Impact of Adenovirus Life Cycle Progression on the Generation of Canine Helper-Dependent Vectors

ORIGINAL ARTICLE Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors

P Fernandes1,2, D Simão1,2, MR Guerreiro1,2, EJ Kremer3, AS Coroadinha1,2 and PM Alves1,2

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~ 4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus–cell interaction.

Gene Therapy (2015) 22, 40–49; doi:10.1038/gt.2014.92; published online 23 October 2014

INTRODUCTION and HV genomes replicate inside the cells, but the encapsidation Adenoviruses (Ads) are non-enveloped, icosahedral particles of the HV genome is minimized, by flanking its Ψ with recognition containing a linear, double-stranded DNA genome. Ad vectors sequences for a recombinase constitutively expressed by the cells, 11,12 13 have received considerable attention as gene transfer tools in such as FLP or Cre. which human Ad serotype 5 is the typical prototype vector Despite their enormous potential, the use of HDVs in preclinical backbone. However, the hurdles facing the clinical use of human and clinical settings is limited because of two bottlenecks: adenoviral vectors, including innate and pre-existing immunity, contamination with HV and production yields. Several efforts to may limit the efficiency and time of transgene expression.1 One of reduce HV contamination have been described. Based on the alternatives to circumvent these drawbacks is using non- alternative vector designs, deletion of the Ψ in the HV genome human Ads, such as canine adenovirus type 2 (CAV-2) vectors.2 In was improved through the development of an inducible self- addition to the paucity of anti-CAV-2 neutralizing antibodies in inactivating vector (vector expressing a recombinase that can 14 humans,2,3 their vectors induce a low level of innate response and block its own genome packaging). Moreover, tweaking the HV 15 no activation of human complement pathways.4,5 Their ability to packaging domain can delay its packaging, or reduce its 16,17 preferentially transduce neurons, combined with a remarkable packaging efficiency in co-infection contexts. Reduced HDV capacity of axonal transport,6,7 make CAV-2 vectors promising production obtained after the initial transfection step, limited by candidates for the treatment of neurodegenerative diseases.8,9 transfection efficiency and initiation of viral DNA replication from Ad genome has been progressively modified from the wild-type plasmid substrates, has also prompted others to improve the to improve vector safety and efficacy in therapeutic applications strategies. With a focus on this step, production of HDV was (reviewed elsewhere Dormond et al.10). Helper-dependent vectors improved by creating cell lines expressing pre-terminal protein (HDVs), devoid of viral coding regions, are powerful tools for gene and adenoviral DNA polymerase,18 optimizing transfection meth- transfer in vivo. HDVs combine high transduction efficacies, long- ods compliant with scalable processes9 and developing an term gene expression and a cloning capacity of 430 kb. These intracellular system to release HDV genome from transfected vectors retain only on their genomes the inverted terminal circular plasmids, which increased transfection efficiencies of repeats, the packaging region (Ψ) and an expression cassette(s). To ~10-fold.20 However, even under improved conditions, multiple produce HDVs, both early and late viral functions need to be amplification steps are needed to rescue HDV amplification to its complemented in a synchronized manner. Standard production maximum and prepare sufficient quantities of vector.14,19,21,22 methods are based on the co-infection of E1-transcomplementing Moreover, most reports show a relatively low infectivity of HDV in cells with HDV and an E1-deleted helper vector (HV), which comparison with physical particles (PPs; optical density based) provides the viral proteins in trans. During production, both HDV titers14,23 and, equally importantly, inconsistent infectious yields

1iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; 2Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal and 3Institut de Génétique Moléculaire de Montpellier, CNRS—Universities of Montpellier I and II, Montpellier, France. Correspondence: Dr PM Alves, Animal Cell Technology Unit, iBET/ITQB- UNL -Av. República, Apt. 12, Oeiras 2781-901, Portugal. E-mail: [email protected] Received 14 April 2014; revised 8 September 2014; accepted 18 September 2014; published online 23 October 2014 Impact of adenovirus life cycle on HDV generation P Fernandes et al 41 obtained in different preparations of the same HDV.14 Although characteristics of each vector (Figure 1b). CAVGFP (herein most studies were conducted with human HDVs, consistently low designated as ΔE1), harboring the same enhanced green infectious titers are also extended to canine HDVs.4,6,9 fluorescent protein (GFP) expression cassette as HD CAVGFP Ads have a regulated infection cycle where multiple viral and (herein designated as HDV), was used as the control. Moreover, cellular proteins interact to enable a productive virus replication considering that to produce HDV, cells are co-infected with HV, co- 24 and generate infectious virus particles (reviewed in Flint et al. ). infection with HV JBΔ5 was also performed in the ΔE1 production Therefore, understanding the Ad replication steps during the control assays. Finally, the propagation of HV alone was also generation of HDV particles could help break the bottlenecks performed. Thus, three infections/production scenarios in Cre- behind the productivity of these vectors. After cell binding, the expressing cells were analyzed: (i) HDV co-infected with HV (HDV mature viral particle is internalized and transported toward the +HV), (ii) ΔE1 co-infected with HV (ΔE1+HV) and (iii) HV. nucleus. During this transport, the particle is dismantled by an ordered elimination of some structural proteins and culminates with the delivery of the viral genome into the nucleus via nuclear Volumetric productivity and viral particles infectivity are impaired pore complexes.25 Once in the nucleus, early genes are expressed in HDV production and viral DNA replication starts. Late phase of infection is then To determine the productivities of HDV, production assays with activated and structural proteins expressed. These proteins are HDV+HV, ΔE1+HV and HV alone were performed (Figure 1a). likely assembled into empty virions, followed by the packaging of Vectors were harvested 48 hours post infection (h.p.i.) and viral genome. Finally, viral particles undergo a protease-mediated quantified for PPs (genome-containing capsids) and infectious maturation process to becomes infectious.26–28 particles (IPs) (Figure 2). We found a similar cell-specific In this study, we investigated the steps of Ad cycle that could be production of PPs for both HDV and ΔE1 vectors (Figure 2a). affected during HD CAV-2 production. As CAV-2 vectors use keeps However, fewer infectious HDV particles were detected. Con- increasing,8,9 larger amounts of high-quality vectors to conduct comitantly, similar PP/cell of HV was obtained in the three preclinical and possibly clinical assays are required. Therefore, infection schemes, but at a lower number of IP in HDV+HV- understanding HDV productivity is critical to identify and hope- producing profile (Figures 1a–i and 2b). From 24 h.p.i. onward, the fully overcome the manufacturing limitations. Here, we provide viability of HDV+HV-producing cells was half of that obtained for evidences of a distinct replication cycle progression during HDV both control infections (Figure 2c), which likely affected the production, which resulted in changes in the cell physiology and volumetric productivity (Figure 2d). vector productivity. Cell binding and trafficking into the nucleus does not affect HDV RESULTS production We initially asked whether the limitations in HDVs production As mentioned above, there are several steps in vector amplifica- were related to differences in Ad propagation kinetics. To address tion. To address the capacity of binding, trafficking and genome this possibility, HD CAVGFP production was characterized and the delivery of HDV, the progression of infection was monitored in main steps of Ad life cycle were evaluated by comparing it with extracellular and intracellular fractions. The decrease of extra- CAVGFP (an E1-deleted vector) and JBΔ5 (a HV containing a loxP cellular vector concentration (that is, culture medium) typically flanked packaging domain). Figure 1 depicts the experimental occurs during the first 24 h.p.i.29 and can be used to quantify design used in this study (Figure 1a) and the relevant binding and/or internalization. In parallel, intracellular genome

Figure 1. (a) Experimental design of the infections performed in this work. (b) Viral vector genome construct that should be considered in each infection scheme. The ampliﬁcation of HDV was analyzed by comparison with ΔE1 and HV, and three infection/production assays were performed: (i) HDV, (ii) ΔE1 (which share same expression cassette than HDV), and (iii) HV. The production of HDV requires co-infection of HDV and HV. As a control of co-infection, ΔE1 assays were performed by co-infecting producer cells with HV. To isolate the role of HV, the third group of production assays was performed by infecting producer cells with only HV. HDV or ΔE1 production assays mentioned throughout the text correspond to (i) HDV+HV or (ii) ΔE1+HV co-infections, respectively. MDCK-E1-Cre cells were used as producer cells and infected using MOI 5 of HDV and ΔE1 and MOI 1 of HV. Ψ, packaging sequence.

Figure 2. Characterization of HDV production. (a) Cell-speciﬁc productivity of physical (genome-containing) and infectious HDV and ΔE1 vectors and the PP:IP ratio. *P = 0.007, given by a single factor analysis of variance (Anova). (b) Cell-speciﬁc productivity of HV and the PP:IP ratio in the three infection schemes. *P = 0.03, **P = 0.01, given by a single factor Anova analysis. (c) Changes in viable cell concentration of HDV − , ΔE1 − and HV-producing cells over time. Viable cell concentration was compared by calculating the fold-change in viable cell number between HDV+HV and ΔE1+HV or HV infections. *Po0.05, given by a single factor Anova analysis against the corresponding cell concentration values of ΔE1+HV and HV infections. (d) Volumetric productivity (PP ml–1) obtained 48 h.p.i. for each viral vector in the three infections. Values are shown as average ± s.d. (n = 3).

accumulation can be accurately measured until the vector Replication of vector genome increases in HDV-producing cells replication starts at approximately 8 h.p.i. After the delivery of viral genomes into the nucleus, transcription of To evaluate the extracellular fraction, IPs during HDV+HV early region gene products and viral DNA replication are the production were quantified from culture medium at 0, 12 and subsequent steps in Ad propagation. To analyze whether viral DNA 24 h.p.i. and the profiles were compared with ΔE1+HV production. replication was altered in HDV+HV-producing cells, intracellular viral fi Δ We found that the pro les of HDV and E1 vectors were similar genomes were quantified from 12 to 48 h.p.i. (Figure 4). Similar (Figure 3a), which suggests that the ability to bind cells was quantities of vector genomes were obtained in the three vector maintained in HDV. production scenarios at 48 h.p.i. (Figures 4a and b). However, the fi The progression from binding to internalization and traf cking quantity of HV genomes in HDV+HV-producing cells (scenario (i) was evaluated by quantifying viral genomes from intracellular from Figure 1a) was higher from 12 to 24 h.p.i., especially at 15 h.p.i.: fractions collected at 3, 6 and 9 h.p.i. in the three infection increases of a 10- and 71-fold were observed when compared with scenarios (Figure 1a). To analyze vector genomes reaching ΔE1+HV − and HV-producing cells, respectively (Figure 4b). These nucleus, nuclear extracts were collected 8 h.p.i. We found that data indicated that HV genome replication occurred faster in HDV the intracellular accumulation of vector genomes was similar for Δ +HV-producing cells. HDV and E1 at the three time points (Figure 3b). Also, we found The transgene expression in terms of mRNA was also evaluated similar levels of HDV and ΔE1 viral genomes in nuclear extracts as an indicator of transcription capacity because to some extent (Figure 3c), indicating that these steps were not affected during may represent the immediate-early transcription. These showed HDV+HV production. To complement these assays, the intracel- lower levels of GFP and slightly higher levels of β-galactosidase in lular accumulation of HV genomes was also evaluated. We found HDV+HV-infected cells when compared with ΔE1+HV and HV similar intracellular HV genomes at 3 and 6 h.p.i. in all production infections (Supplementary Material Figure S1). schemes. However, a slight increase in HV genomes in HDV+HV- producing cells compared with ΔE1+HV and HV productions was observed at 9 h.p.i. (Figure 3d). As vector genome replication Expression of genes implicated in viral DNA replication and late starts at ~ 8 h.p.i., this observation may be connected to phase is observed earlier in HDV-producing cells differences in HV genome replication in HDV+HV production Although early-phase genes are primarily transcribed before scenario, rather than to the capacity to infect cells. genome replication and the corresponding mRNA is transcribed

Figure 3. Characterization of HDV infection and viral genomes reaching cell nucleus. (a) IPs in cell supernatant after infection. (b) Intracellular genomes of HDV and ΔE1 vectors during infection time. Values were normalized to intracellular viral genomes obtained at 3 h.p.i. in control ΔE1 infection. (c) Viral genomes in cell nucleus at 8 h.p.i. (d) Intracelullar genomes of HV in HDV+HV, ΔE1+HV and HV infections during infection time. Values were normalized to intracellular viral genomes obtained at 3 h.p.i. for control HV infection. The legends HDV+HV, ΔE1+HV and HV correspond to infections schemes depicted in Figure 1. Values are shown as average ± s.d. (n = 2).

Figure 4. Analysis of viral genomes replication of viral vectors in each infection scheme. (a) HDV and ΔE1 genomes. (b) HV genomes in each infection. Values were normalized to intracellular viral genomes obtained at 3 h.p.i. for control ΔE1+HV and HV infections. Values are shown as average ± s.d. (n = 2). from the input genomes, late region transcription becomes robust choose at least two viral genes from early and late phases, and once DNA replication starts. To analyze whether the expression of viral genes involved in processes that occurred differently during viral genes was altered during HDV production, transcription and HDV production, namely those involved in DNA replication (E2 translation were analyzed. region)30 and those involved in capsid maturation (protease and Transcription analysis was performed by determining mRNA pVI).28,31 In addition, IVa2 was analyzed because of its role during levels from several regions to have representatives of the different mid-late phase in activating late region expression 32 and later as infection phases. Table 1 describes the viral genes analyzed and key factor in viral genome packaging.33 the corresponding infection phase and biological role. Viral genes Regardless of the phase, the expression of all viral genes to be analyzed were selected according to two main criteria: increased from 12 to 48 h.p.i. (Supplementary Table S2).

© 2015 Macmillan Publishers Limited Gene Therapy (2015) 40 – 49 Impact of adenovirus life cycle on HDV generation P Fernandes et al 44 and ΔE1+HV-producing cells (Figure 7b). By comparison, the Table 1. Viral transcripts analyzed and the corresponding infection quantity of HV-free viral genomes was similar in the three infection phase and biological function on adenovirus amplification schemes. More free viral genomes should be obtained for HV than Δ fi Target Phase Main Role for HDV or E1 vectors, as Cre recombinase speci cally impacts HV genome packaging. This observation was not clear in Figure 7b, Adpol Early (E2) Genome replication30 probably because of the considerable inter-assay variability DBP Early (E2) Genome replication30 (depicted by the error bars) that disguised the expected differences pTP Early (E2) Genome replication30 Δ 55,56 between free genomes percentage of HV and HDV or E1 vector. E4 Orf3 Early (E4) mRNA transport Together, these results showed that despite more viral capsids E4 Orf6 Early (E4) mRNA transport55,56 32 generated, the percentage of packaged HDV genomes did not IVa2 Middle and Activation of late gene expression Δ Late and genome packaging33 increased. Notably, the total DNA of HDV and E1 were similar at Protease Late (L3) Maturation28,31 48 h.p.i. (Figure 4a). pVI Late (L3) Maturation28,31 55 II (hexon) Late (L3) Capsid protein Low cell viability during HDV production is linked to activation of Abbreviations: Adpol, adenoviral DNA polymerase; DBP, DNA-binding autophagy and high levels of viral proteins affecting virus protein; pTP, pre-terminal protein. infectivity When compared with non-infected cells, a 3.32 ± 0.23-fold increase in necrotic cells was obtained at 24 h.p.i. during HDV Notably, we found earlier and higher levels of viral transcripts in production, versus 1.74 ± 0.15 and 1.42 ± 0.01 for ΔE1 and HV, HDV+HV-producing cells. To better understand the gene expres- respectively, which is consistent with the cell viability analysis sion pattern and the corresponding percentage of each viral (Figure 2d). To further understand the cell death during HDV product in the different infections, gene expression values were production, we evaluated the effect of HDV infection on cell further normalized to the overall expression at each time. After viability, the activation of autophagy, an Ad-related programmed this normalization, the results showed the switch of expression cell death,35,36 and the role of virus cycle progression on producer pattern from early-phase (12 h.p.i.) to late-phase transcription (24 cells viability and viral productive outcome. and 48 h.p.i.) (Supplementary Figure S2). Also, at 24 and 48 h.p.i. To determine the effect of infection on cell death, HDV the expression pattern was similar and thus proportional in the infections were performed in MDCK-E1-Cre cells and MDCK cells different infections scenarios. However, an over-representation of at same multiplicity of infection (MOI) (Figure 1a-i), which do not E2 (E2B), IVa2 and L3 gene expression was observed at 12 h.p.i. express the CAV-2 E1 region and are therefore unable to allow (Figure 5) in HDV+HV-producing cells, when compared with ΔE1 Δ − productive infectious cycle of E1 CAV-2 vectors. At 48 h.p.i., +HV and HV-producing cells. This indicates that, in addition to MDCK cell viability was maintained, indicating that HDV transduc- the viral genes involved in genome replication (adenoviral DNA tion (binding, trafficking and delivery of vector genome to the polymerase and pre-terminal protein),30 late-phase activator 32 nucleus) per se did not induce cell death (data not shown). (IVa2) and late genes (L3) were also differentially expressed The decay in cell viability (Figure 2c) should be connected to an during HDV production, earlier in infection. Ad-specific programmed cell death. Autophagy, a form of To evaluate translation, western blot analysis was performed programmed cell death induced by Ads,35,36 was therefore using rabbit sera that detects capsid proteins against cell extracts evaluated via the detection of LC3-incorporated autophagosomes. obtained at 12, 24 and 48 h.p.i. In accordance to transcription One of the major hallmarks of activation of autophagic process is analysis, the results showed that higher production of viral proteins the formation of cellular autophagosome puncta containing LC3-II. was obtained earlier during HDV+HV production (Figure 6). We found an increase in LC3-II autophagosomes in HDV+HV- producing cells (Figure 8). While HDV+HV-producing cells Despite more assembled particles during HDV production, presented 16 ± 4% autophagy-positive cells, ΔE1+HV-producing packaging occurs similarly to ΔE1 control cells presented 2 ± 1%, corresponding to an eightfold difference. Once the translation of late region transcripts is accomplished, the HV-producing cells or mock-infected cells did not present structural proteins are assembled into capsids and genomes are detectable autophagosome-like structures in the six analyzed packaged to generate full virions. However, when producing Ad sections. Moreover, the number of autophagosomes per vectors, two types of viral particles are routinely produced: autophagy-positive cells was higher in cells producing HDV+HV assembled genome-containing or empty capsids, which are (Figure 8). This indicates that autophagy was more strongly assembled particles with little or no viral genome.34 Therefore, induced during the production of these vectors. to understand the type of particles generated during HDV To determine if cell death was due to the viral cycle progression, production, assembly and packaging were further analyzed. the intracellular environment observed during HDV+HV production Assembly was analyzed by quantifying the assembled viral (high levels of HV genomes and viral proteins earlier in time) was particles obtained 48 h.p.i. using the NanoSight technology, which induced during ΔE1 production by increasing the MOI of HV measured assembled particles, the sum of empty capsids and full (Figure 9). Forty-eight h.p.i., cell concentration and viral proteins virions. In addition to a positive correlation between the number levels were determined. Consistent with an increase in viral proteins of assembled viral particles and viral proteins expression in each (Figure 9a), we found a decrease in cell viability when the HDV+HV infection (Figure 6), we found more assembled viral particles in production scenario was pursued (Figure 9b). Furthermore, to HDV+HV production (Figure 7a). analyze the effect of this scenario on production of viral vectors, PP Genome packaging was evaluated by comparing free viral and IP were quantified in the above-mentioned assay (Figure 9b). genomes with total viral genomes (free genomes plus packaged The results showed a relation between cell death and low viral genomes) at 48 h.p.i. To do so, viral DNA was extracted from particles infectivity (increase in PP:IP ratio). However, although a intracellular samples in duplicate, in which one replicate was 1.7 ± 0.3-fold increase in PP:IP ratio of ΔE1 vectors was obtained subjected to benzonase treatment prior DNA extraction; the when co-infecting cells with a HV MOI 40, fewer viable cells decrease of quantified viral genomes in the samples treated with compared with those observed during HDV+HV production were benzonase before DNA extraction, in comparison with non-treated observed. Moreover, even under these circumstances, the decrease sample, indicates the presence of free viral genomes. We found no in viral particles infectivity did not reach the fourfold change when significant changes in the free viral genomes obtained in HDV+HV − comparing HDV with ΔE1 vectors (Figure 2a).

Figure 5. Fold-change in gene expression pattern between HDV+HV and ΔE1+HV (a) and HDV+HV and HV (b) of the different viral genes. Gene expression pattern values, representing the contribution of each viral target to the overall expression, were calculated as the percentage of mRNA level of each viral gene relative to total mRNAs levels obtained at each infection point. Only fold-changes 42 were considered relevant. Values are shown as average ± s.d. (n = 2).

Despite the difficulty in comparing the titers described in the literature, because of the use different protocols, vectors and samples purity, cell-specific productivity of HD CAV-2 vectors (~105 PPs per cell (Figure 2a)) was similar to the production yields reported for human HDV.14,21,22 On the other hand, a ~ 4-fold decrease in IPs of HDV was observed when compared with ΔE1 control vectors (Figure 2a). Canine HDV preparations with higher PP:IP ratios than those obtained with ΔE1 vectors, being already reported,4,9 were consistently obtained in our hands (data not shown). Some studies show a relatively high and/or inconsistent PP:IP ratios among different preparations of same human 14,23 Figure 6. Western blot of cell extracts obtained in each infection HDV. Special attention must be paid to this, as the PP:IP ratio scheme. Cell extracts were normalized to cell concentration and of clinical grade Ads is limited by Food and Drug Administration. each lane was loaded with same sample volume. Image corresponds In addition, we found a reduction in HDV volumetric productivity to a single membrane. β-Actin blot (loading control) was obtained related with an higher producer cell death (Figures 2c and d). by cutting a fragment, which included the 42 kDa size region from To further understand the limitations in HDV amplification, we the original membrane, using the bands of SeeBlue Plus2 Pre- analyzed virus cycle during vectors production. We found that Stained Protein Standard (Invitrogen) as reference. After detection, HDV has a distinct viral cycle progression, which impaired Magic MagicMark XP Western Protein Standard (Invitrogen) was used as molecular weight marker. For a matter of clarity, blot is producer cell homeostasis and vector production and quality. shown cropped. Despite similar internalization and nuclear delivery (Figure 3), a faster vector genome replication of HV from 9 h.p.i. onward was obtained during the co-infection of HDV+HV (Figures 3d and 4b). Any HV-specific effect was ruled out because this was not DISCUSSION observed in HV-producing cells (Figure 1a-iii). Although The production of HDVs presents two important bottlenecks: HV an increase in HV genome replication was observed during contamination and production yields. Several efforts toward the co-infection with ΔE1 vector production (Figure 1a-ii), HV 11–15 reduction of HV contamination and the improvement of genome replication was even faster in HDV+HV-producing cells 18–20 transfection step have been described, however, less atten- (Figure 1a–i). The viral proteins involved in Ad genome replication, tion has been paid to the overall production of HDV. More DNA-binding protein, adenoviral DNA polymerase and specifically, as the interest in CAV-2 vectors for gene therapy pre-terminal protein belong to E2 transcription unit.30 Indeed, increases,8,9 understanding canine HDV productivities is important an upregulation of subunit E2B products was observed in to accomplish a consistent production system, streamline the HDV-producing cells (Figure 5), which was consistent with the bioprocess and debottleneck high-quality preparations. replication profile of HV genome in these conditions. Why this

Figure 7. Assembled viral particles (a) and free viral genomes (b) obtained in each infection at 48 h.p.i. Values are shown as average ± s.d. (n = 2).

Figure 8. Detection of autophagy activation through LC3-integrated autophagosomes (shown as light blue spots) in infected cells 24 h.p.i. White arrowheads point to LC3B-positive cells with punctuate pattern indicating the integration of LC3 into the autophagosomes. High magniﬁcation inset represents the region indicated by the white square. Scale bars: 50 μm, 10 μm for high-magniﬁcation inset.

increase occurred during HDV+HV production, and the mechan- form of programmed cell death.42,43 Recent studies link ism behind this upregulation, is still not clear. First, the activation autophagy to the main mechanisms for Ad-mediated cell of early-phase transcription is amplified by E1 (E1A) expression.37 lysis.35,36 This type of programmed cell death was therefore more However, no differences were detected in E1 levels in the different activated during HDV+HV production, which may be implicated in infections assayed in this work (data not shown). The mechanism a critical physiological state. Still, autophagy may be necessary for behind the upregulation of E2B gene products appears to be a successful Ad replication.36 Together, our results are in line with specific, because this was not observed in E2A subunit, which the importance of a balanced autophagy activation to pursue share same promoter, or E4. Unlike HV, the replication profile of both a sustainable host cell homeostasis and an adequate HDV genome was more modest and in accordance with that environment for virus amplification and maturation. obtained in ΔE1 control vector infection (Figure 4a). Assuming that Consistent with the overall increase in viral protein production genome replication was upregulated in HDV+HV-producing cells, in HDV+HV-producing cells, more viral capsids (empty plus this may indicate that the stuffer sequence, besides affecting genome-containing) were obtained when compared with ΔE1 transgene expression (Supplementary Figure S1) as previously +HV-infected cells (Figure 7a). This demonstrates that assembly described,38,39 may impact HDV genome replication. occurred in proportion to the viral protein content (Figure 6). Faster HV genome replication contributed to an increase in Given the similar number of physical (genome-containing) expression of viral products, namely IVa2 and late-phase proteins particles between HDV and ΔE1 infection (Figure 2a), this also (Figures 5 and 6). IVa2, expressed during DNA replication, is indicates that more empty and/or defective particles were implicated in the activation of major late promoter during obtained during HDV+HV production. Assembly of Ad particles late-phase infection.32 Thus, upregulation of IVa2 can be proceeds packaging: the Ad genome, along with packaging correlated with the overexpression of late-phase transcripts and proteins, interacts with structural and non-structural proteins proteins. Such high levels of viral proteins were likely responsible resulting in the formation of a ‘procapsid’ structure with the viral for the increase in cell death during HDV+HV production, which DNA.44 Then, the genome is encapsidated. The quantification of compromised the volumetric productivity (Figures 2c and d). DNA-containing particles showed that HDV packaging occurred Concomitantly, activation of cell death was more pronounced similarly to control infections (Figure 2a). Empty capsids and free in HDV+HV-producing cells, in which autophagy was evident in viral genomes were obtained in all of the assayed productions these cells (Figure 8). Autophagy, in addition to its role in schemes (Figure 7). This indicates that a threshold for packaging degradation and recycling of cellular constituents,40,41 can act as a was obtained, even under higher levels of viral transcripts

Gene Therapy (2015) 40 – 49 © 2015 Macmillan Publishers Limited Impact of adenovirus life cycle on HDV generation P Fernandes et al 47 intracellular environment similar to HDV+HV-producing cells was induced in ΔE1-producing cells. Similar protein levels and cell death profiles to those obtained during HDV production were detected using this strategy (Figure 9), consistent with a correlation of cell death with viral protein content. However, the impact of earlier cell death on virus maturation is only partial, because the differences in ΔE1 infectivity (PP:IP ratio) (Figure 9b) did not reach the fourfold decrease observed in HDV (Figure 2a). This suggests that the physiological state of HDV+HV-producing cells was not fully simulated using the increasing HV MOI strategy. In fact, higher levels of autophagosomes were always obtained in HDV+HV-producing cells, even in cells co-infected with ΔE1 and HV at MOI 40 (data not shown), which supports the hypothesis of a HDV-specific interaction with producer cell. In addition, other intrinsic feature of HDV may also be implicated in maturation and viral particles infectivity. The maturation process is thought to be independent of DNA sequence,27 thus the effect of HDV genome sequence should be ruled out. However, it is not known how viral genome structurally interacts with core proteins, and if that affects viral capsid assembly and subsequent maturation. In the light of these results, the role of stuffer sequence and the clarification of HDV structure would be important to understand any connection with viral particle maturation state.26 In summary, in HDV+HV-producing cells viral genome replication occurred faster, leading to higher levels of late region proteins. Such high viral protein content was consistent with increased cell death observed during production. Despite similar cell-specific productivities of total HDV and ΔE1 virions, infectious to PPs ratios obtained in HDV were four times lower, indicating lower infectivity. Increased levels of empty capsids and defective Figure 9. Production of ΔE1 vectors by inducing the intracellular particles interfere with the infectious titers. Therefore, factors environment obtained in HDV-producing cells at 48 h.p.i. The affecting the maturation process should be involved. For instance, intracellular environment of HDV+HV-producing cells was induced it is likely that the cell physiological state contributed to reduced by co-infecting cells with MOI 5 of ΔE1 and increasing MOIs of HV. vectors infectivity. Nevertheless, a particular feature of HDV must a ( ) Western blot of cell extracts to compare production of viral be further affecting its maturation, which would fully explain the proteins. Cell extracts were normalized to cell concentration and differences in infectious titers. each lane was loaded with same sample volume. Cell extracts of fi HDV+HV-producing cells were added as a control to show the level This work identi es faster genome replication, viral protein of proteins produced under these conditions. Image corresponds to synthesis, higher production of defective particles and lower a single membrane and was processed as described in Figure 6. maturation as bottlenecks in helper-dependent CAV-2 production, (b) Fold-change on viable cell concentration and PP:IP ratio relative highlighting the effect of vector design on the virus replication to co-infection with MOI 1 of HV. Values are shown as average ± s.d. cycle and virus–cell interaction as an important feature to (n = 2). understand the viral vector productive outcome. involved in packaging, namely IVa2 (Figure 5),33 L1 52/55K,45 L4 46 47 MATERIALS AND METHODS 22K and IIIa, as observed during HDV+HV production (data not Cell lines and culture medium shown). Together, these observations raise the hypothesis that MDCK (ECCAC, Nr 84121903) and MDCK-E1-Cre cells51 were grown in post-transcriptional events or the levels of host cell factors, such as ’ fi ’ 48 fl Dulbecco s modi ed Eagle s medium (Gibco, Paisley, UK) supplemented P-complex, may in uence genome packaging in a rate limiting with 10% (v/v) fetal bovine serum (Gibco). Cells were maintained at 37 °C in manner. a humidified atmosphere air containing 5% CO2. Following assembly and packaging, Ad undergoes a final Cell concentration and viability were determined by the Trypan blue maturation step driven by the virus-encoded protease. This exclusion method using a 0.1% (v/v) solution prepared in phosphate- process, involving the cleavage of several viral proteins and buffered saline (PBS) and counting cells in a Fuchs–Rosenthal haemacyt- increased by the presence of two co-factors (C-terminal of pVI and ometer (Brand, Wertheim, Germany). viral double-stranded DNA), renders virus particles infectious.28,49 High levels of pVI and protease transcripts were obtained during Vectors HDV+HV production (Figure 5), however, the low infectious titer of CAVGFP, JBΔ5 and HD CAVGFP are vectors derived from CAV-2 strain these vectors suggests a defect in capsid maturation. On the other Toronto A 26/61, GenBank J04368. CAVGFP2 and HD CAVGFP7 are hand, late-phase viral proteins produced earlier may compromise E1-deleted (ΔE1) and HD vectors, respectively, and contain a cytomega- cell homeostasis and the success of virus replication progress. For lovirus–enhanced GFP expression cassette. JBΔ5 is a HV containing loxP instance, protease is also responsible for the cleavage of cell flanking the packaging domains and a RSV-lacZ expression cassette.7 cytokeratin system, weakening mechanical integrity of the cell and CAVGFP, JBΔ5 and HD CAVGFP viral stocks were prepared and purified by 2,51 promoting host cell lysis late in infection.50 Thus, such high levels CsCl gradients as described previously. of protease earlier (or any late-phase product) may contribute to impair cell integrity in a way that maturation step is not fully Gene expression analysis accomplished. Taking this into account, we hypothesized a link To assess gene expression, total RNA was extracted from 1 × 106 cells using between cell death/physiological state imposed by HDV infection RNeasy mini kit (Qiagen, Germantown, MD, USA). In infected cells, two and impaired virus maturation. To test this possibility, an rounds of RNA cleaning were performed. For real-time PCR, RPL-22 was

© 2015 Macmillan Publishers Limited Gene Therapy (2015) 40 – 49 Impact of adenovirus life cycle on HDV generation P Fernandes et al 48 chosen as a control gene. Reverse transcription of total RNA was permeabilized with 0.1% (v/v) Triton X-100 solution (Sigma-Aldrich) and performed according to Transcriptor High Fidelity cDNA Synthesis kit processed as described previously.53 Briefly, after blocking with 0.2% (w/v) (Roche Applied Science, Mannheim, Germany) protocol for complementary fish skin gelatin, samples were incubated with anti-LC3B antibody (Sigma- DNA (cDNA) synthesis using 1 μg of total RNA and oligo dT primer for total Aldrich), diluted to 1:100 in 0.125% (w/v) fish skin gelatin+0.1% (v/v) Triton mRNA reverse transcription. The reverse transcribed cDNA was aliquoted X-100 in PBS. Alexa Fluor 647 Goat Anti-Rabbit IgG (H+L) antibody and stored at − 20 °C until further processing. SYBR Green I dye chemistry (Invitrogen, Carlsbad, CA, USA) was used as secondary antibody diluted to was used to detect the PCR products using LightCycler 480 SYBR Green I 1:500. Cell nuclei were counter stained with ProLong Gold antifade reagent Master (Roche Applied Science) according to the manufacturer’s instruc- with 4,6-diamidino-2-phenylindole (Invitrogen). Samples were visualized tions using LightCycler 480 Real Time PCR System (Roche Applied Science). using fluorescence microscopy (DMI6000, Leica Microsystems GmbH, cDNA samples were run with a reverse transcription reaction-free control Wetzlar, Germany). Images were processed using the open source ImageJ and a sample without cDNA template. The primers used for each target are software (Rasband, WS, ImageJ, U. S. National Institutes of Health, shown in Supplementary Table S1. Bethesda, MD, USA, http://imagej.nih.gov/ij/, 1997–2012.). Autophagy- positive cells were quantified by counting the number of cells presenting fi Viral vectors amplification studies autophagosomes in relation to the number of stained nuclei. Quanti ca- 4 –2 tions were performed in two biological replicates, using at least three MDCK-E1-Cre cells were seeded at 1.5 × 10 cells cm , infected the day different sections per replicate. after with medium exchange. Unless stated otherwise, HD CAVGFP production assays were performed using MOI 5 and MOI 1 of HD CAVGFP and JBΔ5, respectively. As control, CAVGFP production assays were Western blot analysis performed by co-infecting JBΔ5 under same conditions. Production assays Cell protein extracts were obtained from 1 × 106 cells using 500 μlof using single infection of JBΔ5 with MOI 1 were also performed to isolate mammalian protein extraction reagent M-PER (Thermoscientific, Rockford, the role of this vector on production kinetics. Intracellular samples were IL, USA) according to the manufacturer’s instructions. Equal volumes of collected using triton (Sigma-Aldrich, St Louis, MO, USA) 0.1% (v/v) in Tris sample were subjected to sodium dodecyl sulfate–polyacrylamide gel HCl, clarified at 3000 g for 10 min at 4 °C and stored at − 85 °C until further electrophoresis gel according to NuPAGE SDS-PAGE gel system (Invitro- analysis. gen), and transfered to Hybond-C extra membranes (GE Healthcare, Uppsala, Sweden) using Trans-blot turbo transfer system (Bio-Rad, Infectious vectors titration Hercules, CA, USA). Membranes were blocked with 5% (w/v) dry milk in tris-buffered saline-Tween 20, and probed with polyclonal rabbit anti- Quantification of infectious HD CAVGFP and CAVGFP was performed by CAV-2 primary antibody54 and anti-β actin abcam8226 (housekeeping monitoring the expression of GFP, whereas titration of infectious JBΔ5 control). Both antibodies were diluted to 1:1000. Western blot membrane vectors was based in lacZ gene expression and β-galactosidase activity as detection was performed using horseradish peroxidase-linked antibodies described previously.51 and Amersham ECL (GE Healthcare) reagents and protocols.

Viral genomes and PPs by real-time PCR Nuclei extracts Viral genomes were extracted and purified by High Pure Viral Nucleic Acid Cells at 80% confluence in four 150 cm2 T-flasks were infected and Kit (Roche Diagnostics, Penzberg, Germany). SYBR Green I dye chemistry harvested 8 h.p.i. in cold PBS using a cell scraper. The extract from nuclei was used to detect PCR products using LightCycler system. Quantification were prepared by adapting the protocol provided in CelLytic NuCLEAR was based on the detection of GFP (for HD CAVGFP and CAVGFP) or extraction kit (Sigma-Aldrich). Briefly, cells were centrifuged and lacZ (for JBΔ5). Previously purified and quantified plasmids coding for suspended in Lysis buffer (5 mM Tris HCl, pH 7.5 with 1 mM MgCl , E1-deleted CAV-2 genome, GFP or lacZ were used as standards. To ensure 2 1.5 mM CaCl and 1 mM DTT), then cells were let to swell on ice for 15 min the removal of free viral genomes from samples and accurately quantify 2 and disrupted using a syringe with a narrow-gauge needle (no. 26). After viral PPs, a benzonase treatment was performed before DNA extraction by –1 the suspension was centrifuged during 20 min at 11 000 g, and the crude adding 2 μl of benzonase (250 U μl ) (Merck Millipore, Darmstadt, nuclei pellet suspended in extraction buffer (20 mM HEPES pH 7.9, with Germany) to the same sample volume and incubating for 1 h at 37 °C. Benzonase was inhibited using the proteinase K step of DNA extraction 1.5 mM MgCl2 and 0.42 M NaCl, 0.2 mM EDTA, 1 mM DTT and 25% (v/v) protocol. GFP and lacZ primers are shown in Supplementary Table S1. glycerol). Finally, 0.1% (v/v) of Triton (Sigma-Aldrich) in Tris HCl was added, the suspended nuclei was agitated for 30 min and centrifuged for 3000 g during 10 min at 4 °C. Extracts were stored at − 85 °C. Viral genomes were Quantification of assembled capsids isolated from nuclei extracts using the High Pure Viral Nucleic Acid Kit After confirming that virions and empty capsids were quantifiable through (Roche diagnostics) and quantified as stated above. Nanoparticle Tracking Analysis (NTA), assembled capsids were titrated using LM10 instrument (NanoSight, Amesbury, UK). Following the manufacturer’s instructions, samples were diluted with Dulbecco’s PBS to CONFLICT OF INTEREST 8 reach a particle concentration suitable for analysis with NTA (1.0 × 10 to The authors declare no conflict of interest. 2.5 × 109 particles ml–1). At least two different dilutions were prepared for each sample and analyzed twice. The NanoSight LM10 recorded 60 s sample videos, which were then analyzed with the NTA 2.0 Analytical ACKNOWLEDGEMENTS software release version build 0125 (NanoSight). The NTA software is then We thank Sandy Ibanes and Aurelie Gennetier for the initial preparations of CAV-2 able to identify and track individual nanoparticles moving under Brownian vectors, Francisca Monteiro, Vanessa Bandeira and Rute Castro for technical support. motion and the results allow particle size and concentration to be This work was supported by Fundação para a Ciência e a Tecnologia (FCT)—Portugal, recovered.52 through the project PTDC/EBB-BIO/118615/2010, and European Commission through FP7 project BrainCAV (222992). Paulo Fernandes acknowledges FCT for his PhD grant Cell death analysis (SFRH/BD/70810/2010). Cells were seeded in 24-well plates, infected under the conditions above mentioned and analyzed for necrosis and autophagy at 20 h.p.i. Necrosis was assessed by staining cells with propidium iodide (Sigma-Aldrich). After REFERENCES 20 h.p.i., cells were collected from wells in duplicates, stained with 2 μlof 1 Lowenstein PR, Mandel RJ, Xiong WD, Kroeger K, Castro MG. Immune responses propidium iodide solution (Sigma-Aldrich) in 2 ml final volume, incubated to adenovirus and adeno-associated vectors used for gene therapy of brain dis- in the dark for 10 min and analyzed by flow cytometry using a CyFlow eases: the role of immunological synapses in understanding the cell biology of Space (Partec, Münster, Germany) equipped with a 561 nm yellow laser neuroimmune interactions. Curr Gene Ther 2007; 7:347–360. and 630 ± 22 nm filter. Non-stained and non-infected controls were also 2 Kremer EJ, Boutin S, Chillon M, Danos O. Canine adenovirus vectors: an alternative analyzed. Autophagy was evaluated through the detection of autophago- for adenovirus-mediated gene transfer. J Virol 2000; 74:505–512. somes by immunostaining. Cells grown in coverslips inside wells were 3 Perreau M, Kremer EJ. Frequency, proliferation, and activation of human memory fixed in 4% (w/v) paraformaldehyde+4% sucrose (w/v) in PBS, T cells induced by a nonhuman adenovirus. J Virol 2005; 79: 14595–14605.

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