E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes.

The journey from plasma membrane to nuclear pore is a critical step in the lifecycle of DNA viruses, many of which must successfully deposit their genomes into the nucleus for replication. Viral capsids navigate this vast distance through the coordinated hijacking of a number of cellular host factors, many of which remain unknown. We performed a gene-trap screen in haploid cells to identify host factors for adenovirus (AdV), a DNA virus that can cause severe respiratory illness in immune-compromised individuals. This work identified Mindbomb 1 (MIB1), an E3 ubiquitin ligase involved in neurodevelopment, as critical for AdV infectivity. In the absence of MIB1, we observed that viral capsids successfully traffic to the proximity of the nucleus but ultimately fail to deposit their genomes within. The capacity of MIB1 to promote AdV infection was dependent on its ubiquitination activity, suggesting that MIB1 may mediate proteasomal degradation of one or more negative regulators of AdV infection. Employing complementary proteomic approaches to characterize proteins proximal to MIB1 upon AdV infection and differentially ubiquitinated in the presence or absence of MIB1, we observed an intersection between MIB1 and ribonucleoproteins (RNPs) largely unexplored in mammalian cells. This work uncovers yet another way that viruses utilize host cell machinery for their own replication, highlighting a potential target for therapeutic interventions that counter AdV infection.

ADAM17 stabilizes its interacting partner inactive Rhomboid 2 (iRhom2) but not inactive Rhomboid 1 (iRhom1).

The metalloprotease ADAM17 (a disintegrin and metalloprotease 17) is a key regulator of tumor necrosis factor α (TNFα), interleukin 6 receptor (IL-6R), and epidermal growth factor receptor (EGFR) signaling. ADAM17 maturation and function depend on the seven-membrane-spanning inactive rhomboid-like proteins 1 and 2 (iRhom1/2 or Rhbdf1/2). Most studies to date have focused on overexpressed iRhom1 and -2, so only little is known about the properties of the endogenous proteins. Here, we show that endogenous iRhom1 and -2 can be cell surface-biotinylated on mouse embryonic fibroblasts (mEFs), revealing that endogenous iRhom1 and -2 proteins are present on the cell surface and that iRhom2 also is present on the surface of lipopolysaccharide-stimulated primary bone marrow-derived macrophages. Interestingly, very little, if any, iRhom2 was detectable in mEFs or bone marrow-derived macrophages lacking ADAM17, suggesting that iRhom2 is stabilized by ADAM17. By contrast, the levels of iRhom1 were slightly increased in the absence of ADAM17 in mEFs, indicating that its stability does not depend on ADAM17. These findings support a model in which iRhom2 and ADAM17 are obligate binding partners and indicate that iRhom2 stability requires the presence of ADAM17, whereas iRhom1 is stable in the absence of ADAM17.

The C Terminus of the Herpes Simplex Virus UL25 Protein Is Required for Release of Viral Genomes from Capsids Bound to Nuclear Pores.

The herpes simplex virus (HSV) capsid is released into the cytoplasm after fusion of viral and host membranes, whereupon dynein-dependent trafficking along microtubules targets it to the nuclear envelope. Binding of the capsid to the nuclear pore complex (NPC) is mediated by the capsid protein pUL25 and the capsid-tethered tegument protein pUL36. Temperature-sensitive mutants in both pUL25 and pUL36 dock at the NPC but fail to release DNA. The uncoating reaction has been difficult to study due to the rapid release of the genome once the capsid interacts with the nuclear pore. In this study, we describe the isolation and characterization of a truncation mutant of pUL25. Live-cell imaging and immunofluorescence studies demonstrated that the mutant was not impaired in penetration of the host cell or in trafficking of the capsid to the nuclear membrane. However, expression of viral proteins was absent or significantly delayed in cells infected with the pUL25 mutant virus. Transmission electron microscopy revealed capsids accumulated at nuclear pores that retained the viral genome for at least 4 h postinfection. In addition, cryoelectron microscopy (cryo-EM) reconstructions of virion capsids did not detect any obvious differences in the location or structural organization for the pUL25 or pUL36 proteins on the pUL25 mutant capsids. Further, in contrast to wild-type virus, the antiviral response mediated by the viral DNA-sensing cyclic guanine adenine synthase (cGAS) was severely compromised for the pUL25 mutant. These results demonstrate that the pUL25 capsid protein has a critical role in releasing viral DNA from NPC-bound capsids.IMPORTANCE Herpes simplex virus 1 (HSV-1) is the causative agent of several pathologies ranging in severity from the common cold sore to life-threatening encephalitic infection. Early steps in infection include release of the capsid into the cytoplasm, docking of the capsid at a nuclear pore, and release of the viral genome into the nucleus. A key knowledge gap is how the capsid engages the NPC and what triggers release of the viral genome into the nucleus. Here we show that the C-terminal region of the HSV-1 pUL25 protein is required for releasing the viral genome from capsids docked at nuclear pores. The significance of our research is in identifying pUL25 as a key viral factor for genome uncoating. pUL25 is found at each of the capsid vertices as part of the capsid vertex-specific component and implicates the importance of this complex for NPC binding and genome release.

Diminished Innate Antiviral Response to Adenovirus Vectors in cGAS/STING-Deficient Mice Minimally Impacts Adaptive Immunity.

UNLABELLED: Infection by adenovirus, a nonenveloped DNA virus, induces antiviral innate and adaptive immune responses. Studies of transformed human and murine cell lines using short hairpin RNA (shRNA) knockdown strategies identified cyclic guanine adenine synthase (cGAS) as a pattern recognition receptor (PRR) that contributes to the antiadenovirus response. Here we demonstrate how the cGAS/STING cascade influences the antiviral innate and adaptive immune responses in a murine knockout model. Using knockout bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMMOs), we determined that cGAS and STING are essential to the induction of the antiadenovirus response in these antigen-presenting cells (APCs) in vitro We next determined how the cGAS/STING cascade impacts the antiviral response following systemic administration of a recombinant adenovirus type 5 vector (rAd5V). Infection of cGAS(-/-) and STING(-/-) mice results in a compromised early antiviral innate response compared to that in wild-type (WT) controls: significantly lower levels of beta interferon (IFN-ß) secretion, low levels of proinflammatory chemokine induction, and reduced levels of antiviral transcript induction in hepatic tissue. At 24 h postinfection, levels of viral DNA and reporter gene expression in the liver were similar in all strains. At 28 days postinfection, clearance of infected hepatocytes in cGAS or STING knockout mice was comparable to that in WT C57BL/6 mice. Levels of neutralizing anti-Ad5V antibody were modestly reduced in infected cGAS mice. These data support a dominant role for the cGAS/STING cascade in the early innate antiviral inflammatory response to adenovirus vectors. However, loss of the cGAS/STING pathway did not affect viral clearance, and cGAS deficiency had a modest influence on the magnitude of the antiviral humoral immune response to adenovirus infections. IMPORTANCE: The detection of viral infection by host sentinel immune cells contributes to the activation of a complex and varied antiviral innate and adaptive immune response, which limits virus replication, spread, and susceptibility to infection. In this study, we have characterized how the cGAS/STING DNA-sensing cascade contributes to early detection of adenovirus infections. cGAS influences APC activation and early innate antiviral inflammatory immune responses, but adaptive immune pathways associated with virus clearance and anti-Ad antibody production were minimally influenced by the loss of the cGAS PRR signaling cascade.

Unabated adenovirus replication following activation of the cGAS/STING-dependent antiviral response in human cells.

UNLABELLED: The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35>Ad7>Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency. IMPORTANCE: This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions. Furthermore, activation of the cGAS cascade occurred in a cell-specific manner. Activation of the cGAS/STING response did not impact viral replication, and viral immune evasion strategies did not target the cGAS/STING/TBK1/IRF3 cascade. These studies provide novel insight into the early innate recognition response to adenovirus.

Adenovirus detection by the cGAS/STING/TBK1 DNA sensing cascade.

Adenovirus (Ad) infection triggers a cell-specific antiviral response following exposure of viral DNA to the intracellular compartment. A variety of DNA sensors (DAI, AIM2, DDx41, RNA polymerase [Pol] III, and IFI16 [p204]) have been identified in recent years; however, the DNA sensor involved in detection of adenovirus has not been established. Cyclic GMP-AMP synthase (cGAS), a DNA sensor that produces a cyclic guanine-adenine dinucleotide (cGAMP) inducer of STING, has been examined to determine its role in generating an antiadenoviral response. Short hairpin RNA (shRNA) lentiviral vectors targeting TBK1, STING, and cGAS were established in murine MS1 endothelial and RAW 264.7 macrophage cell lines. Knockdown of TBK1, STING, and cGAS results in a dramatic reduction in the activation of the primary antiviral response marker phosphorylated interferon (IFN) response factor 3 (IRF3) following exposure to adenovirus. Furthermore, activation of secondary type I IFN signaling targets ((ptyr)STAT1 and (ptyr)STAT2 [(ptyr)STAT1/2]) was also compromised. Consistent with compromised activation of primary and secondary response markers, transcriptional activation of IRF3-responsive genes (beta IFN [IFN-ß], ISG15, ISG54) and secondary response transcripts were diminished in cells knocked down in cGAS, STING, or TBK1. These data establish cGAS as the dominant cytosolic DNA sensor responsible for detection of internalized adenovirus leading to induction of the type I interferon antiviral cascade.

Highly divergent integration profile of adeno-associated virus serotype 5 revealed by high-throughput sequencing.

UNLABELLED: Adeno-associated virus serotype 5 (AAV-5) is a human parvovirus that infects a high percentage of the population. It is the most divergent AAV, the DNA sequence cleaved by the viral endonuclease is distinct from all other described serotypes and, uniquely, AAV-5 does not cross-complement the replication of other serotypes. In contrast to the well-characterized integration of AAV-2, no published studies have investigated the genomic integration of AAV-5. In this study, we analyzed more than 660,000 AAV-5 integration junctions using high-throughput integrant capture sequencing of infected human cells. The integration activity of AAV-5 was 99.7% distinct from AAV-2 and favored intronic sequences. Genome-wide integration was highly correlated with viral replication protein binding and endonuclease sites, and a 39-bp consensus integration motif was revealed that included these features. Algorithmic scanning identified 126 AAV-5 hot spots, the largest of which encompassed 3.3% of all integration events. The unique aspects of AAV-5 integration may provide novel tools for biotechnology and gene therapy. IMPORTANCE: Viral integration into the host genome is an important aspect of virus host cell biology. Genomic integration studies of the small single-stranded AAVs have largely focused on site preferential integration of AAV-2, which depends on the viral replication protein (Rep). We have now established the first genome wide integration profile of the highly divergent AAV-5 serotype. Using integrant capture sequencing, more than 600,000 AAV-5 integration junctions in human cells were analyzed. AAV-5 integration hot spots were 99.7% distinct from AAV-2. Integration favored intronic sequences, occurred on all chromosomes, and integration hot spot distribution was correlated with human genomic GAGC repeats and transcriptional activity. These features support expansion of AAV-5 based vectors for gene transfer considerations.

High-throughput sequencing reveals principles of adeno-associated virus serotype 2 integration.

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics.

Adeno-associated virus type 2 preferentially integrates single genome copies with defined breakpoints.

BACKGROUND: Adeno-associated virus (AAV) serotype 2 prevalently infects humans and is the only described eukaryotic virus that integrates site-preferentially. In a recent high throughput study, the genome wide distribution of AAV-2 integrants was determined using Integrant Capture Sequencing (IC-Seq). Additional insight regarding the integration of AAV-2 into human genomic DNA could be gleaned by low-throughput sequencing of complete viral-chromosomal junctions. FINDINGS: In this study, 140 clones derived from Integrant-Capture Sequencing were sequenced. 100 met sequence inclusion criteria, and of these 39 contained validated junction sequences. These unique sequences were analyzed to investigate the structure and location of viral-chromosomal junctions. CONCLUSIONS: Overall the low-throughput analysis confirmed the genome wide distribution profile gathered through the IC-Seq analysis. We found no unidentifiable sequence inserted at AAV-2 chromosomal junctions. Assessing both left and right ends of the AAV genome, viral breakpoints predominantly occurred in one hairpin of the inverted terminal repeat and AAV genomes were preferentially integrated as single copies.

Sensing adenovirus infection: activation of interferon regulatory factor 3 in RAW 264.7 cells.

We have used the RAW 264.7 murine macrophage-like cell line as a platform to characterize the recognition and early signaling response to recombinant adenoviral vectors (rAdV). Infection of RAW 264.7 cells triggers an early response (2 to 6 h postinfection) that includes phosphorylation of the interferon (IFN) response factor 3 (IRF3) transcription factor, upregulation of IRF3 primary response genes (interferon-stimulated gene 56 [ISG56], beta IFN [IFN-ß]), and subsequent type I IFN secondary signaling (STAT1/2 phosphorylation). Using short hairpin RNA (shRNA) lentiviral vectors, we show an essential role for Tank binding kinase 1 (TBK1) in this pathway. Data also support a role for STING (MITA) as an adaptor functioning in response to rAdV infection. Using UV/psoralen (Ps)-inactivated virus to block viral transcription, Ps-inactivated virus stimulated primary (IRF3) and secondary (STAT1/2) activation events to the same degree as untreated virus. IRF3 phosphorylation was not blocked in RAW 264.7 cells pretreated with the RNA polymerase III inhibitor ML60218. However, they were compromised in the type I IFN-dependent secondary response (phosphorylation of STAT1/STAT2). At 24 h postinfection, ML60218-treated cells were compromised in the overall antiviral response. Therefore, initial sensing of rAdV or viral DNA (vDNA) does not depend on viral template transcription, but ML60218 treatment influences cellular cascades required for an antiviral response to rAdV. Using overexpression or knockdown assays, we examined how four DNA sensors influence the antiviral response. Knockdown of DNA Activator of Interferon (DAI) and p204, the murine ortholog to IFI16, had minimal influence on IRF3 phosphorylation. However, knockdown of absent in melanoma 2 (AIM2) and the helicase DDX41 resulted in diminished levels of (pser388)IRF3 following rAdV infection. Based on these data, multiple DNA sensors contribute to an antiviral DNA recognition response, leading to TBK1-dependent IRF3 phosphorylation in RAW 264.7 cells.

Cell-specific regulation of nucleic acid sensor cascades: a controlling interest in the antiviral response.

In this study, we examined the capacities of non-antigen-presenting cell types to propagate antiviral signals following infection with recombinant adenovirus or by direct nucleic acid transfection. Three murine cell lines (RAW264.7 macrophages as a positive control, FL83B hepatocytes, and MS1 endothelial cells) were assessed following exposure to adenovirus, DNA, or RNA ligands. Based on primary (interferon response factor 3 [IRF3] phosphorylation) and secondary (STAT1/2 phosphorylation) response markers, we found each cell line presented a unique response profile: RAW cells were highly responsive, MS1 cells were modified in their response, and FL83B cells were essentially nonresponsive. Comparative reverse transcription-quantitative PCR (RT-qPCR) of nucleic acid sensing components revealed major differences between the three cell types. A prominent difference was at the level of adaptor molecules; TRIF, MyD88, MAVS, and STING. TRIF was absent in MS1 and FL83B cells, whereas MyD88 levels were diminished in FL83B hepatocytes. These differences resulted in compromised TLR-mediated activation. While the cytosolic adaptor MAVS was well represented in all cell lines, the DNA adaptor STING was deficient in FL83B hepatocytes (down by nearly 3 log units). The absence of STING provides an explanation for the lack of DNA responsiveness in these cells. This hypothesis was confirmed by acquisition of IRF3 activation in Flag-STING FL83B cells following DNA transfection. To consolidate the central role of adaptors in MS1 endothelial cells, short hairpin RNA (shRNA) knockdown of STING and MAVS resulted in a ligand-specific loss of IRF3 responsiveness. In contrast to the requirement for specific adaptor proteins, a requirement for a specific DNA sensor (AIM2, DDx41, or p204) in the IRF3 activation response was not detected by shRNA knockdown in MS1 cells. The data reveal that cell-specific regulation of nucleic acid sensing cascade components influences antiviral recognition responses, that controlling levels of adaptor molecules is a recurring strategy in regulating antiviral recognition response functions, and that comparative RT-qPCR has predictive value for antiviral/innate response functions in these cells.

Clathrin adaptor AP1B controls adenovirus infectivity of epithelial cells.

Adenoviruses invading the organism via normal digestive or respiratory routes require the Coxsackie-adenovirus receptor (CAR) to infect the epithelial barrier cells. Because CAR is a component of tight junctions and the basolateral membrane and is normally excluded from the apical membrane, most epithelia are resistant to adenoviruses. However, we discovered that a specialized epithelium, the retinal pigment epithelium (RPE), anomalously expressed CAR at the apical surface and was highly susceptible to adenovirus infection. These properties of RPE cells correlated with the absence of the epithelial-specific clathrin adaptor AP1B. Furthermore, knockdown of this basolateral sorting adaptor in adenovirus-resistant MDCK cells promoted apical localization of CAR and increased dramatically Adenovirus infectivity. Targeting assays showed that AP1B is required for accurate basolateral recycling of CAR after internalization. AP1B knock down MDCK cells missorted CAR from recycling endosomes to the apical surface. In summary, we have characterized the cellular machinery responsible for normal sorting of an adenovirus receptor and illustrated how tissue-specific variations in such machinery result in drastic changes in tissue-susceptibility to adenoviruses.

Reciprocal impacts of telomerase activity and ADRN/MES differentiation state in neuroblastoma tumor biology.

Telomere maintenance and tumor cell differentiation have been separately implicated in neuroblastoma malignancy. Their mechanistic connection is unclear. We analyzed neuroblastoma cell lines and morphologic subclones representing the adrenergic (ADRN) and mesenchymal (MES) differentiation states and uncovered sharp differences in their telomere protein and telomerase activity levels. Pharmacologic conversion of ADRN into MES cells elicited consistent and robust changes in the expression of telomere-related proteins. Conversely, stringent down-regulation of telomerase activity triggers the differentiation of ADRN into MES cells, which was reversible upon telomerase up-regulation. Interestingly, the MES differentiation state is associated with elevated levels of innate immunity factors, including key components of the DNA-sensing pathway. Accordingly, MES but not ADRN cells can mount a robust response to viral infections in vitro. A gene expression signature based on telomere and cell lineage-related factors can cluster neuroblastoma tumor samples into predominantly ADRN or MES-like groups, with distinct clinical outcomes. Our findings establish a strong mechanistic connection between telomere and differentiation and suggest that manipulating telomeres may suppress malignancy not only by limiting the tumor growth potential but also by inducing tumor cell differentiation and altering its immunogenicity.

Adenovirus induction of IRF3 occurs through a binary trigger targeting Jun N-terminal kinase and TBK1 kinase cascades and type I interferon autocrine signaling.

Pathogen recognition is a critical function of immune sentinel cells. Naïve macrophages or dendritic cells (DCs) undergo pathogen-directed activation and maturation, and as mature antigen-presenting cells (APCs), they contribute essential functions to both innate and adaptive immunity. Using recombinant adenovirus (rAdV) as a model for murine APC activation by DNA viruses, we demonstrate a critical role for stress kinase activation in cell intrinsic and extrinsic antiviral signaling cascades. We propose two viral triggers, viral capsid and viral DNA, are required for APC activation. Endosomal escape and presentation of cytosolic rAdV DNA induces phosphorylation of TANK-binding kinase 1 (TBK1) at serine 172 but does not induce IkappaB kinase epsilon activity as determined by in vitro kinase assays. However, induction of TBK1 alone is not sufficient for interferon regulatory factor 3 (IRF3) phosphorylation. We show that capsid-dependent activation of Jun N-terminal kinase (JNK) stress kinase is a necessary step, licensing TBK1 phosphorylation of IRF3 at Ser 396. A second later phase of JNK activity is required to coordinate phosphorylation of JNK-dependent transcription factors (c-Jun/ATF2) with activated IRF3 in the induction of primary IRF3-responsive transcripts. Finally, we demonstrate that maximal JNK/TBK1/IRF3 stimulation by rAdV depends on an intact type I interferon (IFN) signaling cascade. By requiring multiple viral triggers and type I IFN autocrine regulation, APCs have an inherent fail-safe mechanism against inappropriate activation and maturation.

Adenovirus capsid chimeras: fiber terminal exon insertions/gene replacements in the major late transcription unit.

The adenovirus major late transcription unit (MLTU) encodes the main structural capsid proteins. Expression from the MLTU is accomplished through alternative mRNA processing and use of a terminal exon coding strategy. The capsid proteins hexon, penton, and fiber contribute to efficient infection by adenovirus, and each contributes in some manner to the antiviral immune response against adenovirus infection. The ability to manipulate these genes affords one the opportunity to "detarget" adenovirus, to retarget adenovirus, and to alter immune recognition. In this chapter, we are presenting a terminal exon-replacement strategy that can be used to genetically manipulate capsid proteins expressed from the MLTU. An emphasis will be placed on manipulations of fiber as an intact terminal exon.

Parvovirus B19 integration into human CD36+ erythroid progenitor cells.

The pathogenic autonomous human parvovirus B19 (B19V) productively infects erythroid progenitor cells (EPCs). Functional similarities between B19V nonstructural protein (NS1), a DNA binding endonuclease, and the Rep proteins of Adeno-Associated Virus (AAV) led us to hypothesize that NS1 may facilitate targeted nicking of the human genome and B19 vDNA integration. We adapted an integration capture sequencing protocol (IC-Seq) to screen B19V infected human CD36+ EPCs for viral integrants, and discovered 40,000 unique B19V integration events distributed throughout the human genome. Computational analysis of integration patterns revealed strong correlations with gene intronic regions, H3K9me3 sites, and the identification of 41 base pair consensus sequence with an octanucleotide core motif. The octanucleotide core has homology to a single region of B19V, adjacent to the P6 promoter TATA box. We present the first direct evidence that B19V infection of erythroid progenitor cells disrupts the human genome and facilitates viral DNA integration.

Transgene expression after rep-mediated site-specific integration into chromosome 19.

We have used a plasmid-based transfection model of the adeno-associated virus (AAV) Rep-mediated site-specific integration (RMSSI) pathway to characterize the stability and expression of a site-specifically integrated transgene (either green fluorescent protein [GFP] or chloramphenicol acetyltransferase [CAT]). Three plasmids containing the AAV p5 integration efficiency element (p5IEE) have been used to study integration and transgene expression in HeLa cells: (1) pRepGFP(itr+) contains both AAV ITRs, rep, and p5IEE and can be used as either a plasmid or rAAV vehicle for integration; (2) pRepGFP(itr-) contains the AAV rep gene and the p5IEE; (3) pAd-p5CAT contains only the 138-bp p5IEE of AAV. The data presented demonstrate that in the absence of drug selection, all three constructs undergo site-specific integration (efficiencies of between 10 and 40% of transduced cell lines). At 6 weeks posttransfection most cell lines that underwent RMSSI also expressed the appropriate transgene product. By 18 weeks posttransfection cell lines that were established with rep in cis to the transgene showed a decline in transgene expression as well as a loss of transgene DNA. In many cell lines, there appears to be transgene-containing DNA that does not contribute to gene expression. Data support a model of gene expression and transgene instability through a Rep-mediated pathway. In contrast to rep-containing cell lines, clonal cell lines containing p5IEECAT (with Rep provided in trans) maintained both the integrated transgene and transgene expression throughout the entire experimental time course (18 weeks).

Serotype 5 Adenovirus fiber (F7F41S) chimeric vectors incur packaging deficiencies when targeting peptides are inserted into Ad41 short fiber.

Adenovirus is a well-established viral gene transfer model system that presents two major hurdles when being considered for cell-specific targeting applications. First is the need to detarget the vector from inherent host binding mechanisms, and second is the need to establish a productive and stable method to retarget the vector to a desired cell receptor. In previous studies we had generated an adenovirus vector platform that lacks the normal targeting attributes derived from the fiber and penton capsid proteins. In the current study we characterized our detargeted Ad5-based vectors (Ad5.F7F41S and Ad5.F7F41SDeltaRGD) as platforms for novel retargeted viruses. The experimental strategy relied on incorporating small peptide ligands into several sites of the Ad 41short fiber knob domain (AB, CD, HI, G and Cterm). Reengineering of Ad41 short fiber resulted either in a bypass to fiber 7 usage, or in a dominant negative packaging/production deficiency phenotype. Under specific growth conditions we could remedy some of the capsid deficiencies and generate high titer viruses. However when examined by Western blot analysis, the resulting viruses were still defective in capsid content. The tandem fiber F7F41S platform has revealed an unanticipated sensitivity of Adenovirus packaging to fiber 41short structural modifications. These studies indicate fiber assembly into an intact virion or fiber influenced capsid stability as a bottleneck to efficient particle production. We also demonstrate that virus particles characterized as mature virions following CsCl banding can vary significantly in capsid protein content. Considering the complexity of virus entry into a target cell, modified "mature virions" may be compromised at the level of transduction not only through the intended modification, but also by virtue of secondary structural packaging conflicts.

Adeno-associated virus type 2 p5 promoter: a rep-regulated DNA switch element functioning in transcription, replication, and site-specific integration.

The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.

Sensing infection by adenovirus: Toll-like receptor-independent viral DNA recognition signals activation of the interferon regulatory factor 3 master regulator.

Infection with adenovirus vectors (AdV) results in rapid activation of innate immunity, which serves the dual purpose of stimulating inflammatory antiviral host defenses and the adaptive immune system. Viral recognition by macrophages, dendritic cells, and other cell types requires an ability to sense the presence of a foreign molecular pattern by "pattern recognition receptors." The nature of the adenoviral sensor, the target ligand of the sensor, and the downstream antiviral signaling response triggered by virus infection have not been defined for this nonenveloped double-stranded DNA (dsDNA) virus. We have identified four critical links involved in AdV recognition by murine antigen-presenting cells (APC) and primary lung fibroblasts: (i) viral recognition occurs chiefly via a Toll-like receptor (TLR)-independent nucleic acid-sensing mechanism recognizing the viral dsDNA genome, (ii) the intact viral particle and capsid proteins are required for efficient intracellular delivery of the viral genome, (iii) delivery of the viral genome triggers interferon regulatory factor 3 (IRF3) phosphorylation, and (iv) IRF3 activation is the required dominant antiviral signaling pathway used by APC, whereas the "primary" involvement of NF-kappaB, mitogen-activated protein kinase, or Akt pathways is less prominent. In this study we provide the first direct evidence that infection by a dsDNA virus stimulates an IRF3-mediated interferon and proinflammatory response through a TLR-independent DNA-sensing mechanism.