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Adeno-associated virus (AAV) serotypes are being tailored for numerous therapeutic applications, but the parameters governing the subcellular fate of even the most highly characterized serotype, AAV2, remain unclear. To understand how cellular conditions control capsid trafficking, we have tracked the subcellular fate of recombinant AAV2 (rAAV2) vectors using confocal immunofluorescence, three-dimensional infection analysis, and subcellular fractionation. Here we report that a population of rAAV2 virions enters the nucleus and accumulates in the nucleolus after infection, whereas empty capsids are excluded from nuclear entry. Remarkably, after subcellular fractionation, virions accumulating in nucleoli were found to retain infectivity in secondary infections. Proteasome inhibitors known to enhance transduction were found to potentiate nucleolar accumulation. In contrast, hydroxyurea, which also increases transduction, mobilized virions into the nucleoplasm, suggesting that two separate pathways influence vector delivery in the nucleus. Using a small interfering RNA (siRNA) approach, we then evaluated whether nucleolar proteins B23/nucleophosmin and nucleolin, previously shown to interact with AAV2 capsids, affect trafficking and transduction efficiency. Similar to effects observed with proteasome inhibition, siRNA-mediated knockdown of nucleophosmin potentiated nucleolar accumulation and increased transduction 5- to 15-fold. Parallel to effects from hydroxyurea, knockdown of nucleolin mobilized capsids to the nucleoplasm and increased transduction 10- to 30-fold. Moreover, affecting both pathways simultaneously using drug and siRNA combinations was synergistic and increased transduction over 50-fold. Taken together, these results support the hypothesis that rAAV2 virions enter the nucleus intact and can be sequestered in the nucleolus in stable form. Mobilization from the nucleolus to nucleoplasmic sites likely permits uncoating and subsequent gene expression or genome degradation. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics and have identified cellular parameters that mobilize virions in the nucleus and significantly influence AAV infection.
Adeno-associated virus (AAV) is classified as a dependovirus because it requires the presence of a helper virus, such as adenovirus or herpesvirus, in order to enter into a productive lytic cycle (6). Because of its nonpathogenicity and ability to promote sustained, long-term transgene expression in a wide variety of tissues such as the brain, liver, muscle, retina, and vasculature (51), several recombinant AAV (rAAV) serotypes are emerging as attractive vectors for gene therapy. Despite many advances in AAV vector design, barriers such as a preexisting immune response and off-target binding have necessitated administration of high viral titers to achieve efficient transduction (24, 51).
Beyond the barriers of the immune response (9, 42) and cell surface targeting (52), researchers are becoming increasingly aware that subcellular processing is a significant barrier to infection (16, 29, 52). Subcellular processing may include conformational changes within the endosome or similar compartments, endosomal escape, nuclear targeting, and uncoating, but the factors that control these events are not well defined. Understanding how cellular conditions affect subcellular processing of virions will lead to improved gene delivery through exploitation of these parameters and promote better vector design.
Given that the virion is an icosahedral particle only 25 nm in diameter, rAAV must contain all of the molecular components required to navigate through the subcellular environment in a remarkably small structure. Wild-type AAV is a nonenveloped parvovirus that packages a single-stranded DNA genome of approximately 4.7 kb in length. The viral genome is flanked by two inverted terminal repeats and contains two open reading frames, one that codes for replication proteins and another that codes for capsid proteins. Three capsid proteins (VP1, VP2, and VP3) are encoded in the second overlapping reading frame, each beginning with a different start codon but sharing a common C terminus and stop codon. Capsids are comprised of 60 copies of V1, VP2, and VP3 in a ratio of approximately 1:1:10, respectively (11, 43). During production, AAV capsids are known to assemble at early time points in the nucleolus (64), a subdomain of the nucleus and one of the oldest known cellular structures. Intact capsids have been shown to interact with nucleolar proteins such as nucleolin (NCL) and B23/nucleophosmin (NPM1) in the context of assembly (8, 46), but how these proteins affect infection or vector delivery is currently unknown.
Initial cell surface binding of AAV capsids is mediated by expression of glycoprotein receptors and specified by residues in VP3 (45, 58, 59). After binding receptors on the host cell plasma membrane, AAV serotype 2 (AAV2) is endocytosed from the cell surface in a clathrin- and dynamin-dependent process (3, 5, 19). Following endocytosis, many AAV particles accumulate in late endosomes, lysosomes, or other compartments and do not deliver their genome to the nucleus (17). This impediment to gene delivery is exacerbated when particles lack VP1 or contain specific mutations in the unique N terminus of VP1 (23). The N terminus of VP1 is normally folded inside the capsid, harboring a phospholipase domain and putative nuclear localization signals necessary for infection (13, 23, 74). These regions of VP1 are thought to translocate to the capsid exterior during subcellular processing of the virus (10, 35, 57). Even with proper capsid composition, the vast majority of internalized particles remain clearly outside the nuclear membrane, and although recent evidence suggests that successful infection occurs when the capsid uncoats inside the nucleus (57, 61), whether AAV can enter the nucleus as an intact capsid is still vehemently debated.
In general, it has proven difficult to discern whether infectious particles truly cross the nuclear membrane, due to the limitations of fluorescence microscopy (5, 67). In an in vitro setting it has been demonstrated that unmodified AAV capsids are capable of entering purified nuclei (28), yet these conditions do not accurately represent what occurs physiologically, since virus directly microinjected into cytoplasm will not enter the nucleus or efficiently transduce the cell (17, 57). In one instance, single-particle tracking of AAV has been used to follow capsids in a live-cell imaging paradigm and has found that they can be quickly and directly transported to the nucleus (54). However, another recent study has parsed confocal images of green fluorescent protein-tagged AAV2 particles during infection and has reported that few if any particles enter the nucleus during infection (38).
Although it is unclear whether capsids enter the nucleus intact, it has been well established that nuclear delivery of the genome is highly inefficient and significantly limits transduction. Several studies have identified agents that surmount subcellular barriers to transduction (20, 22, 69). Two of the most well-documented agents known to improve subcellular processing are proteasome inhibitors and hydroxyurea (HU); however, their mechanisms of action remain unknown. Therefore, we set out to determine what effect, if any, these agents had on subcellular trafficking of rAAV2 in the hope of identifying specific cellular parameters that promote efficient transduction.
Here we report that rAAV2 capsids accumulate in the nucleolus during infection. Proteasome inhibitors were found to potentiate nucleolar accumulation, while HU reduced nucleolar accumulation and appeared to mobilize capsids to the nucleoplasm. Acting independently, both proteasome inhibitors and HU increased transduction, and together they were cooperative, which suggests that these treatments operate through separate pathways to improve gene delivery. In addition, we found that small interfering RNA (siRNA) knockdown of nucleolar proteins NCL and NPM1 had effects similar to those of proteasome inhibition or HU and increased transduction. Based on our results, we have proposed a model wherein AAV virions initially enter the nucleus intact and can be sequestered in the nucleolus in stable form. Disruption of the nucleolus subsequently mobilizes virions from the nucleolus to nucleoplasmic sites and likely permits uncoating.
HeLa cells and HEK-293 cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium that was supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin.
Virus was produced in HEK-293 cells as previously described (68). Briefly, using polyethylenimine (linear molecular weight, ~25,000), cells were triple transfected with pXR2, the pXX6-80 helper plasmid, and pTR-CMV-luciferase containing the luciferase reporter transgene flanked by inverted terminal repeats. At 60 h posttransfection, cells were harvested and virus purified by cesium chloride gradient density centrifugation for 5 h at 65,000 rpm or overnight at 55,000 rpm. Fractions that contained peak virus titers were dialyzed against 1× phosphate-buffered saline (PBS) supplemented with calcium and magnesium. Viral titers were determined in triplicate after treating dialyzed fractions with DNase, digesting the capsid with proteinase K, and purifying viral DNA by use of a DNeasy column (Qiagen) according to the manufacturer's protocol. Viral DNA was applied via a dot blot manifold to a Hybond-XL membrane (Amersham) and detected with a [γ-32P]CTP-labeled probe complementary to the luciferase transgene. Empty capsids were harvested from HEK-293 cells after transfection of only pXR2 and XX6-80 without a transgene plasmid and dialyzing from low-density cesium chloride fractions that displayed peak monoclonal antibody (MAb) A20 reactivity. For this study, consistent results were obtained for many different virus preparations and could also be reproduced when virus was purified by use of an iodixanol gradient.
Similarly to what we have previously described (25), HeLa cells (5 × 104/well) were plated on poly-l-lysine-coated 12-mm glass coverslips (no. 1.5) at 24 h before infection. Recombinant virions were added to cell media (2 × 104 vector genomes [vg]/cell). No virus was added to control wells. Where indicated, HU was added at 10 mM, left for 12 h, and washed off extensively prior to virus administration. A proteasome inhibitor (MG132; Calbiochem) was present for the duration of infection at 2 μM, when used. At the indicated time points, cells were washed three times with PBS and then fixed with 2% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Following four washes with PBS, the permeabilized cells were blocked with immunofluorescence buffer (IFB) (20 mM Tris [pH 7.5], 137 mM NaCl, 3 mM KCl, 1.5 mM MgCl2, 5 mg/ml bovine serum albumin, 0.05% Tween) for 30 min at room temperature. The cells were incubated with primary antibody to detect intact capsids (MAb A20 [1:10], NCL [Abcam 22758, 1:1,000], or laminB1 [Abcam 16048, 1:750]), diluted in IFB, for 1 h at 37°C or overnight at 4°C. The cells were then incubated in secondary antibody, diluted 1:5,000 in IFB (anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 568 [Molecular Probes]), for 1 h at 37°C. After six washes in PBS, coverslips were mounted cell side down on glass slides with mounting medium (Prolong antifade Gold with DAPI [4′,6′-diamidino-2-phenylindole]; Molecular Probes). Images were captured on a Leica SP2 AOBS upright laser scanning confocal microscope and processed using Adobe Photoshop.
Confocal z-stack sections of HeLa cells fixed 16 h after infection with either rAAV2 or empty capsids in the presence of 2 μM MG132 were processed and rendered in three dimensions using Volocity software (Improvision).
Nucleoli were isolated from cell fractionations as previously described (41), with minor modifications allowing for viral infection. Briefly, five 15-cm plates of HeLa cells at 90% confluence were used for each preparation. Empty or full rAAV2 particles were incubated with cells for 16 h at 37°C (2,000 vg/cell, with empty capsids normalized to A20 reactivity of full capsids). Cells were washed three times with ice-cold PBS and harvested by centrifugation at 218 × g for 5 min. The cell pellet was resuspended in hypotonic buffer and homogenized on ice using a tight pestle to the point where >90% of cells were burst and leaving nuclei intact. The homogenate was spun at 218 × g for 5 min to separate the crude nuclear pellet from the postnuclear supernatant (PNS, ~5 ml). Nuclei were further purified from cytoplasmic contaminants by spinning through a 0.35 M sucrose cushion to give a nucleus-associated (NA) pellet. Nuclei were lysed by limited sonication on ice, and the nuclear suspension in 0.35 M sucrose was layered over a 0.88 M sucrose cushion to separate nucleolar (NU) fractions (200 μl) from NA fractions (~5 ml). Nucleoli were further purified by another spin through sucrose, and their integrity and purity were verified by phase-contrast microscopy and Western blotting.
At least 4 h prior to infection or drug treatment, HeLa cells were plated in 24-well plates at a density of 105cells/well. For drug studies, cells were handled as stated above. Cells were infected with purified rAAV2 at the designated number of vector genomes per cell or 5 μl of the indicated samples after cell fractionation and were typically harvested after 24 h unless otherwise noted. Luciferase activity was measured in accordance with the manufacturer's instructions (Promega). Luciferase activity was measured with a Wallac 1420 Victor2 plate reader. Error bars in the figures represent standard deviations from samples scored in triplicate. Graphs are for representative data sets from at least three independent assays.
Samples from purified virus or cell fractionations were loaded onto NuPage 10% bis-Tris gels and typically run for 3 h at 150 V in 1× NuPage MOPS (morpholinepropanesulfonic acid) buffer. Electrophoresis was performed in an XCell SureLock minicell (Invitrogen). Proteins were transferred to a Hybond ECL membrane utilizing the XCell II Blot module (Invitrogen) for wet transfer according to the manufacturer's protocol. Membranes were blocked for at least 30 min at room temperature in 5% nonfat dry milk (NFDM) dissolved in 1× PBS-Tween (0.1%). For detection of capsid proteins, primary antibody (MAb B1) was diluted 1:20 with 2.5% NFDM in 1× PBS-Tween (0.1%) and incubated for 1 h at room temperature or overnight at 4°C. After washing with 1× PBS-Tween (0.1%), blots were incubated for 1 h at room temperature with anti-mouse-horseradish peroxidase secondary antibody, diluted 1:5,000 in 2.5% NFDM with 1× PBS-Tween (0.1%). Following multiple washes with 1× PBS-Tween (0.1%), SuperSignal West Femto Maximum Sensitivity substrate (Pierce) was added to each membrane according to the manufacturer's protocol for developing. Each membrane was then exposed to Amersham Hyperfilm MP.
Identically to methods stated earlier, cells were fractionated into PNS, NA, and NU fractions as previously described (41). DNA was isolated from fractions using DNeasy columns (Qiagen) according to the manufacturer's protocol. Quantitative PCR was performed on a LightCycler 480 using Sybr green (Roche) and primers designed against the firefly luciferase transgene: 5′ AAA AGC ACT CTG ATT GAC AAA TAC 3′ (forward) and 5′ CCT TCG CTT CAA AAA ATG GAA C 3′ (reverse). Conditions used for the reaction were as follows: 1 cycle at 95°C for 10 min; 45 cycles at 95°C for 10 s, 62°C for 10 s, and 72°C for 10 s; and 1 cycle at 95°C for 30 s, 65°C for 1 min, and 99°C for acquisition.
Confluent 10-cm plates of HeLa cells were pulsed with rAAV2 (2,000 vg/cell) for 2 h at 37°C, washed with medium, and harvested at 0, 12, and 24 h postinfection in 5 ml PBS. DNA from 200 μl of the cell suspension was purified using DNeasy columns and applied to a Hybond XL membrane using a dot blot manifold. DNA specific to the viral genome was detected as mentioned above by hybridization of a radiolabeled probe created against the luciferase transgene.
Knockdown of NCL, NPM1, and vallosin-containing protein (VCP) was obtained by transfecting validated siRNA sequences using Hiperfect (Qiagen) according to the manufacturers' protocol for fast-forward transfection of adherent cells. Briefly, 80,000 HeLa cells were plated into 24well plates and mixed immediately with preincubated Dulbecco's modified Eagle's medium, transfection reagent, and siRNA at a final concentration of 5 nM. Two days after transfection, cells were trypsinized and replated at appropriate densities for transduction assays or confocal immunofluorescence. Target sequences were as follows: Hs_NPM1_7, 5′ AAA GGT GGT TCT CTT CCC AAA 3′; HS_NPM1_8, 5′ AAT GTC TGT ACA GCC AAC GGT 3′; Hs_VCP_6, 5′ AAG ATG GAT CTC ATT GAC CTA 3′; Hs_VCP_7, 5′ AAC AGC CAT TCT CAA ACA GAA 3′; Hs_NCL_10, 5′ AAG GAA ATG GCC AAA CAG AAA 3′; and Hs_NCL_5, 5′ AAG CTA TGG AGA CTA CAC CAG 3′.
Identifying and overcoming subcellular barriers that prevent the majority of virions from delivering their payload to the nucleus are critical steps toward developing successful vectors for gene therapy applications. As previously reported, proteasome inhibitors and genotoxic agents affect events of subcellular processing to enhance transduction of rAAV2 in a cell type- and serotype-specific manner (16, 20, 27, 29). The proteasome inhibitor MG132 is known to reduce the rate of degradation of ubiquitin-conjugated proteins by the 26S complex of the proteasome. In contrast, the primary target of the genotoxic agent HU is thought to be ribonucleotide reductase. It is unclear precisely how transduction of rAAV is enhanced following these treatments, but MG132 and HU are postulated to increase efficiency of nuclear entry (20, 22, 69). Here we demonstrate that the aforementioned pharmacological agents can regulate accumulation and mobilization of intact AAV virions in the nucleus following infection using a variety of techniques.
To establish the trafficking profile of rAAV2, we tracked the subcellular fate of virions by confocal immunofluorescence microscopy. Using a MAb (MAb A20) that recognizes only intact capsids, we were able to identify virions within the nucleus by 16 h after infection when virus was continuously present in the medium (Fig. (Fig.1A).1A). The distinct pattern of accumulation that we observed for rAAV2 with no drug treatment was potentiated when MG132 was present. Capsids could be detected in the nucleus as early as 4 h after infection, which corresponds to the earliest time that we could detect gene expression. A markedly different pattern was observed during infection following HU pretreatment, with capsids showing a diffuse distribution in the nucleoplasm. At 16 and 24 h, MG132 and HU had clearly contrasting effects on capsid localization in the nucleus. This finding was intriguing considering that both MG132 and HU increase infection efficiency over a wide range of particle numbers (Fig. 1B to D), as measured by luciferase assays of transduction. HU increased transduction from 5- to 10-fold, and MG132 increased transduction by almost 2 log orders of magnitude.
It is noteworthy that despite the need for high particle numbers (20,000 vg/cell) in immunofluorescence studies using AAV (38, 57, 67), a linear relationship between transduction and dose was observed (Fig. 1B to D). This supports the notion that similar trafficking patterns are occurring at a low multiplicity of infection (MOI) (200 vg/cell), despite these doses being below the limit of visual detection. Nonetheless, the striking nuclear accumulation pattern that we observed with proteasome inhibition in immunofluorescence studies prompted further exploration.
The nucleolus has been termed the gateway to viral infection because of its seemingly ubiquitous involvement in the replication dynamics of many viruses (31). For wild-type AAV2, capsid assembly has been reported to occur in nucleoli (8, 46, 64), but the roles of the nucleolus or nucleolar proteins during infection of an exogenously applied vector have not been studied. To more clearly determine whether rAAV capsids accumulate in the nucleolus, we carried out immunofluorescence experiments to identify intact capsids and the nucleolar marker NCL at 16 h after infection with 20,000 vg/cell in the presence of MG132 (Fig. (Fig.2A).2A). Cross-sectional analysis of z-stacked confocal images shows that rAAV2 colocalizes with NCL in nucleoli, although the majority of virions remain outside the nuclear membrane.
Remarkably, in contrast to rAAV2 virions, empty capsids were not found to accumulate in nucleoli or to colocalize with NCL, even in the presence of MG132. Only with samples where DNA-containing particles were administered were we able to detect nucleolar accumulation (Fig. (Fig.2A2A versus B) at any time point after infection, despite similar levels of internalization for rAAV2 and empty capsids as indicated by A20 staining. Nucleolar accumulation of rAAV2 could also be potentiated by another proteasome inhibitor, ALLN, and was demonstrated in HEK-293 cells (data not shown), suggesting that this phenomenon is not unique to one cell type.
Additionally, we were able to explore nucleolar accumulation of rAAV2 in three dimensions by rendering z-stacked images acquired by confocal microscopy using Volocity software (Fig. (Fig.2C).2C). This enabled us to digitally subtract the nucleus by channel gating to reveal more precisely the localization of capsids in its interior. Detection of NCL was observed diffusely in the nucleoplasm (Fig. (Fig.2C,2C, ii and vi), and when the channel was gated to show only intense staining, it was found confined to nucleoli (Fig. (Fig.2C,2C, iii and vii). After completely subtracting the NCL channel, a collection of virions localized to the nucleolar interior could be clearly seen for rAAV2 (Fig. (Fig.2C,2C, iv), yet empty capsids were absent from these regions (Fig. (Fig.2C,2C, viii).
To further support the evidence from immunostaining that rAAV2 virions are capable of accumulating in the nucleolus, we infected cells and attempted to isolate nucleoli containing virus by subcellular fractionation. To this end, we infected HeLa cells with 2,000 vg/cell rAAV2 or an equivalent amount of empty capsids for 16 h in the presence of MG132. Cells were fractionated into PNS, which contained mostly cytoplasmic material; an NA fraction, which may have included material attached to the outside of the nuclear membrane; and a fraction enriched with highly purified nucleoli. When analyzed by Western blotting, capsid proteins from rAAV2 and empty capsids were represented at similar levels in NA fractions (diluted 25-fold over fraction NU [Fig. [Fig.2B]).2B]). Identical to what was found with immunostaining, a population of capsids or capsid proteins was detected in NU fractions after rAAV2 infection but not after empty capsid infection. The fact that empty capsids could not be detected in nucleoli by immunostaining or subcellular fractionation suggests that accumulation of rAAV2 in the nucleus is not an artifact but rather represents a property unique to genome-containing rAAV2 that is not demonstrated by empty capsids (see Discussion).
Since the localization of intact capsids or capsid protein may not reflect the localization of genomes if virions have already uncoated, we chose to quantify relative amounts of capsid proteins and genomes after subcellular fractionation. We have also analyzed whether proteasome inhibition or HU treatment affects capsid and genome localization using subcellular fractionation, since these treatments appear to have different effects as observed by immunofluorescence. It has been reported that uncoating is a slow and inefficient process for rAAV2 (56, 61, 75), so we predicted that the amount of genomes in nucleolar fractions should roughly correspond to the amount of capsid protein detected in those fractions if the majority of virions have not released their DNA. As expected, MG132 increased the amount of capsid protein associated with fractions NA and NU (Fig. (Fig.3A,3A, lanes 7 and 11). In contrast, HU reduced the amount of capsid protein most notably in fraction NU (lane 12), relative to untreated control infection (lane 10). These findings further support our immunofluorescence data that suggest proteasome inhibitors increase nucleolar accumulation of capsids whereas HU mobilizes capsids into the nucleoplasm, away from the nucleolus.
Quantifying the number of genomes in these locations is just as important in analyzing capsid distribution, since capsids could have released their genomes prior to this point in trafficking. The nucleolus may either be a site where potentially infectious virions are sequestered given certain cellular conditions or represent a site for depositing empty capsids after genome release that would have no bearing on transduction efficiency. To determine if genomes could be detected in fraction PNS, NA, or NU, we purified DNA from these fractions after infecting cells and subjected the samples to quantitative PCR. The trends observed for genome copy number (Fig. (Fig.3B)3B) closely follow those observed for capsid protein levels, with more genomes detected in fraction NU following proteasome inhibition (Fig. (Fig.3B,3B, lane 11) and fewer detected after HU treatment (lane 12), compared to an untreated control infection (lane 10). It should be noted that fraction NU contains highly purified nucleoli and during the isolation is concentrated roughly 25-fold over fractions PNS and NA. Thus, it is important to realize that the majority of capsids and genomes in the cell do not enter the nucleolus. Since most virions are likely associated with the outside of the nuclear membrane, nuclear entry is an inefficient process even in the presence of proteasome inhibitors.
Detecting viral capsids and viral genomes in the same subcellular compartment does not necessitate that the former still carry the latter. When these are observed to accumulate in the nucleolus, the capsid could have remained intact after already releasing its genome or the capsid could still contain the genome if the virus has not yet completed infection. To test whether vector genomes detected in the nucleolus were protected in intact capsids or had been released during capsid uncoating, we assayed whether virions isolated from nucleoli after infection could successfully transduce cells in secondary infections (Fig. (Fig.4A).4A). In one condition, rAAV2 (2000 vg/cell) was spiked into HeLa cell medium for roughly 1 min. This short incubation period is not long enough for the majority of virions to bind and internalize into cells. As shown in Fig. Fig.4A,4A, condition 1 serves as an internal control to verify that the procedure is not vulnerable to contamination from infectious particles in the medium during wash steps and nucleolar fractionation. Conditions 2 and 3 were 16-h infections (2,000 vg/cell) with rAAV2 or rAAV2 plus MG132, respectively. This time interval was chosen based on the immunofluorescence evidence that prominent nucleolar accumulation is seen by 16 h after infection (Fig. (Fig.1A1A).
After the primary infection under the conditions listed above, HeLa cells were fractionated into PNS, NA, and NU fractions (Fig. (Fig.4A).4A). To determine if virions accumulating in these fractions remained infectious, small volumes of these fractions were administered to fresh HeLa cells and transduction was scored 24 h later by luciferase assay. Compared to purified rAAV2 (10,000 vg/cell), negligible levels of transduction were observed from the PNS fraction for all conditions, from the NA fraction for condition 1, or from the NU fraction from condition 1 (Fig. (Fig.4B).4B). Low transduction was observed in the NA fraction for conditions 2 and 3, and moderate transduction was detected with the NU fraction for conditions 2 and 3. When these samples for conditions 2 and 3 from the NU fraction were heated to 70°C for 10 min to disassemble capsids, transduction levels dropped to baseline, as they did for heat-treated purified rAAV2. These data strongly suggest that capsids accumulating in nucleoli during infection still contain their genomes and remain infectious entities.
The central tenet that inefficient subcellular processing of rAAV significantly reduces transduction has driven research toward discovering ways to enhance processing and subcellular trafficking of the virus. To date, no effect on capsid degradation has been observed during proteasome inhibition, despite capsid ubiquitination being demonstrated in vitro (20, 70). Additionally, knowing the total amount of virion accumulation in cells treated with MG132 or HU would establish a baseline for comparing the effects of each treatment. To determine if proteasome inhibitors or genotoxic agents affect the amount of vector genomes internalized and to follow how these drugs affect the persistence of rAAV2 DNA, we performed a viral genome pulse-chase to compare control, MG132, and HU conditions. Cells were infected for 2 h at 37°C, washed with medium, and harvested at 0, 12, and 24 h postinfection. The remaining viral genomes were purified and detected by dot blot hybridization (Fig. (Fig.5A).5A). As previously documented for AAV2 (18), proteasome inhibition slows clearance of viral genomes compared to untreated control samples, though the mechanism of action remains unknown. Degradation of viral genomes is not impeded in samples pretreated with HU, as the pattern of genome disappearance closely matches that of controls.
Both MG132 and HU are pluripotent agents and have numerous downstream effects, but since these drugs are thought to act through different pharmacological mechanisms, we hypothesized that together they may act synergistically to increase rAAV transduction. Thus, we performed a luciferase assay of transduction, comparing the effects of MG132, HU, or the combination of the two to determine if they operate through separate pathways. As expected, when combined, MG132 and HU enhance transduction more than either one independently (Fig. (Fig.5B).5B). It should be noted that in these experiments the concentration of MG132 was reduced twofold compared to concentrations used in earlier assays, since some toxicity was observed when it was combined with HU. These results further corroborate the ability of MG132 and HU to act through independent mechanisms that manifest as enhanced nucleolar accumulation and nucleoplasmic mobilization of AAV capsids, respectively.
The fact that AAV capsids are known to assemble in the nucleolus during replication has important ramifications when considering steps leading to nucleolar accumulation during infection (64). Two reports have identified nucleolar proteins that colocalize and immunoprecipitate with intact capsids (8, 46). These proteins, NCL and NPM1, are predominant components of the nucleolus. While traditionally known for their roles in ribosome assembly, they have also been found to exist outside of the nucleolus, having roles as chaperone proteins, and are known to relocate in response to stresses such as HU. It is clear that NCL and NPM1 are implicated in the life cycles of many viruses in addition to AAV, including human immunodeficiency virus, herpes simplex virus, and coronavirus (31). Yet, the impact of such proteins on AAV infection has not been established.
To test whether NCL and NPM1, which bind the AAV capsid during assembly, can also sequester virions in the nucleolus after nuclear entry postinfection, we knocked down NCL and NPM1 by transfection of corresponding siRNA in HeLa cells. Significant reductions of targeted protein levels were verified by Western blot analysis relative to β-actin loading controls (Fig. (Fig.6A).6A). When transduction efficiency was compared for controls, mock-transfected cells, cells transfected with scrambled nontargeted siRNA, and cells transfected with targeted siRNAs, we observed significant increases in samples with NCL or NPM1 knocked down (Fig. (Fig.6B).6B). Samples that were treated with siRNAs targeting a protein unrelated to nucleolar function, VCP, showed a slight decrease in transduction. No discernible change in capsid localization was apparent in mock-transfected samples, cells transfected with scrambled siRNA, or cells transfected with VCP siRNA compared to controls (data not shown). Remarkably, the localization of capsids in samples treated with NCL siRNA was dramatically altered and appeared similar to that in samples treated with HU (Fig. (Fig.6C).6C). In contrast, samples treated with NPM1 siRNA had prominent nucleolar accumulation of capsids, similar to what is observed during proteasome inhibition.
Many agents that augment AAV infection, such as adenovirus, herpesvirus, and genotoxic agents, are known to redistribute nucleolar proteins away from the nucleolus (37, 40, 49). By changing the localization of NCL or NPM1, these treatments might essentially be analogous to siRNA-mediated protein depletion if the primary function of these proteins in the AAV life cycle is associated with the nucleolus. Although expression levels are relatively unaffected by MG132 or HU up to 24 h after administration (Fig. (Fig.6D),6D), HU treatment resulted in partial redistribution of NCL from the nucleolus to the nucleoplasm (Fig. (Fig.6E,6E, iii). Because capsid localization appears similar in HU and NCL siRNA samples, we expect these treatments to operate through the same pathway. Since both NCL siRNA and NPM1 siRNA treatments increase transduction but they have different effects on capsid localization, we hypothesized that at least two nucleus-associated pathways can be exploited to increase transduction of rAAV2.
To test whether MG132 or HU will synergistically increase transduction in NCL siRNA or NPM1 siRNA samples, we performed luciferase assays of transduction following various combination treatments (Fig. (Fig.7).7). In support of our hypothesis, HU was found to have reduced efficacy of increasing transduction in NCL siRNA cells, suggesting that both treatments influence the same pathway. Similarly, the efficacy of MG132 appeared to decrease in NPM1 siRNA samples. Synergy was observed in samples treated with MG132 and NCL siRNA or samples treated with HU and NPM1 siRNA, which supports that these treatments operate through separate pathways in the nucleus to enhance transduction. It is noteworthy that a linear relationship between transduction efficiency and dose was observed across MOIs ranging from 200 to 20,000 vg/cell (Fig. 7A to C).
With these experiments we have sought to elucidate steps during subcellular trafficking as rAAV2 traverses from the cell surface to the nucleus. We have demonstrated that rAAV2 virions enter the nucleus intact and have explored through confocal immunofluorescence and biochemical fractionation how changing of cellular parameters influences nucleolar accumulation. Our results suggest virions that accumulate in nucleoli remain infectious and are sequestered there in stable form. These data prompt us to speculate that mobilization from the nucleolus to nucleoplasmic sites enables capsid transition from a stable environment to one where uncoating and subsequent gene expression or genome degradation can occur (Fig. (Fig.8).8). This model is particularly striking in light of the helper-dependent nature of AAV, as the virus has likely evolved to utilize nucleolar proteins for sequestration in a stable compartment and exploit nucleolar disruption during mitosis, genotoxic stress, or coinfection to trigger genome release under favorable conditions.
In these studies we employed multiple techniques to substantiate our conclusions. Initially with immunofluorescence experiments, we examined the subcellular trafficking profile of rAAV2 and detected accumulation of intact capsids in the nucleolus. We also showed through secondary infections that rAAV virions isolated from nucleoli retain infectivity. Moreover, disruption of nucleoli by genotoxic agents or siRNA knockdown of nucleolar proteins mobilizes virions and increases transduction. Our trafficking studies, like all immunofluorescence experiments, were subject to limitations with respect to detection sensitivity, as high particle numbers are required for visualization. However, we are able to detect capsid proteins in nucleoli postinfection at 10-fold-lower doses by biochemical analyses. Our conclusions are also supported by the fact that transduction assays display a linear trend between 200 and 20,000 vg/cell in control, drug treatment, and siRNA studies (Fig. (Fig.11 and and7).7). Although we cannot empirically visualize rAAV2 in nucleoli at 200 vg/cell, transduction studies strongly argue for effects on nucleolar accumulation and nucleoplasmic mobilization that are identical to those that the aforementioned treatments would have at lower MOIs.
Along with documenting accumulation of rAAV in the nucleus, a significant finding from these studies is that empty capsids are internalized into cells but cannot be detected in the nucleus, even in the presence of proteasome inhibitors (Fig. (Fig.2).2). This observation suggests that the phenotype of nuclear accumulation is linked to infectious particles, or to virions that can at least pass beyond the nuclear membrane. A potential reason for the distinct trafficking patterns observed for empty and full virions is that empty capsids fail to expose the N terminus of VP1 during infection (data not shown). Indeed, Kronenberg et al. have demonstrated similar results after limited heat treatment and have shown that full and empty capsids display different cryoelectron microscopy profiles (35). Those studies, in conjunction with our observations, suggest that empty capsids are unable to expose the phospholipase domain in VP1 that is thought to be required for endosomal escape or subsequent steps during infection. In addition to the implications regarding cell entry mechanisms, this finding poses an interesting caveat concerning vector preparations for clinical use. High numbers of empty particles in vector preparations may compete with genome-containing particles for cell attachment and uptake, effectively reducing the chances of gene delivery to the nucleus. Other effects, such as the potential for empty particles to trigger cellular recycling or degradation pathways or the possibility that empty particles saturate extracellular and/or subcellular binding sites, in turn could either inhibit or augment transduction. Thus, care must be taken to control the level of empty capsids in vector preparations and to understand their effects in laboratory and clinical studies.
Although it is unlikely, capsids detected in the nucleolus during infection could represent reassembled particles. This could occur if capsids disassembled in the cytoplasm during entry and subunits were transported into the nucleolus for assembly. Similar examples of this concept have been found in cellular systems, such as with the nuclear pore complex, which disassembles in the cytoplasm and reassembles in the nuclear membrane over the course of mitosis (34). However, for several reasons it is unlikely that the virus is subject to this system. During replication, AAV monomers and subunits can be readily detected in cytoplasm and nuclei by antibody B1 (64, 65), but in our studies and others, researches have failed to detect significant B1 staining during infection (references 38 and 57 and data not shown). Thus, the supply of free monomers and subunits would likely not be great enough to support reassembly during infection. Moreover, after isolating nucleoli from cells infected with rAAV2, we found that particles in these fractions retained infectivity (Fig. (Fig.4).4). These particles could not have reassembled during infection, since replication proteins are needed for repackaging of the genome, and these proteins are absent from current vectors. For these reasons it is unlikely that capsids detected in the nucleus represent a reassembly artifact. More importantly, the most striking observation in this study is that infectious virions are sequestered in nucleoli, supporting the existence of an “accumulation pathway” for rAAV in the nucleus (Fig. (Fig.8A8A).
Roughly 40 years ago, AAV serotypes were first shown to accumulate in nucleolar structures in the context of replication (2, 30). Our results highlight the apparent paradox of how AAV could assemble and disassemble in the same cellular location. The process of uncoating can be defined as the separation of the genome from the capsid and might not require complete disassembly of the particle. But even in this light, the prospect of virions uncoating in the nucleus during infection seems perplexing. Why wouldn't all newly assembled capsids be driven to uncoat during production? There are at least two plausible explanations for this paradox. One possibility is that during production, capsids will in fact uncoat as they are assembled. Since these processes obey principles of hysteresis (55), uncoating is likely to occur more slowly than assembly, because an external impetus is needed to overcome the collective contact energies of the subunits. A few groups have suggested that uncoating is not a rapid process for AAV2 (56, 61, 75). As progeny virions are assembled and packaged, a certain number may always be uncoating, albeit at a lower rate. Although this would explain how virions could assemble and disassemble in the nucleus, it would not be efficient from an evolutionary perspective, since unnecessary energy would be expended during replication and slow uncoating during infection would place limits on viral survival.
A second possible explanation for this paradox has been suggested by Sonntag et al. (57), relying on the premise that incoming virions undergo modifications during infection that promote uncoating. These modifications may include conformational changes, such as the extrusion of the N terminus of VP1/2 from the capsid interior to its exterior (35, 57), partial proteolytic cleavage (1, 63), ubiquitination, or phosphorylation. Considering that these changes may confer some instability to the capsid, we could expect the AAV genome to become more accessible to the cell as the virion matures during entry. Indeed, with the parvoviruses minute virus of mice and B19, viral genomes are found to be exposed during infection but prior to capsid disassembly (15, 39). It remains to be seen whether AAV genomes become more accessible after VP1 exposure or during subcellular processing; however, in such a scenario the cell could distinguish between a virion assembled in the nucleus and one entering from outside.
Exploring how drug treatments influence rAAV virions in the nucleus became a major focus of this study. Proteasome inhibitors have repeatedly been shown to increase transduction efficiency in a cell type- and tissue-specific manner (17). Fittingly, when proteasomal degradation is inhibited, we see an increase in capsid accumulation in the nucleolus (Fig. (Fig.11 and and3)3) and a decrease in the rate of genome degradation (Fig. (Fig.5A).5A). In one report, rAAV capsids were shown to accumulate in nucleoli after microinjection into the nucleus in the presence of proteasome inhibitors (57). It is unlikely that capsid degradation is occurring in the nucleolus, since no proteasome activity has been detected there (32, 47, 48). However, proteasome activity in the nucleoplasm could indirectly influence processing of AAV genomes and affect their degradation. An increase in genome availability would serve to enhance transduction intensity and also lead to more rapid transgene expression. Studies in addition to ours have demonstrated prolonged genome persistence following administration of proteasome inhibitors (18), and several reports provide evidence that interfering with proteasomal degradation increases AAV transduction primarily by improving nuclear uptake of genomes (16, 71). Certainly, both subcellular trafficking and the kinetics of transduction are positively influenced by proteasome inhibitors (Fig. 1B to D). In broad terms these results lend credence to the hypothesis that a nuclear accumulation pathway can be exploited to increase rAAV transduction (Fig. (Fig.8A),8A), but we cannot assume accumulation is due to inhibition of capsid degradation. While it is tempting to speculate that proteasome inhibitors block degradation of AAV capsids, the results from these studies, or in fact from any other report to date, do not support the conclusion that proteasomes directly operate on intact capsids in the context of infection.
Since AAV2 is known to assemble in the nucleolus and interact with nucleolar proteins (8, 46), it is intriguing that recombinant virions can be detected in this compartment after exogenous administration. In this study we have demonstrated that in addition to being involved in AAV replication, NCL and NPM1 are involved in pathways that influence rAAV2 trafficking and transduction. We found these pathways to intersect with accumulation and mobilization pathways, which are influenced by proteasome inhibitors and HU, respectively. Studies of minute virus of mice, a parvovirus related to AAV, have shown that NCL interacts directly with the viral genome during infection (4). Although we see effects on capsid localization following knockdown of NCL and NPM1, we cannot discount the possibility that these pathways affect the genome as well. Both NCL and NPM1 display single-stranded DNA and RNA binding capability (36, 72), in the context of transcription (73), DNA attachment to the nuclear matrix, or chromatin decondensation (21). NCL and NPM1 also have roles in the DNA damage response (33, 60, 72). Recent work by Cervelli et al. indicates that rAAV genome single-strand-to-double-strand conversion can be detected at nuclear foci where DNA damage response components, the MRN complex or MDC1 protein, are recruited (14). It has been demonstrated that DNA damage or genotoxic stress agents such as HU reduce the associations between rAAV genomes and these components, suggesting that machinery involved in the DNA damage response negatively affects genome processing (14, 53), which would be consistent with our results.
Understanding why some cells are readily transduced by viruses while other cells are recalcitrant to infection is a cornerstone to general virology and research pertaining to viral vectors. Evolutionarily speaking, it would be highly advantageous for a virus to lie dormant in a cellular compartment until favorable conditions arose that would promote successful gene delivery. This resonates particularly well in the case of AAV, where coinfection by a helper virus is necessary for productive replication. Perhaps helper-dependent viruses have learned to exploit nucleolar proteins to sequester themselves in the nucleolus and use nucleolar disruption during mitosis, genotoxic stress, or coinfection as a trigger to release of their genetic contents into the nucleoplasm. In support of this view, a common theme emerges when studying agents that augment rAAV infection and affect the nucleolus. Components of adenovirus bind nucleolar proteins (50), and adenovirus protein V induces the redistribution of NCL and NPM1 from the nucleolus to the cytoplasm (40). Herpesvirus, another AAV helper, also modulates the spatial distribution of NCL (7, 12, 37). Additionally, inactivation of NCL and NPM1 by siRNA results in nucleolar disruption and cell cycle arrest (26, 62), and these phenotypes are remarkably similar to those caused by genotoxic agents such as HU (49). This information corroborates our hypothesis that rAAV2 virions, initially sequestered in the nucleolus, are subject to a “mobilization pathway” (Fig. (Fig.8B)8B) whereby capsids are released from a stable, protective environment to one where the genome becomes accessible.
In summary, our results support a model wherein at least two pathways are in play to influence AAV trafficking and transduction in the nucleus, an accumulation pathway and a mobilization pathway (Fig. (Fig.8).8). In cases where negligible or low transduction is observed, we expect few if any virions reach the nucleus, and those that do may be sequestered in the nucleolus in a dormant state. This may help explain why some investigators observe a threshold effect with AAV vectors following in vivo administration (44, 66). Favorable conditions may be created during cell division or cell stress that would permit virus or vector mobilization within the nucleus to sites that promote uncoating in the nucleoplasm (Fig. (Fig.8B).8B). Externally, these conditions can be manipulated using physical or pharmacological treatments to force virions to adopt productive infectious pathways. Transduction is most dramatically potentiated under conditions where more than one pathway is engaged to overcome subcellular barriers, such as in the case of MG132 and HU cooperation or synergy (Fig. (Fig.55 and and7).7). It remains to be seen whether nucleolar trafficking is an obligatory step in the cascade of infectious events, yet it is clear that by disrupting the integrity of the nucleolus using genotoxic agents or by affecting the expression and localization of nucleolar proteins, transduction can be improved. In summary, with these studies we have refined our understanding of AAV2 trafficking dynamics, separating two pathways that accumulate or mobilize virions in the nucleus and significantly enhance AAV infection.
We thank members of the Samulski lab for productive discussions and Aravind Asokan for critically reading the manuscript. We thank Nina DiPrimio for help with generating empty rAAV2 capsids used in this study, and we thank Swati Yadav for calculating titers by quantitative PCR. Microscopy equipment and analysis software used in this study were provided courtesy of the Michael Hooker Microscopy Facility.
This research was supported by NIH fellowships 1F31NS060688-01A1 and 5F31NS060688-02.
Published ahead of print on 24 December 2008.