These studies describe how subcellular stress increases susceptibility to infection with the parvovirus, AAV, in cellular models of CF. We report that AAV infection efficiency is significantly higher in HAE when cultures are stressed by exposure to SMM (), an inflammatory stimulant derived from diseased lungs that models physiological conditions present in CF airways. Granting that SMM is a pleiotropic model of subcellular stress, we demonstrate independently that expression of misfolded ΔF508-CFTR is sufficient to increase infection efficiency by 5- to 10-fold ( & ). A similar opportunistic phenomenon also occurs with the human respiratory pathogens, Ad and RSV (), which are known to have a disproportionately greater impact on CF patients than on normal individuals 
. Although it remains to be seen if the same mechanisms are responsible for enhancing virus infection in all cases, effects related to protein misfolding were primary factors involved in the increased susceptibility to AAV. Rescuing misfolded ΔF508-CFTR with low temperature conditioning blocked the elevation in rAAV2 infection efficiency. Additionally, using a panel of mutant CFTR proteins we show this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity, since the G551D-CFTR mutant did not confer increased susceptibility to AAV infection. While we did not find direct colocalization between AAV and CFTR proteins, we were able to detect virus capsids in regions of the secretory pathway that correspond to retrograde trafficking out of the Golgi apparatus. Furthermore, we have demonstrated that infection efficiency can be modulated by siRNA-mediated knockdown of components in the secretory pathway or the proteasome. With this work we have provided evidence that factors in the secretory pathway are enlisted to process misfolded proteins and protect against infection.
Manipulation and subversion of stress responses is a common thread in virus replication. Hepatitis B virus is known to activate an ER stress response in order to optimize production of its surface proteins 
. Japanese encephalitis virus infection similarly triggers ER stress and activates an unfolded protein response, thus promoting apoptosis and progeny dissemination 
. Other viruses, such as human cytomegalovirus and herpes viruses, have evolved mechanisms that activate chaperone upregulation and expand the ER, while controlling unwanted consequences of eIF2α phosphorylation, which would normally inhibit protein translation 
. In our studies, the AAV reporter virus has been stripped of its wild-type viral genome and does not replicate. Taken together with the fact that no difference was observed in ΔF508-CFTR-expressing cells with respect to virus binding, and the effect on transduction was not observed in promoter transgene cassette transfection studies (supplementary Fig. S1
), our focus became more narrowed toward studying how exploitation of subcellular stress may occur during virus entry.
A variety of drug treatments and physical stressors are known to increase infection efficiency of AAV through effects on capsid processing or genome amplification 
. It should be noted that AAV capsids are trafficked to the MTOC early during infection (supplementary Fig. S2A
), a location where misfolded proteins are targeted for degradation 
. Typically, select cytoplasmic proteins are marked for degradation by ubiquitin, a signaling hallmark that can direct either a change in substrate trafficking or promote substrate degradation via the proteasome. Previous research has demonstrated AAV serotypes -2 and -5 can be ubiquitinated during in vitro reactions and during infection 
. Likewise, pharmacological inhibition of the proteasome results in a potent increase in AAV infection efficiency in a cell- and serotype-specific manner 
, although the mechanisms responsible are not completely understood. This prompts the question of whether enhanced infection of ΔF508-CFTR cells is due to proteasome inhibition. Surprisingly, we have found that proteasomes function at relatively the same rate in ΔF508-CFTR-expressing cells as in control cells and CFTR-expressing cells, according to a luciferase assay of proteasome activity (supplementary Fig. S7A
). Yet, supporting our hypothesis, the use of proteasome inhibitors in control cells or cells expressing wild type CFTR increases AAV transduction to just over that of untreated cells expressing ΔF508-CFTR (see supplementary Fig. S7B
). Treating ΔF508 cells with proteasome inhibitors results in a partial increase in transduction that is not as effective as in control cells (a three-fold increase compared to 10-fold in controls). These data point to the proteasomal pathway as having a significant impact on viral transduction in this setting. While it is tempting to speculate that dysfunction of the ubiquitin/proteasome system is a primary factor leading to AAV's ability to exploit the stressed subcellular environment, our siRNA results () suggest that other changes in the secretory pathway are additionally at play. Together, our findings suggest the increase in susceptibility to AAV infection in ΔF508 cells is not strictly due to a direct inhibition of the proteasome, but could also be due to saturation or sequestration of pathway components upstream of catalytic action.
Maintaining homeostasis in the secretory pathway is paramount for proper folding or disposal of misfolded proteins. Throughout its biogenesis and degradation, ΔF508-CFTR interacts with a multitude of proteins in the cell 
and may influence the effectiveness of quality control machinery in the post-ER secretory pathway. One hypothesis is that during stressed conditions, expression of ΔF508-CFTR or other misfolded proteins may saturate or sequester proteins with chaperone and quality control function. AAV infection may be enhanced if these proteins would otherwise impede virus trafficking or processing. Indeed, we have identified overlapping systems that process ΔF508-CFTR and influence AAV. Proteins known to play a role in ΔF508-CFTR processing include the chaperones calnexin, Hsp90, and the Hsc-Hsp40/Hsp70 complexes 
. In one report, Hsp90 has been shown to inhibit AAV infection 
, and similarly, we see that the Hsp90 inhibitor radicicol potently increases transduction (supplementary Fig. S1B
), as does siRNA-mediated knockdown of calnexin (). Although siRNA-mediated knockdown of calnexin increases AAV transduction in HEK-293 and HeLa cells, this does not necessitate an interaction between calnexin and AAV capsids. Loss of calnexin has been shown to be associated with an increased constitutively active unfolded protein response 
. While many viruses have been shown to manipulate the unfolded protein response during infection, it is unclear whether this influences AAV transduction. It is noteworthy to mention that activating or inactivating a major axis of the unfolded protein response through IRE1/XPB-1, which is linked to ER expansion and Ca2+
-mediated inflammation in human airway epithelium 
, did not impact rAAV2 transduction in a human bronchial epithelial cell line (supplementary Fig. S8
). However, we cannot rule out that other arms of the unfolded protein response (ATF6 or PERK pathways) could be involved, or that factors in addition to, and/or superseding an unfolded protein response might be engaged to mediate increased AAV transduction during subcellular stress.
Interestingly, siRNA-mediated knockdown of HDAC6 or dynactin did not significantly impact AAV infection under the conditions tested. These proteins are putatively required for microtubule-based transport of misfolded proteins like ΔF508-CFTR to aggresomes and autophagosomes 
. In agreement with these siRNA studies, we did not find AAV capsids to colocalize with cellular markers for aggresomes or autophagosomes in HeLa cells (vimentin, atg5, atg7, LC3; data not shown). However, we cannot conclude that AAV infection is unaffected by autophagic processes, especially since we observe 3-methyladenine, an inhibitor of autophagy to increase AAV-mediated gene delivery (supplementary Fig. S1B
The most significant siRNA-mediated effect on AAV infection in these studies was found with knockdown of PSMB3 (), a proteasome subunit that has been reported to interact with ΔF508-CFTR, but not wild-type CFTR 
. PSMB3 knockdown increased the number of GFP-positive cells, but more impressively augmented expression intensity in both HEK-293 and HeLa cells. Knocking down KDEL-R or β-COP also modulates AAV infection efficiency. It is not clear what role these proteins may play in AAV trafficking, but classically KDEL-R and β-COP are retrograde transport proteins, which serve as ER-retention devices for misfolded proteins that transition between the Golgi, ER-intermediate compartment, and the ER 
. KDEL-R was recently identified as a potential AAV capsid binding partner in a yeast two-hybrid screen 
. Capsid mutagenesis studies may in the future shed light on how AAV is recognized by cellular sensors in the post-ER secretory pathway, and potentially elucidate how these factors could restrict viral trafficking. Additionally, we'd like to note that the endocytic and secretory systems in polarized cells are quite different from that in non-polarized cells. In polarized airway epithelium, the nucleus is positioned near the basolateral membrane, whereas the ER and Golgi are oriented sub-apically at the minus end of microtubules. It is currently unclear whether AAV traffics differently in polarized and non-polarized cells, and how this would pertain to viral exploitation of subcellular stress. Considering that viral exploitation of stress may not be related to a trafficking block occurring at a specific subcellular location, an attractive hypothesis is that altered capsid processing underlies enhanced transduction (such as ubiquitination, phosphorylation, proteolytic processing, etcetera).
The facets of subcellular stress discussed here, such as expansion of the ER, changes in the secretory pathway, activation of the unfolded protein response, and dysfunction of the ubiquitin/proteasome system, will likely have a profound effect on a cell's ability to combat infection. In this report we have illustrated that the consequences of these subcellular stresses, elicited by exposure to SMM or the presence of misfolded proteins, can render cells more susceptible to AAV in cellular models of CF. Irrespective of whether all of these effects contribute to pathogenesis in diseased airways, it is clear they are associated with numerous misfolded protein diseases 
. In one instance, it has been shown that adaptation to constitutive expression of misfolded surfactant protein C, led to increased susceptibility to RSV infection in vitro 
, corresponding with enhanced cytotoxicity. It will be of great interest to uncover if exploitation of subcellular stress by pathogens is a generalized phenomenon that occurs in prion diseases, Parkinson's disease (α-synuclein), amyotrophic lateral sclerosis (superoxide dismutase-1), hereditary emphysema (α-1-antitrypsin), familial neurohypophyseal diabetes insipidus (arginine vasopressin), or many other disorders that stem from misfolded or unfolded proteins 
. Even more striking, would be to discover that there is a viral component to the progression of some of these diseases. If the presence of misfolded proteins or other subcellular insult, might render certain cells more susceptible to infection, this parameter should also hold true for viral-mediated therapies. These may be important angles to consider as future research shapes new ways to understand, prevent, and provide treatment for cells under threat from internal or external stressors.