Search

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., Yu et al. identify marked differences in the telomere and immunologic profiles of neuroblastoma cell lines displaying MES and ADRN lineage characteristics. They show that pharmacologically converting ADRN into MES cells triggers associated switch in telomere/immunologic protein profiles whereas inhibiting telomerase activity in ADRN cells induces reversible conversion into MES cells.

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.

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 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.

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.

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.

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.

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-β) 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.

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-β]), 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.

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.

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 IκB kinase ε 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.

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.

Fiber and penton base capsid proteins of adenovirus type 5 (Ad5) mediate a well-characterized two-step entry pathway in permissive tissue culture cell lines. Fiber binds with high affinity to the cell surface coxsackievirus-and-adenovirus receptor (CAR), and penton base facilitates viral internalization by binding αv integrins through an RGD motif. In vivo, the entry pathway is complicated by interactions of capsid proteins with additional cell surface molecules and blood factors. When administered systemically in mice, adenovirus vectors (Adv) localize primarily to hepatic tissue, resulting in efficient gene transduction and potent activation of the host antiviral immune response. The goal of the present study was to detarget Adv uptake through fiber and penton base capsid protein manipulations and determine how detargeted vectors influence transduction efficiency, inflammatory activation, and activation of the adaptive arm of the immune system. By manipulating fiber and the penton base, we have generated highly detargeted vectors (up to 1,200-fold reduction in transgene expression in vivo) with reduced macrophage stimulatory activity in vitro and in vivo. In spite of the diminished transduction and macrophage activation, the detargeted vectors induce strong neutralizing immunity as well as efficient antitransgene antibody. Three of the modified vectors produce antitransgene humoral immunity at levels that exceed or are equal to that seen with an unmodified Ad5-based vector. The fiber-pseudotyped and penton base constructs with RGD deleted have attributes that could be important enhancements in a number of vaccine applications.

The major adenovirus (Ad) capsid proteins hexon, penton, and fiber influence the efficiency and tropism of gene transduction by Ad vectors. Fiber is the high-affinity receptor binding protein that serves to mediate cell attachment in vitro when using coxsackie-adenovirus receptor (CAR)-containing cell lines. This contrasts with transduction efficiency in macrophages or dendritic cells that lack high concentrations of CAR. To determine how fiber influences gene transduction and immune activation in a murine model, we have characterized Ad type 5 (Ad5) vectors with two classes of chimeric fiber, CAR binding and non-CAR binding. In a systemic infection, Ad5 fiber contributes to DNA localization and vector transduction in hepatic tissue. However, the majority of vector localization is due to Ad5 fiber-specific functions distinct from CAR binding. CAR-directed transduction occurs but at a modest level. In contrast to CAR binding vectors, the F7 and F7F41S non-CAR-binding vectors demonstrate a 2-log decrease in hepatic transduction, with a 10-fold decrease in the amount of vector DNA localizing to the hepatic tissue. To characterize the innate response to early infection using fiber chimeric vectors, intrahepatic cytokine and chemokine mRNAs were quantified 5 hours postinfection. Tumor necrosis factor alpha mRNA levels resulting from Ad5 fiber infections were elevated compared to viruses expressing serotype 7 or 41 fiber. Levels of chemokine mRNA (gamma interferon-inducible protein 10, T-cell activation gene 3, and macrophage inflammatory protein 1β) were 10- to 20-fold higher with CAR binding vectors (Ad5 and F41T) than with non-CAR-binding vectors (F7 and F7F41S). In spite of quantitative differences in vector localization and innate activation, fiber pseudotyping did not significantly change the outcome of anti-Ad adaptive immunity. All vectors were cleared with the same kinetics as wild-type Ad5 vectors, and each induced neutralizing antibody. Although non-CAR-binding vectors were impaired in transduction by nearly 2 orders of magnitude, the level of antitransgene immunity was the same for each of the vectors. Using primary bone marrow-derived macrophages and dendritic cells, we demonstrate that transduction, induction of cytokine/chemokine, and phenotypic maturation of these antigen-presenting cells are independent of fiber content. Our data support a model where fiber-mediated hepatic localization enhances innate responses to virus infection but minimally impacts on adaptive immunity.

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).

The initial aim of this study was to combine attributes of adeno-associated virus (AAV) and adenovirus (Ad) gene therapy vectors to generate an Ad-AAV hybrid vector allowing efficient site-specific integration with Ad vectors. In executing our experimental strategy, we found that, in addition to the known incompatibility of Rep expression and Ad growth, an equally large obstacle was presented by the inefficiency of the integration event when using traditional recombinant AAV (rAAV) vectors. This study has addressed both of these problems. We have shown that a first-generation Ad can be generated that expresses Rep proteins at levels consistent with those found in wild-type AAV (wtAAV) infections and that Rep-mediated AAV persistence can occur in the presence of first-generation Ad vectors. Our finding that traditional rAAV plasmid vectors lack integration potency compared to wtAAV plasmid constructs (10- to 100-fold differences) was unexpected but led to the discovery of a previously unidentified AAV integration enhancer sequence element which functions in cis to an AAV inverted terminal repeat-flanked target gene. rAAV constructs containing left-end AAV sequence, including the p5- rep promoter sequence, integrate efficiently in a site-specific manner. The identification of this novel AAV integration enhancer element is consistent with previous studies, which have indicated that a high frequency of wtAAV recombinant junction formation occurs in the vicinity of the p5 promoter, and recent studies have demonstrated a role for this region in AAV DNA replication. Understanding the contribution of this element to the mechanism of AAV integration will be critical to the use of AAV vectors for targeted gene transfer applications.

The innate immune response against replication-defective adenoviruses (Ad) is poorly defined. We and others have previously observed striking differences in the rate at which the Ad vector itself or the virus encoding a variety of transgenes is eliminated in different mouse strains. Here, we report that Ad infection of BALB/ mice is associated with sixfold-higher levels of serum alanine aminotransferase and that Ad transgenes induce two- to threefold-higher levels of intrahepatic NK cells and NK activity compared to C57BL/6 mice. The increase in NK activation in BALB/c mice was associated with ∼4-fold higher level of mRNA expression of a newly described NKG2 receptor activator, H-60, as well as increased expression of interleukin-12 and gamma interferon mRNAs in BALB/c mice compared to C57BL/6 mice. NK depletion in BALB/c mice or defective NK function in C3H beige mice extended transgene expression compared to their appropriate controls, and attenuation of NK together with CD8 T-cell function had a synergistic effect. These findings indicate that there are intrinsic differences in the innate immune responses of different mouse strains to Ad and Ad transgenes and that NK cells, in cooperation with CD8 T cells, play a pivotal role in the early extinction of transgene expression in BALB/c mice.

To study DNA double-strand break (DSB) repair in mammalian cells, the Saccharomyces cerevisiae HO endonuclease gene, or its recognition site, was cloned into the adenovirus E3 or E1 regions. Analysis of DNA from human A549 cells coinfected with the E3::HO gene and site viruses showed that HO endonuclease was active and that broken viral genomes were detectable 12 h postinfection, increasing with time up to approximately 30% of the available HO site genomes. Leftward fragments of approximately 30 kbp, which contain the packaging signal, but not rightward fragments of approximately 6 kbp, were incorporated into virions, suggesting that broken genomes were not held together tightly after cleavage. There was no evidence for DSB repair in E3::HO virus coinfections. In contrast, such evidence was obtained in E1::HO virus coinfections of nonpermissive cells, suggesting that adenovirus proteins expressed in the permissive E3::HO coinfection can inhibit mammalian DSB repair. To test the inhibitory role of E4 proteins, known to suppress genome concatemer formation late in infection (Weiden and Ginsberg, 1994), A549 cells were coinfected with E3::HO viruses lacking the E4 region. The results strongly suggest that the E4 protein(s) inhibits DSB repair.

Following receptor binding and internalization, intracellular trafficking of adenovirus (Ad) among subgroups B and C is different, with significant amounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly translocate to the nucleus. To evaluate the role of the fiber in these differences, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimeric vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fiber) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated viruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly translocated to the nucleus (within 1 h of infection), while Ad7 virions were widely distributed in the cytoplasm at the same time point. Interestingly, chimeric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h following infection were 72% ± 4%, 32% ± 6%, and 38% ± 2%, respectively. Consistent with these observations, fluorescence in situ hybridization demonstrated that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucleus and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demonstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 DNA at 1 h postinfection were 80% ± 13% and 43% ± 1%, respectively. Although it has been generally accepted that Ad fiber protein mediates attachment of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surface, fiber likely modulates intracellular trafficking as well.

Robust expression of genes transferred by adenovirus (Ad) vectors depends upon efficient entry of vectors into target cells. Cells deficient in the coxsackie/adenovirus receptor (CAR) are difficult targets for Admediated gene transfer. We hypothesized that low levels of CAR expression may be responsible, in part, for the relative inefficiency of Ad-mediated gene transfer to human alveolar macrophages (AMs). CAR gene expression was detected in human AMs by reverse transcription‐polymerase chain reaction and at low levels by Northern analysis. Indirect immunofluorescence showed specific, low-intensity surface staining for CAR, but at levels below those found on the positive-control A549 human lung epithelial cell line. Consistent with this, AMs expressed Ad vector transgenes 100 to 1,000-fold less efficiently than A549 cells, as assessed using the b -galactosidase reporter (chemiluminescence assay) and green fluorescent protein (fluorescence microscopy and flow cytometry). At high multiplicity of infection, AMs from an HIV 1 individual could be transduced with an AdIFN g vector to secrete detectable human interferon- g . Ad transgene expression by AMs was blocked by capsid fiber protein, suggesting that CAR is required in the pathway for productive Ad entry into alveolar macrophages. To confirm that Ad transgene expression by AMs is limited by low levels of CAR expression, cells were infected with an Ad vector containing the CAR complementary DNA (cDNA). Enhanced expression of CAR protein was demonstrated by indirect immunofluorescence, and the CAR cDNA-transduced cells showed 5-fold enhancement of subsequent Ad transgene expression. These observations demonstrate that human AMs can be targets for Ad-mediated gene transfer, but that efficiency of transgene expression is limited, at least in part, by low levels of CAR expression. Kaner, R. J., S. Worgall, P. L. Leopold, E. Stolze, E. Milano, C. Hidaka, R. Ramalingam, N. R. Hackett, R. Singh, J. Bergelson, R. Finberg, E. Falck-Pederson, and R. G. Crystal. 1999. Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. Am. J. Respir. Cell Mol. Biol. 20:361‐370.

A strategy for constructing replication-defective adenovirus vectors from non-subgroup C viruses has been successfully demonstrated with adenovirus type 7 strain a (Ad7a) as the prototype. An E1A-deleted Ad7a reporter virus expressing the chloramphenicol acetyltransferase (CAT) gene from the cytomegalovirus promoter enhancer was constructed with DNA fragments isolated from Ad7a, an Ad7a recombination reporter plasmid, and the 293 cell line. The Ad7a-CAT virus particle transduces A549 cells as efficiently as Ad5-based vectors. Intravenous infections in a murine model indicate that the Ad7a-CAT virus infects a variety of tissues, with maximal levels of CAT gene expression found in the liver. The duration of Ad7a-CAT transgene expression in the liver was maximally maintained 2 weeks postinfection, with a decline to baseline activity by the week 4 postinfection. Ad7a-CAT represents the first example of a non-subgroup C E1A- adenovirus gene transfer vector.

Gene expression in complex transcription units can be regulated at virtually every step in the production of mature cytoplasmic mRNA, including transcription initiation, elongation, termination, pre-mRNA processing, nucleus-to-cytoplasm mRNA transport, and alterations in mRNA stability. We have been characterizing alternative poly(A) site usage in the adenovirus major late transcription unit (MLTU) as a model for regulation at the level of pre-mRNA 3'-end processing. The MLTU contains five polyadenylation sites (L1 through L5). The promoter proximal site (L1) functions as the dominant poly(A) site during the early stage of adenovirus infection and in plasmid transfections when multiple poly(A) sites are present at the 3' end of a reporter plasmid. In contrast, stable mRNA processed at all five poly(A) sites is found during the late stage of adenovirus infection, after viral DNA replication has begun. Despite its dominance during early infection, L1 is a comparatively poor substrate for 3'-end RNA processing both in vivo and in vitro. In this study we have investigated the basis for the early L1 dominance. We have found that mRNA containing an unprocessed L1 poly(A) site is compromised in its ability to enter the steady-state pool of stable mRNA. This inhibition, which affects either the nuclear stability or nucleus-to-cytoplasm transport of the pre-mRNA, requires a cis-acting sequence located upstream of the L1 poly(A) site.

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-β], 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.

Recombinant, replication-deficient adenovirus (Ad) vectors have been successfully used to transfer and express the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA in vivo in the respiratory epithelium of experimental animals and humans with cystic fibrosis (CF). Since Ad-directed gene expression wanes over time, repeat administration is necessary to achieve an effective treatment for CF. A major hurdle to such a strategy is the possibility that anti-Ad humoral immunity may prevent gene expression in individuals with pre-existing anti-Ad immunity or following repeat administration. One strategy to circumvent such a problem would be alternating the use of Ad vectors belonging to different subgroups. Neutralizing antibodies developed with the administration of one Ad serotype do not cross-react with an Ad belonging to a second serotype in a manner that blocks infection and gene expression. To test this hypothesis, an immunizing dose of wild-type Ad5 (subgroup C), Ad4 (subgroup E), or Ad30 (subgroup D) was administered intratracheally to experimental animals, followed by an intratracheal administration of a replication-deficient subgroup C-derived vector coding for marker genes (chloramphenicol acetyl transferase or beta-galactosidase) or for the normal human CFTR cDNA. As expected, studies with vectors coding for marker genes or for CFTR cDNA demonstrated that airway administration of a vector does not yield efficient gene transfer, if there has been prior recent airway administration of the same Ad subgroup. In contrast, effective expression from the second administration can be achieved with an adenovirus vector belonging to a subgroup different from the first adenovirus administered. These data support the paradigm of alternating Ad vectors derived from different subgroups as strategy to circumvent anti-Ad humoral immunity, thus permitting the use of Ad vectors as a means to treat the respiratory manifestations of CF.

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.F7F41SΔRGD) 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.

Temporal regulation of poly(A) site choice occurs in an adenovirus recombinant encoding a miniature version of the major late transcription unit with two poly(A) sites, L1 and L3. Using deletion mutagenesis, we have looked directly for cis-acting elements regulating poly(A) site choice in this recombinant. From this work, we draw two main conclusions. First, elements other than the AAUAAA and downstream sequences of the L1 poly(A) site are required for temporal regulation of poly(A) site choice during infection. Second, these regions function in two distinct modes during infection. The two regions enhance selection of the L1 poly(A) site in an additive manner during an early infection, but deletion of either element abolishes the switch in poly(A) site choice during a late infection. This work documents the first example of a regulatory element downstream of a core poly(A) region.

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.

Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 (≈100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.

Adeno-associated virus (AAV) undergoes site-specific integration into human chromosome 19 through a deletion-substitution mechanism at the well characterized AAVS1 site. We have shown previously that a cis element within the left end of the AAV genome enhances the efficiency of Rep-mediated site-specific integration into chromosome 19 when present in inverted terminal repeat-containing recombinant AAV (rAAV) plasmids. We now demonstrate that a 138-bp cis element, the p5 integration efficiency element (p5IEE), mediates efficient integration. The p5IEE is not only required for efficient site-specific integration, it is also sufficient. Integration mediated by the p5IEE occurs in the absence of the AAV inverted terminal-repeat elements. The data presented in this study demonstrate that the p5IEE is a multifunctional element, serving as the highly regulatable Rep promoter and the primary substrate for targeted integration.

A general paradigm in signal transduction is ligand-induced feedback inhibition and the desensitization of signaling. We found that subthreshold concentrations of interferon-gamma (IFN-gamma), which did not activate macrophages, increased their sensitivity to subsequent IFN-gamma stimulation; this resulted in increased signal transducer and activator of transcription 1 (STAT1) activation and increased IFN-gamma#150;dependent gene activation. Sensitization of IFN-gamma signaling was mediated by the induction of STAT1 expression by low doses of IFN-gamma that did not effectively induce feedback inhibition. IFN-gamma signaling was sensitized in vivo after IFN-gamma injection, and STAT1 expression was increased after injection of lipopolysaccharide and in rheumatoid arthritis synovial cells. These results identify a mechanism that sensitizes macrophages to low concentrations of IFN-gamma and regulates IFN-gamma responses in acute and chronic inflammation.

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.

This study has used the strategy of gene replacement to characterize the contribution of the adenovirus (Ad) capsid protein hexon to serotype definition. By replacing the Ad type 5 (Ad5) hexon gene with sequences from Ad2, we have changed the type specificity of the chimeric virus. The type-determining epitopes are primarily associated with loop 1 of hexon and, to a much lesser degree, with loop 2. In spite of the serotype distinctiveness of the chimeric hexon viruses, epitope similarity between the vectors resulted in a low level of cross-reactive neutralizing antibody, which in combination with activated cellular and innate arms of the immune system is sufficient to suppress gene transduction following readministration in vivo.

Precursor cells found in the subventricular zone (SVZ) of the adult brain can undergo cell division and migrate long distances before differentiating into mature neurons. We have investigated the possibility of introducing genes stably into this population of cells. Replication-defective adenoviruses were injected into the SVZ of the lateral ventricle of adult mice. The adenoviruses carried a cDNA for the LacZ reporter or the human p75 neurotrophin receptor, for which species-specific antibodies are available. Injection of the viruses into the SVZ led to efficient labeling of neuronal precursors. Two months after viral injection, infected cells were detected in the olfactory bulb, a significant distance from the site of injection. Labeled periglomerular and granular neurons with extensive dendritic arborization were found in the olfactory bulb. These results demonstrate that foreign genes can be efficiently introduced into neuronal precursor cells. Furthermore, adenovirus-directed infection can lead to long-term stable gene expression in progenitor cells found in the adult central nervous system.

The efficient uptake of adenovirus into a target cell is a function of adenovirus capsid proteins and their interaction with the host cell. The capsid protein fiber mediates high-affinity attachment of adenovirus to the target cell. Although the cellular receptor(s) for adenovirus is unknown, evidence indicates that a single receptor does not function as the attachment site for each of the 49 different serotypes of adenovirus. Sequence variation of the fiber ligand, particularly in the C- terminal knob domain, is associated with serotype-specific binding specificity. Additionally, this domain of fiber functions as a major serotype determinant. Fiber involvement in cell targeting and its function as a target of the host immune response make the fiber gene an attractive target for manipulation, both from the perspective of adenovirus biology and from the perspective of using adenovirus vectors for gene transfer experiments. We have constructed a defective chimeric adenovirus type 5 (Ad5) reporter virus by replacing the Ad5 fiber gene with the fiber gene from Ad7A. Using the chloramphenicol acetyltransferase reporter gene, we have characterized this virus with respect to infectivity both in vitro and in vivo. We have also characterized the role of antifiber antibody in the host neutralizing immune response to adenovirus infection. Our studies demonstrate that exchange of fiber is a strategy that will be useful in characterizing receptor tropism for different serotypes of adenovirus. Additionally, the neutralizing immune response to Ad5 and Ad7 does not differentiate between two viruses that differ only in their fiber proteins. Therefore, following a primary adenovirus inoculation, antibodies generated against fiber do not constitute a significant fraction of the neutralizing antibody population.

The adenovirus major late transcription unit is a well-characterized transcription unit which relies heavily on alternative pre-mRNA processing to generate distinct populations of mRNA during the early and late stages of viral infection. In the early stage of infection, two major late transcription unit mRNA transcripts are generated through use of the first (L1) of five available poly(A) sites (L1 through L5). This contrasts with the late stage of infection when as many as 45 distinct mRNAs are generated, with each of the five poly(A) sites being used. In previous work characterizing elements involved in alternative poly(A) site use, we showed that the L1 poly(A) site is processed less efficiently than the L3 poly(A) site both in vitro and in vivo. Because of the dramatic difference in processing efficiency and the role processing efficiency plays in production of steady-state levels of mRNA, we have identified the sequence elements that account for the differences in L1 and L3 poly(A) site processing efficiency. We have found that the element most likely to be responsible for poly(A) site strength, the GU/U-rich downstream element, plays a minor role in the different processing efficiencies observed for the L1 and L3 poly(A) sites. The sequence element most responsible for inefficient processing of the L1 poly(A) site includes the L1 AAUAAA consensus sequence and those sequences which immediately surround the consensus hexanucleotide. This region of the L1 poly(A) site contributes to an inability to form a stable processing complex with the downstream GU/U-rich element. In contrast to the L1 element, the L3 poly(A) site has a consensus hexanucleotide and surrounding sequences which can form a stable processing complex in cooperation with the downstream GU/U-rich element. The L3 poly(A) site is also aided by the presence of sequences upstream of the hexanucleotide which facilitate processing efficiency. The sequence UUCUUUUU, present in the L3 upstream region, is shown to enhance processing efficiency as well as stable complex formation (shown by increased binding of the 64-kDa cleavage stimulatory factor subunit) and acts as a binding site for heterogeneous nuclear ribonucleoprotein C proteins.

To optimize the use of modified adenoviruses as vectors for gene delivery to the myocardium, we have characterized infection of cultured fetal and adult rat cardiac myocytes in vitro and of adult cardiac myocytes in vivo by using a replication-defective adenovirus carrying the chloramphenicol acetyltransferase (CAT) reporter gene driven by the cytomegalovirus promoter (AdCMVCATgD). In vitro, virtually all fetal or adult cardiocytes express the CAT gene when infected with 1 plaque-forming unit of virus per cell. CAT enzymatic activity can be detected in these cells as early as 4 hr after infection, reaching near-maximal levels at 48 hr. In fetal cells, CAT expression was maintained without a loss in activity for at least 1 week. Using in vitro studies as a guide, we introduced the AdCMVCATgD virus directly into adult rat myocardium and compared the expression results obtained from virus injection with those obtained by direct injection of pAdCMVCATgD plasmid DNA. The amount of CAT activity resulting from adenovirus infection of the myocardium was orders of magnitude higher than that seen from DNA injection and was proportional to the amount of input virus. Immunostaining for CAT protein in cardiac tissue sections following adenovirus injection demonstrated large numbers of positive cells, reaching nearly 100% of the myocytes in many regions of the heart. Expression of genes introduced by adenovirus peaked at 5 days but was still detectable 55 days following infection. Adenoviruses are therefore a very useful tool for high-efficiency gene transfer into the cardiovascular system.

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

For the majority of mRNA encoding eukaryotic transcription units, there is little or no knowledge of the elements responsible for transcription termination or how they may interact with RNA polymerase. In this report, we have used recombinant adenovirus reporter vectors to characterize the mouse beta maj globin sequence elements that cause transcription termination. Within the globin 3' termination region, we have identified at least three sequence elements which induce significant levels of transcription termination (> 50%). The smallest functionally active element (64% termination) is 69 bp in length. The natural arrangement of these elements results in a cumulative termination which is greater than 90%. Recognition of the termination elements by RNA polymerase II depends on the presence of a functional poly(A) signal sequence. We demonstrate that efficient transcription termination depends on appropriate spacing between the poly(A) signal sequence and the termination element.

In induced erythroleukemia cells, transcription of the beta-globin gene terminates in a region 600-1500 nucleotides downstream of the poly(A) site. To determine whether this region of the mouse DNA functions to terminate transcription when moved to another genomic site, portions of the putative termination region have been inserted into the E1A transcription unit of the adenovirus (type 5) chromosome. Analysis of RNA labeled either in isolated nuclei or in whole cells early after infection with reconstructed viruses indicated that transcription is terminated if the inserted DNA is oriented in the same direction as in the beta-globin transcription unit and contains the globin poly(A) site plus an additional 1395 nucleotides downstream. In addition to halting transcription within the E1A unit, the insertion of the terminator region had a negative cis effect on the E1B transcription unit, which normally initiates 363 bp downstream of the site of the globin insert. The E1B transcription unit was the only early gene affected, and complementation of the terminator virus with a wild-type E1A gene did not restore transcription of the E1B gene.