Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6, 40, 47, 70, 75, 77, 87, 93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 heterodimer. DNA-PK functions with XRCC4/DNA ligase IV to repair breaks during NHEJ, and works with Artemis to process DNA hairpin structures during VDJ recombination and during a subset of DNA DSB events (46, 50, 86). While the kinase activity of DNA-PKcs leads to phosphorylation of a large number of substrates in vitro as well as autophosphorylation of specific residues (reviewed in references 16 and 85), it is currently unclear how DNA-PKcs contributes to signaling in cells upon different types of damage.The adeno-associated virus (AAV) genome consists of a molecule of single-stranded DNA with inverted terminal repeats (ITRs) at both ends that form double-hairpin structures due to their palindromic sequences (reviewed in reference 52). The ITRs are important for replication and packaging of the viral genome and for integration into the host genome. Four viral Rep proteins (Rep78, Rep68, Rep52, and Rep40) are also required for replication and packaging of the AAV genome into virions assembled from the Cap proteins. Although the Rep and Cap genes are replaced in recombinant AAV vectors (rAAV) that retain only the ITRs flanking the gene of interest, these vectors can be replicated by providing Rep in trans (reviewed in reference 7). Productive AAV infection requires helper functions supplied by adenovirus (Ad) or other viruses such as herpes simplex virus (HSV) (reviewed in reference 27), together with components of the host cell DNA replication machinery (54, 55, 58). In the presence of helper viruses or minimal helper proteins from Ad or HSV, AAV replicates in the nucleus at centers where the viral DNA and Rep proteins accumulate (35, 76, 84, 89). Cellular and viral proteins involved in AAV replication, including replication protein A (RPA), Ad DNA-binding protein (DBP), and HSV ICP8, localize with Rep proteins at these viral centers (29, 33, 76).A number of published reports suggest associations between AAV and the cellular DNA damage machinery. For example, transduction by rAAV vectors is increased by genotoxic agents and DNA damaging treatments (1, 62, 91) although the mechanisms involved remain unclear. Additionally, the ATM kinase negatively regulates rAAV transduction (64, 92), and we have shown that the MRN complex poses a barrier to both rAAV transduction and wild-type AAV replication (11, 67). UV-inactivated AAV particles also appear to activate a DDR involving ATM and ATR kinases that perturbs cell cycle progression (39, 60, 88). It has been suggested that this response is provoked by the AAV ITRs (60) and that UV-treated particles mimic stalled replication forks in infected cells (39). In addition to AAV genome components, the viral Rep proteins have been observed to exhibit cytotoxicity and induce S-phase arrest (3, 65).The role of cellular repair proteins in AAV genome processing has also been explored by examining the molecular fate of rAAV vectors, which are converted into circular and concatemeric forms that persist episomally (18, 19, 66). Proteins shown to regulate circularization in cell culture include ATM and the MRN complex (14, 64), while in vivo experiments using mouse models have implicated ATM and DNA-PK in this process (14, 20, 72). Additionally, DNA-PKcs and Artemis have recently been shown to cleave the ITR hairpins of rAAV vectors in vivo in a tissue-dependent manner (36). Despite these studies, it is not clear how damage response factors function together and how they impact AAV transduction and replication in human cells.In this study we examined the cellular response to AAV replication in the context of Ad infection or helper proteins. We show that coinfection with AAV and Ad activates a DDR that is distinct from that seen during infection with Ad alone. The ATM and DNA-PKcs damage kinases are activated and signal to downstream substrates, but the response does not require the MRN complex and is primarily mediated by DNA-PKcs. Although expression of the large Rep proteins induced some DDR events, full signaling appeared to require AAV replication and was accompanied by accumulation of DNA-PK at viral replication compartments. Our results demonstrate that AAV replication induces a unique DNA damage signal transduction response and provides a model system for studying DNA-PK.     

A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases

It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.During the past decade protein degradation has become increasingly recognized as a critical mechanism by which cells regulate a number of fundamental processes (reviewed in references 37, 57, and 59). Degradation frequently involves one of a variety of E3 ubiquitin ligase complexes in which a substrate recognition component introduces the target protein for ubiquitination and subsequent degradation by proteasomes (reviewed in reference 59). Several types of these complexes involve a member of the cullin family (reviewed in reference 59), and a considerable amount of information is known about those containing Cul2 or Cul5. In these cases the substrate recognition module is linked via elongins B and C to a subcomplex containing Cul2 or Cul5 and the RING protein Rbx1 (34, 58). This complex interacts with an E2 conjugating enzyme, often either Cdc34 or Ubc5, to conjugate ubiquitin chains to the substrate (44). With both Cul2- and Cul5-based complexes interaction with elongins B and C occurs via a single BC box sequence (42). The presence of either Cul2 or Cul5 is generally determined through the presence in the substrate recognition protein of specific Cul2- or Cul5-box sequences (35).Many viruses have evolved to encode products that inhibit cellular E3 ligases to protect important viral or cellular species or, in some cases, that highjack these cellular complexes to target key substrates for degradation, including components of cellular host defenses, to facilitate the infectious cycle (reviewed in reference 4). These strategies are quite common among the small DNA tumor viruses (7), and one of the most studied examples is the complex formed by the human adenovirus E4orf6 and E1B55K proteins. These proteins have been known for some time to interact (69) and to reduce the levels of the p53 tumor suppressor in infected cells (14, 47, 48, 62, 72, 73). In addition, they were shown to function in concert to block nuclear export of cellular mRNAs late in infection (2, 6, 29, 60) and to enhance the selective export of late viral mRNAs (2, 26, 29, 60, 78). Our group showed that the human adenovirus serotype 5 (Ad5) E4orf6 product interacts with several proteins (13), including components of what was at the time a unique Cul5-based E3 ubiquitin ligase containing elongins B and C and Rbx1 that degrades p53 (61). Curiously, Ad5 E4orf6 contains three BC boxes that we believe make it highly efficient in highjacking cellular elongin B/C complexes (8, 17, 41). The mechanism of selective recruitment of Cul5 by the Ad5 complex remains unknown as E4orf6 lacks a Cul5-box (17, 41). E1B55K seems to function as the substrate recognition module and, of considerable interest, both its association with E4orf6 and induction of selective late viral mRNA transport was found to depend on formation of the E3 ubiquitin ligase complex, suggesting that additional degradation substrates must exist (8, 9). This idea is not surprising since viruses, especially the small DNA tumor viruses, often evolve gene products that target multiple critical cellular pathways (32). In fact two additional E1B55K-binding substrates have now been identified, Mre11 from the MRN DNA repair complex (8, 75), and DNA ligase IV (3), the degradation of which prevent formation of viral genome concatemers, thus enhancing packaging of progeny DNA. Degradation of p53 has been suggested to promote enhanced progeny virus production by preventing the early apoptotic death of infected cells due to the stabilization of p53 by the viral E1A products (reviewed in reference 66). Nevertheless, degradation of these substrates seems unlikely to explain the observed effects on mRNA transport, suggesting that still more substrates remain to be identified. Although the studies described in the present report were in part launched to identify such substrates, as will become clear below, these targets remain to be identified.In an attempt to identify new substrates of the Ad5 E4orf6/E1B55K E3 ubiquitin ligase complex, a proteomics-based approach was initiated involving two-dimensional difference gel electrophoresis (2D-DIGE) analysis and subsequent mass spectrometry. As is well known, this technique has the advantage of improved sensitivity and accuracy provided by its ability to separate samples under two different conditions on a single gel together with a reference sample, thus reducing significantly the analytical coefficient of variation. It allows the quantification of differentially abundant proteins in complex biological samples, providing a tool to detect decreases in the levels of proteins in the cell due to targeted proteolytic degradation. We report here our attempts to identify substrates of the Ad5 E4orf6/E1B55K complex by comparing the proteomes of human non-small cell lung carcinoma H1299 cells expressing, by means of adenovirus vectors, both E1B55K and E4orf6 proteins or E4orf6 protein alone. Ten candidate proteins were identified, most having functions seemingly unrelated to our current understanding of the roles of the E4orf6/E1B55K complex. At least three showed promising features characteristic of substrates, and one has now been confirmed in a parallel study to be a bone fide E4orf6/E1B55K substrate (20). We suggest that this approach could be utilized to identify candidate substrates, among relatively high abundance proteins, that are degraded by other viral cullin-based E3 ubiquitin ligase complexes.     

The Adenovirus E4orf4 Protein Induces G2/M Arrest and Cell Death by Blocking Protein Phosphatase 2A Activity Regulated by the B55 Subunit

Human adenovirus E4orf4 protein is toxic in human tumor cells. Its interaction with the Bα subunit of protein phosphatase 2A (PP2A) is critical for cell killing; however, the effect of E4orf4 binding is not known. Bα is one of several mammalian B-type regulatory subunits that form PP2A holoenzymes with A and C subunits. Here we show that E4orf4 protein interacts uniquely with B55 family subunits and that cell killing increases with the level of E4orf4 expression. Evidence suggesting that Bα-specific PP2A activity, measured in vitro against phosphoprotein substrates, is reduced by E4orf4 binding was obtained, and two potential B55-specific PP2A substrates, 4E-BP1 and p70^S6K, were seen to be hypophosphorylated in vivo following expression of E4orf4. Furthermore, treatment of cells with low levels of the phosphatase inhibitor okadaic acid or coexpression of the PP2A inhibitor I₁^PP2A enhanced E4orf4-induced cell killing and G₂/M arrest significantly. These results suggested that E4orf4 toxicity results from the inhibition of B55-specific PP2A holoenzymes, an idea that was strengthened by an observed growth arrest resulting from treatment of H1299 cells with Bα-specific RNA interference. We believe that E4orf4 induces growth arrest resulting in cell death by reducing the global level of B55-specific PP2A activity, thus preventing the dephosphorylation of B55-specific PP2A substrates, including those involved in cell cycle progression.Our research group and others have shown that the 114-residue product of early region E4 of human adenoviruses, termed E4orf4, induces p53-independent cell death in human tumor cells (24, 25, 34-36, 55) and in Saccharomyces cerevisiae (23, 53). E4orf4 protein, which shares no obvious homology with other viral or cellular products, kills a wide range of human cancer cells but is believed to have reduced activity against normal human primary cells (6, 55, 56). Although in some cases E4orf4-expressing cells exhibit characteristics typical of apoptosis, including the presence of irregularly shaped and shrunken nuclei, cytoplasmic vacuolization, and membrane blebbing (24, 25, 50, 55), cell death may more typically be independent of caspase activation (24, 25, 30, 32, 50). With H1299 human non-small-cell lung carcinoma cells, death is characterized by rapid cell rounding, enlargement, release from the surface of culture plates, cell cycle arrest in G₂/M and possibly G₁, and eventually, after an extended period, loss of membrane integrity (30). Both cytoplasmic and nuclear pathways have been observed, the former involving interactions with c-Src family kinases, activation of calpain, and remodeling of the actin cytoskeleton (7, 24, 50, 51, 58). Little is known about the nuclear pathway, which may represent the predominant death-inducing process. Our current evidence suggests that H1299 cells die following prolonged irreversible cell cycle arrest leading to mitotic catastrophe and death by a necrosis-like process (30).E4orf4 is known to associate with the Bα regulatory subunit of protein phosphatase 2A (PP2A) (22, 34), and this interaction appears to be necessary for the majority of E4orf4 toxicity in both yeast (23, 53) and human tumor cells (34, 56). PP2A is an abundant serine-threonine phosphatase involved in regulation of metabolism, splicing, translation, morphogenesis, development, and cell cycle progression (15, 19, 27, 43, 59). PP2A holoenzymes exist as multiple heterotrimeric complexes composed of a catalytic C subunit, an A subunit that functions as a scaffold, and a B-type regulatory subunit. Two forms each of the A and C subunits exist in mammalian cells; however, more than 20 B-type subunits have been identified in three unique classes (B/B55, B′/B56, B″/PR72), plus striatin/SG2NA (sometimes called B‴) (10, 19, 26). Although one group has suggested that E4orf4 protein interacts with one or more members of the B′/B56 class (57), it is generally accepted that interaction with the Bα/B55 subunit (Cdc55 in yeast) is important for induction of cell death in both human tumor cells and yeast (53, 57). Interestingly, a recent report has also suggested that in yeast, growth suppression induced by E4orf4 is mediated only in part by the catalytic C subunit of PP2A (31).In the present report, we show that E4orf4 protein interacts uniquely with members of the B55 class of PP2A B-type subunits, and at sufficient concentrations, it appears to become toxic by reducing dephosphorylation of substrates of B55-containing PP2A holoenzymes. As cell death is preceded by cell cycle arrest, we believe that key substrates may include proteins required for cell cycle progression.     

Identification of Integrin α3 as a New Substrate of the Adenovirus E4orf6/E1B 55-Kilodalton E3 Ubiquitin Ligase Complex

The human adenovirus E4orf6 and E1B55K proteins promote viral replication by targeting several cellular proteins for degradation. The E4orf6 product has been shown by our group and others to form an E3 ubiquitin ligase complex that contains elongins B and C and cullin family member Cul5. E1B55K associates with this complex, where it is believed to function primarily to introduce bound substrates for degradation via proteasomes. In addition to p53, its first known substrate, the E4orf6/E1B 55-kDa complex (E4orf6/E1B55K) was shown to promote the degradation of Mre11 and DNA ligase IV; however, additional substrates are believed to exist. This notion is strengthened by the fact that none of these substrates seems likely to be associated with additional functions shown to be mediated by the E4orf6-associated E3 ubiquitin ligase complex, including export of late viral mRNAs and blockage of export of the bulk cellular mRNAs from the nucleus. In an attempt to identify new E4orf6/E1B55K substrates, we undertook a proteomic screen using human p53-null, non-small-cell lung carcinoma H1299 cells expressing either E4orf6 protein alone or in combination with E1B55K through infection by appropriate adenovirus vectors. One cellular protein that appeared to be degraded by E1B55K in combination with the E4orf6 protein was a species of molecular mass ∼130 kDa that was identified as the integrin α3 subunit (i.e., very late activation antigen 3 alpha subunit). Preliminary analyses suggested that degradation of α3 may play a role in promoting release and spread of progeny virions.Viruses are well known to promote replication by inhibiting or enhancing endogenous cellular machinery or, in some cases, by reprogramming key cellular pathways. Human adenoviruses have developed effective ways to modulate the immune response, apoptosis, double-strand break repair, mRNA export, and translation to optimize virus replication and the spreading of progeny virions. The expression of adenovirus E1A proteins stabilizes p53 and induces apoptosis (8, 33); however, this effect is reversed in infected cells by the action of two early products: the E1B 55-kDa (E1B55K) and E4orf6 proteins (35, 36). We and others have shown that these proteins act through the formation of an E3 ubiquitin ligase complex analogous to the SCF and VBC complexes but which contains, in addition to elongins B and C and the RING protein Rbx1, the cullin family member Cul5 (18, 41, 43). This E4orf6-mediated E3 ligase complex blocks p53-induced apoptosis (35, 36) by promoting the ubiquitination of p53, followed by its degradation by proteasomes (41, 43). E4orf6 protein mediates the assembly of the complex by its interaction with elongin C through its three BC boxes (11, 41, 43). E1B55K, which appears to associate with the E4orf6 protein only when present in the ligase complex (4), is thought to function as a substrate recognition factor that brings substrates to the complex because, although both E4orf6 and E1B55K bind p53 independently, interaction of E1B55K with p53 is essential for the efficient degradation of p53 (41, 48). In addition to protecting infected cells from early lysis via p53-induced apoptosis, the E4orf6/E1B55K ligase complex performs other functions essential for virus replication. Two other substrates of the complex have been identified: a member of the MRN DNA repair complex, Mre11, and the central component of the nonhomologous end-joining DNA repair system, DNA ligase IV (2, 56). Degradation of both of these proteins prevents viral genome concatenation, which interferes with the packaging of viral DNA into virions (2, 56). E1B55K binds to p53, Mre11, and DNA ligase IV and has been demonstrated to colocalize with p53 and Mre11 in perinuclear cytoplasmic bodies termed aggresomes (1, 2, 32). More recently, we and others have obtained results that suggest that the E4orf6-associated E3 ligase complex regulates viral and cellular mRNA export (5, 66). The Cul5-based ligase activity was shown to be essential for selective viral mRNA export and the block of cellular mRNA export from the nucleus (66), thus contributing to the shutoff of cellular protein synthesis initiated by L4-100K (20). The actual substrates of the complex responsible for regulating mRNA export are currently unknown.As discussed in detail below, our efforts to identify substrates of the E4orf6/E1B55K complex led us to consider a member of the integrin family as a potential substrate. Integrins are members of a family of surface receptors that function in several ways through the formation of cell-extracellular matrices and cell-cell interactions (reviewed in references 21, 26, and 63). Integrins are typically composed of two transmembrane glycoproteins forming heterodimers of α and β subunits each of approximately 80 to 150 kDa. There are at least 18 α subunits and 8 β subunits in mammals that can dimerize in limited combinations to form more than 20 functionally distinct integrins with different ligand specificities. Integrin heterodimers function as transmembrane receptors that link external factors to intracellular signaling pathways. In addition to roles in cell adhesion, these communication events are implicated in a large range of cellular processes, including proliferation, differentiation, translation, migration, and apoptosis. Some of these processes depend on the intracellular trafficking pathways of the integrins (reviewed in references 9, 24, 40, and 44), including the long-loop recycling pathway in which integrins present in clathrin-coated endosomes move first to the perinuclear recycling center, where some accumulate, including the β1 integrin subunit (31), before returning to the plasma membrane. The integrin α3β1 is a member of the β1 integrin subfamily in which the α3 subunit (VLA-3a) is coupled to the β1 subunit to form the very late activation antigen (VLA-3 or CD49c) (21, 59, 60). α3β1 is expressed in a wide range of tissues in which it binds a variety of extracellular matrix substrates, including fibronectin, collagen, thrombospondin 1, and laminins 1, 5, 8, 10, and 11 (13). These associations allow the integrin α3β1 to fill its primary role in cell adhesion. α3β1 also participates in intercellular adhesion through several protein-protein interactions (10, 27, 53, 55, 58), making it a major contributor in the regulation of cellular adhesion.Human adenovirus type 5 (Ad5) particles interact with cell surface receptors to facilitate internalization into target cells. In the high-affinity interacting model (reviewed in reference 29), the viral fiber knob polypeptide binds the coxsackie adenovirus receptor (CAR) protein on the surface of cells as the primary cell binding event (primary receptor). The penton base polypeptide then binds a cell surface integrin (secondary receptor), leading to entry of the capsid into the cell by a process termed receptor-mediated endocytosis or clathrin-mediated endocytosis. Several types of integrins have been identified as being used by Ad5 to mediate virus internalization: αMβ1, αMβ2, αVβ1, αVβ3, αVβ5, and α5β1 (22, 30, 49, 65). Salone et al. have shown that α3β1 serves as an alternative cellular receptor for adenovirus serotype 5 (49). It promotes entry of the virus into cells, transduction of DNA, and mediates adenovirus infection in both CAR-positive and CAR-negative cell lines. Thus, in addition to functions related to cell adhesion, integrin α3β1 plays an important role in the adenovirus infection cycle.To identify new targets for degradation by the E4orf6/E1B55K ubiquitin ligase, we used a proteomic screen covering most cellular proteins to look for any polypeptide that exhibited a significant decrease in amount following the coexpression from appropriate adenovirus vectors of the E4orf6 protein and E1B55K. This screen revealed several interesting candidates, including integrin α3, a species of 130 kDa that also was found to be reduced in wild-type (wt) virus infection. The degradation of α3 was seen to be dependent on the Cul5-based ligase complex driven by E4orf6 and E1B55K. We also found evidence that the E4orf6/E1B55K ligase complex appears to be involved in cell detachment from the extracellular matrix, a function that could play a role in virus spread.     

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10⁴ PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r² value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r² value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.Waterborne viral infection is one of the most important causes of human morbidity in the world. There are hundreds of different types of human viruses present in human sewage, which, if improperly treated, may become the source of contamination in drinking and recreational waters (6, 12, 19). Furthermore, as water scarcity intensifies in the nation, so has consideration of wastewater reuse as a valid and essential alternative for resolving water shortages (31).Currently, routine viral monitoring is not required for drinking or recreational waters, nor is it required for wastewater that is discharged into the environment. This lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples. In the past 20 years, tremendous progress has been made in detection of viruses in the environment based on molecular technology (32, 33, 35). PCR and quantitative real-time PCR (qPCR) methods have improved both the speed and sensitivity of viral detection compared with detection by the traditional tissue culture method (2, 11, 17, 18). However, they provide little information on viral infectivity, which is crucial for human health risk assessment (22-24, 35). Our previous work using a real-time PCR assay to detect human adenoviruses (Ads) in sewage could not differentiate the infectious viruses in the secondary treated sewage from those killed by chlorination disinfection (15). In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29, 34, 39).FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4, 25, 34, 38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14, 16, 34, 47). The FACS viral assay is based on the expression of viral protein inside the recipient cell during viral replication (16). Specific antibody labeled with fluorescence is bound to the target viral protein, which results in fluorescence emission from infected cells. Viral particles outside the cell will not be captured, because the size of virus is below the detection limit of flow cytometry. Therefore, detection of cells, which can be captured with fluorescently labeled viral antibody, is a definitive indication of the presence of infectious virus.This research used human Ads as the target for development of the FACS method. The rationale for this choice is as follows. (i) Ads are important human pathogens that may be transmitted by water consumption and water spray (aerosols) (26, 32). The health hazard associated with exposure to Ads has been demonstrated by epidemiological data and clinical research (1, 7, 9, 35, 40, 43). (ii) Ads are among the most prevalent human viruses identified in human sewage and are frequently detected in marine waters and the Great Lakes (17, 32, 33, 35). (iii) Ads are more resistant to UV disinfection than any other bacteria or viruses (3, 5, 10, 24, 41, 42, 44). Thus, they may survive wastewater treatment as increasing numbers of wastewater treatment facilities switch from chlorination to UV to avoid disinfection by-products. (iv) Some serotypes of Ads, including enteric Ad 40 and 41, are fastidious. They are difficult to detect by plaque assay, and a routine assay of infectivity takes 7 to 14 days (8, 20).In this study, recombinant Ad serotype 5 (rAd5) with the E1A gene (the first transcribed gene after infection) replaced by a green fluorescent protein (GFP) gene was first used to test for sensitivity and speed of the assay. Two other viral proteins were then used as targets for development of FACS assays using Ad serotype 2 (Ad2) and Ad41. This study demonstrated the feasibility, sensitivity, and reliability of the assay for detection of infectious Ads in environmental samples.     

In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.A large number of inherited or acquired diseases remain promising targets for human gene therapy. One vector that has shown outstanding potential thus far in numerous preclinical and clinical evaluations is based on nonpathogenic adeno-associated virus (AAV). A unique asset among various properties that make AAV especially attractive over its competitors, such as adenoviral or lentiviral vectors, is the availability of a vast number of natural isolates which differ significantly in their properties (24). We and others have shown previously that the function of an AAV vector particle is determined mainly by the capsid protein and that viral Rep proteins and genomic packaging elements are largely interchangeable (24, 27, 85). Paradoxically, the ever-increasing repertoire of naturally occurring and synthetically generated AAV capsid sequences (>300 to date) is currently creating a dilemma for the rational selection of the optimal serotype for a given application. The importance of finding the ideal capsid for efficient and safe gene transfer has been exemplified in many preclinical studies, as well as in a clinical trial using the AAV type 2 (AAV-2) prototype in human liver tissue (36, 47). In one previous study, the treatment of patients with severe hemophilia B with recombinant AAV-2 expressing human factor IX (hFIX) resulted in mildly elevated, yet therapeutic, levels of this blood coagulation factor. However, expression was short lived, and the hFIX decline was accompanied by a transient asymptomatic increase of liver transaminases, due to a T-cell immune response against the AAV-2 capsid (47). Also, preexisting neutralizing anti-AAV-2 antibodies (frequent in humans) in these individuals likely inhibited the linear vector dose response previously observed in animals.We and others have suggested previously that the use of novel AAV serotypes, in particular, nonhuman isolates, will help to overcome some of these problems (19, 24, 63). Important examples are AAV-8 and AAV-9, which can transduce mouse liver far better than AAV-2, albeit the difference in dogs or primates is less clear (17, 52, 54, 75). The potential for the complete transduction of liver tissue and perhaps other tissues makes these two non-AAV-2 serotypes also particularly interesting for therapeutic RNA interference (RNAi) (28). We recently demonstrated the feasibility of efficiently and persistently suppressing hepatitis B virus with RNAi from a double-stranded AAV-8 vector (28). On the other hand, a potential drawback of AAV-8 and AAV-9 is their lack of specific tissue tropism (34, 52). The resulting frequent vector dissemination into all organs, including the brain, even from low peripheral doses in mice or monkeys (52, 54) is a particular concern for RNAi therapies in which control over vector biodistribution and the limitation of off-target effects will be imperative for the success of the approach (28).In order to overcome the constraints of wild-type AAV serotypes, numerous groups have recently begun to develop novel strategies to engineer “designer” AAVs tailored for the therapeutic transduction of clinically relevant organs (reviewed in detail in references 9, 12, 35, 41, 51, and 85). Briefly, the variety of strategies can be grouped into indirect or chemical approaches and direct physical modification strategies. In the indirect approaches, specific molecules (e.g., bispecific antibodies [6] or avidin-coupled ligands [4]) are allowed to react with the viral surface (biotinylated in the case of avidin [4]), as well as a cellular receptor, forming a conjugate ideally able to retarget the capsid to a refractory cell type. Yet, numerous pharmacological problems, such as concerns about in vivo complex stability and difficulties in upscaling complex manufacturing, continue to prevent the broad adaptation of these approaches. Alternative, more powerful strategies rely on the direct physical modification of the AAV capsid protein and gene. Early examples include the generation of mosaic AAV capsids via the mixing of helper plasmids carrying capsid genes from distinct serotypes, such as AAV-1 and AAV-2 (30) and pairwise combinations of AAV-1 through AAV-5 (62). Similar mosaics were generated previously via a marker rescue approach, yielding AAV-2/AAV-3 recombinants with unique properties (8). A related strategy is the rational creation of chimeric virions via domain swapping among multiple parental serotypes, involving either entire capsid loops or parts thereof or individual residues. Notable examples include AAV-1/AAV-2 chimeras with improved tropism in muscle tissue (31), with one of these chimeras presently being studied in a phase I clinical trial for the treatment of Duchenne muscular dystrophy (85). Most recently, our own group described a battery of unique chimeras comprising elements from serotypes 2 and 8, which were exploited to identify capsid subdomains responsible for efficient AAV transduction in murine liver tissue in vivo (64).A special type of chimeric capsids are those containing foreign proteins or peptides inserted into various positions of the virion shell. The methods and strategies used are widely diverse, and again, we refer to comprehensive reviews (12, 35, 41). Noteworthy here are approaches to fuse targeting ligands to the N termini of AAV capsid proteins (ideally, VP2 [45, 83]), or more powerful, to insert short peptides (up to 14 amino acids [21], typically 7) into exposed regions of the assembled virion. This strategy is referred to as viral display, in analogy to phage display, and has already been used extensively to retarget AAV-2 virions to a multitude of refractory or hard-to-infect cell types, such as vascular endothelial, smooth muscle, and pancreatic islet cells (43, 55, 77, 81, 82) and various tumor lines (22, 58, 65, 66). It has particularly benefited from comprehensive mutational analyses by various groups (e.g., references 21, 33, 56, and 83) that have resulted in the identification of prominent locations within the AAV-2 capsid tolerating peptide insertion. Most notable is the heparin binding domain (HBD), consisting of a total of four arginine (R) residues and one lysine residue, with R585 and R588 representing the most crucial components (37, 56). Numerous groups have now consistently shown that the insertion of 7-mer peptides into this region not only is frequently well tolerated and efficiently mediates virus retargeting, but also provides the extra benefit that the endogenous AAV-2 tropism can be abolished, thus enhancing target specificity (e.g., reference 21).In addition to identifying sites for vector engineering, some of the mutational AAV studies directly yielded novel capsid variants with potential benefits for clinical use. A remarkable case was a recent study by Lochrie et al. (42) in which a set of 127 AAV-2 variants with point or insertion mutations were generated and screened for multiple properties. Several capsids were isolated which differed from the wild-type AAV-2 capsid in having better in vitro transduction efficiencies (albeit being equally efficient in vivo) or, clinically most relevant, higher-level resistance to individual or pooled human antisera. Nonetheless, the limitations of the approach also became clear, most notably, the extreme effort required to generate and manually screen a large number of mutants, which in fact prevented the interesting analyses of all possible combinations of beneficial point mutations in further capsids.Indeed, the factors of time and labor are the main reasons why an increasing number of groups have recently begun to develop novel means for AAV vector evolution that no longer rely on the rational modification of the AAV-2 capsid. Instead, the new combinatorial methodologies allow for the far more efficient creation and selection of interesting candidates in a library-based high-throughput format. Thus far, two different strategies have been reported, both principally expanding on previously developed techniques. One is the use of viral display libraries, in which random 7-mer peptides are inserted into the AAV-2 HBD (at amino acid 587 or 588), yielding between 4 × 10⁶ and 1.7 × 10⁸ capsids potentially exposing new ligands on their surfaces (50, 58, 76). Subsequent iterative selection on diverse cell types refractory to the wild type, e.g., coronary artery endothelial cells, cardiomyoblasts, and carcinoma, leukemia, and megakaryocytic cell lines, led to enrichment with peptide mutants with increased target specificities and efficacies (48, 50, 58, 76). The second library type, independently described by two groups in 2006, relies on error-prone PCR amplification of the AAV-2 capsid gene (46, 59). Similar to the methods in earlier mutational studies, this approach resulted in the identification of AAV-2 point mutations (usually up to two per capsid) which yielded mutants that differed from the wild type in having mildly enhanced efficacies in vitro and/or improved transduction efficiencies in the presence of neutralizing anti-AAV-2 antibodies either generated in rabbits or preexisting in individual human sera.Here, for the first time, we introduce the technology of DNA family shuffling into the realm of AAV vector evolution. The basic concept of this technology is the in vitro recombination of related parental genes with >50% homology, which are first fragmented and then reassembled based on partial homology, resulting in libraries of chimeric genes. Iterative amplification under pressure can then yield hybrids not only combining parental assets, but also ideally exhibiting novel and synergistic properties (70, 71). DNA family shuffling has been used extensively in recent years to evolve and improve all types of proteins, from markers and enzymes to vaccines (e.g., references 10, 13-15, and 39). Importantly, a set of reports also suggested its power to enhance viral gene therapy vectors by creating retro- or lentiviruses with improved stability or efficacy compared to that of the parental wild types (57, 61, 69). Here, we describe the novel use of DNA family shuffling for the highly efficient molecular interbreeding of eight multispecies AAVs to create chimeric capsids and, moreover, document its compatibility and synergism with existing AAV vector evolution technology.     

Effect of Preexisting Immunity on Oncolytic Adenovirus Vector INGN 007 Antitumor Efficacy in Immunocompetent and Immunosuppressed Syrian Hamsters

Immune responses against adenovirus (Ad) vectors pose a possible concern for the outcome of treatment efficacy. To address the role of preexisting immunity in oncolytic Ad vector antitumor efficacy following intratumoral injection of vector as well as tumor-to-tissue spread of the vector, we employed the Syrian hamster model. These animals are immunocompetent, and their tumors and tissues are permissive for replication of Ad type 5 (Ad5). We used the adenovirus death protein-overexpressing Ad5-based vector INGN 007. Subcutaneous tumors were established in groups of hamsters that were or were not immunized with Ad5. Half of the hamsters in these groups were immunosuppressed with cyclophosphamide. For all groups, tumors injected with INGN 007 grew significantly more slowly than those injected with buffer. Under immunocompetent conditions, there was no significant effect of preexisting immunity on vector antitumor efficacy. Soon after the tumors in naïve animals were injected with vector, the hamsters developed neutralizing antibody (NAb) and the difference in NAb titers between the naïve and immunized groups diminished. Under immunosuppressed conditions, preexisting NAb did significantly reduce vector efficacy. Thus, NAb do reduce vector efficacy to some extent, but immunosuppression is required to observe the effect. Regarding vector toxicity, there was spillover of vector from the tumor to the liver and lungs in naïve immunocompetent hamsters, and this was nearly eliminated in the immunized hamsters. Thus, preexisting immunity to Ad5 does not affect INGN 007 antitumor efficacy following intratumoral injection, but immunity prevents vector spillover from the tumor to the liver and lungs.Oncolytic (replication-competent) viral vectors are being investigated as a treatment for cancer (2, 19, 25, 27). Recently, an oncolytic adenovirus serotype 5 (Ad5)-based vector was approved for cancer therapy in humans for the first time (14, 42). Oncolytic vectors based on Ad, reovirus, herpes simplex virus type 1 (HSV-1), poxvirus, poliovirus, Newcastle disease virus, measles virus, and vesicular stomatitis virus (VSV) are being studied extensively in both preclinical and clinical settings (16, 20, 24). Oncolytic Ad vectors are popular due to the Ad safety profile and ease of manipulation and handling (6, 13, 18, 23).Oncolytic Ad vectors infect and kill cancer cells as a result of the normal Ad life cycle by replicating in cells and releasing progeny viruses. These vectors rely on replication and spread through the tumor to achieve efficacy. A majority of the human population is seropositive for Ad5, which is acquired as a childhood infection (4, 15, 39). Elimination of the vector by preexisting immunity to Ad or vector elimination by the adaptive immune response generated after administration of the vector poses a possible concern with respect to achieving significant antitumor efficacy. A key question is whether the oncolytic Ad vector can efficiently eliminate tumor cells faster than its own clearance by the immune system. Several studies show that suppressing the immune system enhances the efficacy of oncolytic vectors (10, 12, 31).Alternatively, studies show that activation of the adaptive immune system by the vector might increase tumor cell killing, thereby increasing vector antitumor efficacy (11, 21, 27, 34). Studies with oncolytic HSV and VSV show that these vectors induce long-term antitumor immunity (11, 21, 27, 34). Therefore, apart from direct cell lysis, oncolytic vectors may be able to achieve antitumor efficacy by activating the antitumor immune response. Therefore, induced or preexisting immunity to the vector can be either a hurdle or beneficial for vector efficacy.Most efforts to address the effect of preexisting immunity were performed by gene transfer studies with replication-defective Ad vectors (28, 41). These studies showed that preexisting immunity significantly reduces gene transfer and expression in the target organ. In contrast, other studies showed that preexisting immunity does not prevent gene transfer (26) and does not affect vector antitumor efficacy (1). Little work has been done to address the role of induced or preexisting immunity on the efficacy and toxicity of oncolytic Ad vectors (3, 39). Studies with these vectors have been difficult because of a lack of immunocompetent and permissive animal models. Ad replication is generally species specific, and human Ads replicate poorly in cells from most nonhuman species. Consequently, Ad vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model cannot adequately address the effect of the host immune system on the vector-infected tumor or the toxicity of the vector in normal tissues.We recently developed a novel Syrian hamster model for the study of oncolytic Ad5-based vectors (30). These animals are both replication permissive for Ad5 and immunocompetent. In the present study, we modeled the effect of preexisting immunity to Ad5 on the efficacy of an oncolytic Ad vector, INGN 007, and the spillover of the vector from the site of injection to the liver and lungs.     

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus

Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV^CAR) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV^CAR to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV^CAR-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV^CARGFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV^CAR-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV^CAR to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV^CAR-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV^CAR in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV^CAR-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV^CAR-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV^CAR-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV^CAR-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.Adenoviruses (Ads) are extensively used today as gene transfer vectors for in vitro, ex vivo, and in vivo gene transfer protocols (reviewed in reference 65). Cell entry of human Ad type 5 (Ad5), the serotype most widely used as a gene vector, occurs most efficiently by the receptor-mediated endocytosis pathway (reviewed in references 64 and 65), via the coxsackievirus B-adenovirus receptor (CAR) (3, 77) and αvβ3/αvβ5 integrins (84, 85), although alternative receptors have been described (11, 12, 14, 27). Cell surface expression of CAR differs with different cell types, and this represents one of the major determinants of the efficiency of Ad5-mediated transduction (43). The ubiquitous nature of CAR is responsible for transduction of nontarget tissues by Ad vectors. Paradoxically, many target cells such as dermal fibroblasts, synoviocytes, mesenchymal stem cells (MSCs), peripheral blood mononuclear cells (PBMCs), and dendritic cells (DCs), express no or very low levels of CAR at their surface and are relatively resistant to Ad transduction (14, 15, 19). Much work has been done with different strategies to promote the entry of Ad5 into CAR-defective cells. These strategies include (i) the genetic modification of Ad capsid proteins to carry cell ligands (2, 15, 20, 28, 49, 50), (ii) pseudotyping Ad5 vectors with fibers from other serotypes (13, 57, 74, 86), (iii) using bispecific adapters or peptides (25, 40), (iv) chemical modification of Ad (9, 42), and (v) tethering on nanoparticles (7). The limitations to these strategies are that modifications of the Ad capsid are susceptible to negatively affecting the virus growth or viability, due to an alteration of virion assembly, stability, the viral uncoating process, and/or intracellular trafficking (13, 51).Other viruses which are gaining popularity as gene transfer vectors are the baculoviruses (BVs). Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) is an insect virus with a large double-stranded DNA genome packaged in a membrane-enveloped, rod-shaped protein capsid (70). Since the 1980s, the BV-insect cell expression system has been highly exploited for the production of recombinant proteins. In the mid-1990s, it was shown that recombinant BVs carrying reporter genes under cytomegalovirus (CMV) or retroviral Rous sarcoma virus promoter efficiently expressed reporter genes in mammalian cells (6, 22, 38, 41, 44, 69), as well as in avian cells (72) and fish cells (45). Since then, BVs have been reported to transduce numerous cells originating from species as various as humans, bovines, and fish (8, 32, 41, 73). As gene transfer vectors, BVs have been found to be rapidly inactivated by human serum complement (23), but exposing decay-accelerating factor (DAF) at the surface of BV by fusion with the baculoviral envelope glycoprotein can overcome this inactivation (33). BVs also have a good biosafety profile due to their incapacity to replicate in mammalian cells (31).Taking advantage of the ability of BVs to transduce a large repertoire of cells of invertebrate and vertebrate origins, including human primary cells, we investigated whether a recombinant AcMNPV could act as a carrier or macroadapter for Ad5 vectors to enter Ad5-refractory cells. To this aim, we pseudotyped AcMNPV virions with the high-affinity receptor for Ad5, the human CAR glycoprotein (BV^CAR), to enable the formation of complexes between vector particles of BV^CAR and Ad5-green fluorescent protein (Ad5GFP) mediated by Ad5 fiber and CAR interaction. We found that transduction of cell lines which were poorly permissive to Ad5, including human cancer cells and primary cells, was significantly improved using this strategy of piggybacking Ad5 vector on BV^CAR. More importantly, the increase in BV^CAR-Ad5-mediated transduction was obtained with a low range of Ad5 inputs, i.e., at multiplicities of infection (MOI) of less than 50 Ad5 vector particles per cell. We also found that the cell transduction enhancement observed with BV^CAR-Ad5 required the direct coupling of Ad5 to BV^CAR via fiber-CAR binding and that the cell attachment of the complex was mediated by the baculoviral envelope glycoprotein gp64. Kinetic analysis of virus-cell binding showed that the presence of BV^CAR in the complex was beneficial to Ad5 vector, not only in terms of tropism but also in terms of number of cell-bound virions and rate of cell attachment. In addition, the endocytic pathway of BV^CAR-Ad5 did not require Ad5 penton base RGD-integrin interaction. When administered in vivo to nude BALB/c mice, BV^CAR-Ad5 complex showed the same biodistribution as that of control Ad5 vector injected alone. In vitro, transduction of CAR-negative cells by BV^CAR-Ad5 was insensitive to coagulation factor X (FX), in contrast to Ad5 vector alone.Our novel strategy of gene delivery using the BV^CAR-Ad5 duo could be advantageously applied to various situations in vitro or ex vivo, e.g., for transducing Ad5-refractory cells when Ad5 capsid modifications cannot be envisaged, when oncolytic Ads need to be delivered to tumors via nonpermissive cell carriers belonging to the immune system, or when the simultaneous delivery of two transgenes by two separate vectors might be beneficial in terms of timing and/or level of cellular expression of the transgene products.