa cell lysate from each condition was precipitated with GST-PBD and evaluated by Western blot against anti-Rac1 antibodies. The p21 band marked by an arrow is Rac1. The filters were also probed with anti-GST antibodies as a loading control. The position of GST-PBD protein is marked by an arrow below the Rac1 Western blot.

Efficient rAAV trafficking requires PI3K activation. Endocytosis and sorting of integrin-linked receptors have been previously suggested to require PI3K activity (41). For example, following endocytosis of vitronectin, inhibition of PI3K prevents vesicle sorting and movement to lysosomes (37). We hypothesized that the activation of Rac1 during endocytosis might be critical for signaling PI3K to initiate intracellular movement of endocytosed virus to the nucleus. Direct interactions between small GTP-binding proteins (i.e., Rac1, Rho, and Ccd42) and PI3K have also been reported (8, 59). For adenovirus, PI3K pathways also appear to be critical for viral entry and endocytosis (35). In addition, given the evolutionary similarities between the shared V5 integrin coreceptor of adenovirus and AAV (55) and the fact that both viruses appear to be endocytosed through clathrin-coated pits (61), we hypothesized that PI3K pathways might also be involved in AAV-2 endocytosis.

View larger version (68K): [in this window] [in a new window]   FIG. 5.   Trafficking of Cy3AAV to the nucleus is sensitive to wortmannin treatment. HeLa cells were either untreated (A) or treated with 0.1 µM (B) or 1 µM (C) wortmannin for 1 h at 37°C prior to infection with Cy3AAV. Following wortmannin treatment, Cy3AAV was bound to cells for 1 h at 4°C, then the cells were washed of excess virus and incubated at 37°C for an additional 2 h in the continued presence of wortmannin. Representative confocal photomicrographs are given for each condition, each representing six 0.5-µm stacked layers that intersect the nucleus. Each panel illustrates the confocal phase contrast (left), gray scale stack of Cy3AAV images (middle), and superimposed images (right). The nucleus (nu) of each cell is marked for clarity. The effect of rAAV infection on PI3K activation was evaluated as described in Materials and Methods (D and E). Cells were infected with unlabeled tgAAVCF virus at an MOI of 5,000 DNA particles/cell for 0, 5, and 15 min at 37°C. The PI3K complex was immunoprecipitated from cell lysates and assayed in the presence of [-32P]ATP and L--phosphatidylinositol followed by TLC and autoradiography (D, top). Faster migrating bands above the origin of migration (ori) indicate the product (PIP3). Western blot analysis was also performed using anti-PI3K p85 antibody, which indicates that equal amounts of PI3K complex were used for each reaction (lower panel of D). To evaluate whether functional Rac1 was necessary for PI3K activation, cells were infected with either Ad.N17Rac1 or Ad.CMVLacZ virus at an MOI of 1,000 particles/cell for 48 h prior to assaying PI3K activity (E). Conditions for infection and times of harvest are marked above each lane of the TLC autoradiogram. The bottom panel is a Western blot using anti-PI3K p85 antibody.

Cytoskeletal involvement in AAV movement to the nucleus. Cytoskeletal changes involving the polymerization of monomeric actin can be induced by the activation of PI3K as well as small GTP-binding proteins such as Rho, Rac, and CDC42 (58). Cytoskeletal elements such as microtubules and microfilaments have long been recognized as important in controlling the intracellular movement of viruses (11, 12, 36). It has also been suggested that microtubules may have a preferential function in the intracellular movement of adenovirus (57), although some reports have also implicated microfilaments in adenovirus infection (46, 49). Therefore, we next sought to characterize the cytoskeletal elements important in the movement of AAV to the nucleus.

View larger version (82K): [in this window] [in a new window]   FIG. 6.   Nocodazole and cytochalasin B treatment reduces nuclear targeting of Cy3AAV. HeLa cells were either untreated (A and D) or treated with 30 µM nocodazole (B), 100 µM nocodazole (C), 5 µM cytochalasin B (E), or 20 µM cytochalasin B (F) for 1 h at 37°C prior to infection with Cy3AAV. Following nocodazole and cytochalasin B treatment, Cy3AAV was bound to cells for 1 h at 4°C, and the cells were then washed of excess virus and incubated at 37°C for an additional 2 h in the continued presence of inhibitor. Representative confocal photomicrographs are given for each condition, each representing six 0.5-µm stacked layers that intersect the nucleus. Each of the panels shows the confocal phase contrast (left), gray scale stack of Cy3AAV images (middle), and superimposed images (right). The nucleus (nu) of each cell is marked for clarity. Quantitative analysis of AAV nuclear trafficking is shown in panel G. The percentages of cytoplasmic and nuclear associated Cy3AAV particles were quantified by morphometric image analysis, as explained in Materials and Methods, following treatment of cells with various chemical agents. Solid bars indicate nucleus-associated Cy3AAV, while open bars indicate cytoplasmic Cy3AAV particles. Values are the mean ± SEM of 10 cells quantified for each condition.

View larger version (35K): [in this window] [in a new window]   FIG. 7.   Wortmannin, nocodazole, and cytochalasin B inhibit intracellular transport of AAV-2 to the nucleus but not endocytosis. HeLa cells were infected with unlabeled AV.GFP3ori virus (MOI of 1,000 DNA particles/cell) following treatment with wortmannin (W), nocodazole (N), or cytochalasin B (C). Protocols for chemical treatment were as described in Materials and Methods. Cells were infected for 1 h at 4°C, washed, and shifted to 37°C for 2 h. To determine the extent of viral endocytosis, cells were harvested after both 4 and 37°C incubation by either direct scraping to determine viral binding ( trypsin) or by trypsinization to remove extracellular virus (+ trypsin) (A). Alternatively, following 37°C incubations, cells were harvested by trypsinization and washed, and cytoplasmic and nuclear fractions were purified (B). Hirt DNA was prepared from cells or subcellular fractions following the various treatment conditions (as indicated above each lane), and Southern blots were hybridized with 32P-labeled EGFP cDNA probe. Single-stranded rAAV genomes are marked by a 1.6-kb hybridizing band.

Inhibition of rAAV endocytosis and nuclear trafficking reduce transgene expression from recombinant virus. AV.GFP3ori virus carrying the enhanced green fluorescent protein (EGFP) reporter gene (15) was used to determine whether rAAV-mediated transgene expression was affected by inhibitors of either endocytosis (Ad.N17Rac1, AdK44Adynamin, and V5 integrin blocking antibody) or nuclear trafficking (nocodazole, cytochalasin B, and wortmannin). The dominant mutant dynamin-expressing adenovirus (K44A) was used as a comparative control in these studies and has been previously demonstrated to inhibit endocytosis of rAAV2 and transgene expression in HeLa cells (13). HeLa cells were infected with Ad.N17Rac1, Ad.K44Adynamin, or Ad.CMVLacZ virus 48 h prior to infection with AV.GFP3ori virus at an MOI of 1,000 particles/cell. In addition, HeLa cells were treated with V5 integrin-blocking antibody, nocodazole, cytochalasin B, or wortmannin for 1 h prior to infection with AV.GFP3ori virus. The percentage of cells expressing GFP was determined 35 h postinfection with rAAV by fluorescence-activated cell sorting (FACS) analysis. As seen in Fig. 8, all inhibitors of AAV-2 endocytosis and nuclear trafficking significantly reduced the percentage of GFP-expressing cells compared to untreated and Ad.CMVLacZ-infected cells. The extent of inhibition under each of these conditions was not as complete as observed by morphologic analysis of Cy3AAV or Southern blotting of viral DNA. This may be attributed to the longer time course needed to evaluate transgene expression and the reversibility of the inhibitors used. Furthermore, each of the inhibitors used to block either endocytosis or intracellular transport likely reduces the rate of AAV movement to the nucleus but does not completely inhibit these processes. Removal of nocodazole 2 h after the infection restored rAAV transduction to 90% of that achieved in the absence of inhibitor, as determined by EGFP expression (data not shown).

View larger version (23K): [in this window] [in a new window]   FIG. 8.   Inhibitors of AAV-2 endocytosis and nuclear trafficking also reduce rAAV-mediated gene expression. HeLa cells were treated with various agents (indicated below the graph) which modulate AAV-2 endocytosis or nuclear trafficking, and the extent of AV.GFP3ori-mediated GFP expression was analyzed at 35 h postinfection by FACS analysis. For conditions involving infection with recombinant adenoviruses Ad.N17Rac1, Ad.K44Adynamin, and Ad.CMVLacZ, cells were infected at MOIs of 1,000 particles/cell 48 h prior to AV.GFP3ori infection. Treatments with nocodazole, cytochalasin B, wortmannin, anti-IgG, and anti-integrin were performed as described for Cy3 analyses. All conditions included infection with AV.GFP3ori virus at an MOI of 1,000 DNA particles/cell. FACS analysis data represent the mean ± SEM of four independent data points.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Mechanisms underlying rAAV transduction have gained considerable interest due to the increasing development of this virus as a vector for gene therapy. Cy3-labeled rAAV has recently become a valuable tool to analyze rAAV transduction (5, 6, 24, 51). In the present study, we have focused on elucidating early steps in the AAV-2 infection process that occur prior to gene conversion of the single-stranded genome to an expressible form in the nucleus. For these analyses, we have utilized Cy3-labeled rAAV virus to enable delineation of the processes controlling AAV-2 entry and intracellular trafficking to the nucleus.

Previous studies have shown that an HSPG receptor mediates binding of AAV-2 to the surface of HeLa cells. The present study has now demonstrated that internalization of HSPG-bound virus occurs through a Rac1-dependent process. Although a direct link between HSPG receptors and Rac1 activation has not been previously identified, integrin and integrin-linked receptors have been shown to play important roles in organizing the actin cytoskeleton through Rac1-dependent intracellular signal transduction pathways (30, 31, 40). Our evidence for the involvement of V5 integrin in rAAV endocytosis further supports this hypothesis. Although no direct evidence exists to date for an interaction between V5 integrin and Rac1, it is reasonable to speculate that AAV-2 interactions with the V5 integrin coreceptor may be responsible for the activation of Rac1 seen during AAV-2 infection. Hence, as shown in Fig. 9, we hypothesize that HSPG receptors on the cell surface may bind AAV-2 and interact with V5 integrin to activate the internalization process, which is facilitated by Rac1-V5 integrin interactions.

View larger version (39K): [in this window] [in a new window]   FIG. 9.   Schematic representation of AAV-2 endocytic and nuclear trafficking mechanisms. Receptor-mediated endocytosis of AAV-2 in HeLa cells is facilitated through binding to its receptor HSPG and interactions with V5 integrin. Activation of Rac1, potentially by V5 integrin, is required for efficient endocytosis of AAV virus. Following endocytosis, we propose that Rac1 activation leads to stimulation of PI3K pathways, which facilitate the functional rearrangements of the cytoskeleton (microfilaments and microtubules) required for the efficient nuclear targeting of AAV.

Once AAV particles are internalized, triggering steps must occur to start their movement to the nucleus. It is currently unknown whether the virions move to the nucleus within endosomes or whether they break out of endosomes shortly after endocytosis. As previously reported (13) and reproduced in this study (data not shown), persistent colocalization of FITC-labeled transferrin and Cy3AAV within intracellular vesicles near the nucleus suggests that escape of virions from endosomes may occur at or near the nuclear membrane pores. Additionally, confocal localization of Cy3AAV particles inside the nucleus suggests that AAV-2 may be transported through the nuclear pore, a finding which is in stark contrast to studies with adenovirus. Regardless of whether virions reside within endosomes shortly after endocytosis, transport of AAV-2 to the nucleus was greatly inhibited by wortmannin, nocodazole, and cytochalasin B. In contrast, none of these agents appeared to significantly inhibit the binding or endocytosis of AAV-2. These findings suggest that PI3K activation may be necessary to direct virus or virus-containing vesicles along microfilaments and microtubules to the nuclear pores (Fig. 9). Given the link between Rac1 and PI3K pathways in cytoskeleton reorganization, these findings are intriguing.

Several reports have previously suggested that the GTPase Rac1 is a downstream target of PI3K (38, 60). However, in the current study, Rac1 but not PI3K appeared to be necessary for endocytosis. Given the fact that N17Rac1 expression inhibited PI3K activation following rAAV infection, it appears that Rac1 must act proximal to PI3K in the infectious process. Taken together with previous studies in this area, our findings suggest that interactions between Rac1 and PI3K may be bidirectional in nature, depending on the environmental circumstances and stimuli.

In summary, these studies have laid the foundation for a better understanding of AAV-2 infection and the molecular processes that mediate its endocytosis and movement to the nucleus. Given the recent findings that rAAV-2 infection from the apical surface of polarized airway epithelia is significantly impaired by a postendocytic block involving ubiquitination and reduced movement of virus to the nucleus (17), delineation of the intracellular pathways controlling rAAV infection may lead to methods for improving gene delivery with this vector for diseases such as cystic fibrosis.