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. ). 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.
FIG. 8. Illustrated model of two nuclear trafficking paradigms. Virions entering the nucleus are subject to an accumulation pathway (A) and a mobilization pathway (B). Low or negligible transduction occurs when virions do not enter nuclei at high efficiency. (more ...)
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. and ). 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. ). 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
), but in our studies and others, researches have failed to detect significant B1 staining during infection (references 38
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. ). 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. ).
Roughly 40 years ago, AAV serotypes were first shown to accumulate in nucleolar structures in the context of replication (2
). 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
). 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
), partial proteolytic cleavage (1
), 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
). 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. and ) and a decrease in the rate of genome degradation (Fig. ). 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
). 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
). Certainly, both subcellular trafficking and the kinetics of transduction are positively influenced by proteasome inhibitors (Fig. ). In broad terms these results lend credence to the hypothesis that a nuclear accumulation pathway can be exploited to increase rAAV transduction (Fig. ), 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
), 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
), 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
). 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
), 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
). Additionally, inactivation of NCL and NPM1 by siRNA results in nucleolar disruption and cell cycle arrest (26
), 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. ) 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. ). 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
). 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. ). 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. and ). 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.