Ad-infected Cells Research Articles

Disruption of NBS1/MRN Complex Formation by E4orf3 Supports NF-κB That Licenses E1B55K-Deleted Adenovirus-Infected Cells to Accumulate DNA>4n.

ABSTRACTCells increase their DNA content greater than the G2/M (DNA > 4n) phases along the path to cancer. The signals that support this increase in DNA content remain poorly understood. Cells infected with adenovirus (Ad) similarly develop DNA > 4n and share a need to bypass the DNA damage response (DDR) signals that trigger cell cycle arrest, and/or cell death. Ads with deletion in early region 1B55K (ΔE1B Ad) are oncolytic agents that are currently being explored for use in vaccine delivery. Interestingly, they promote higher levels of DNA > 4n than Ads that contain E1B55K. Existing in these and almost all Ads that are being explored for clinical use, is early region 4 (E4). The Ad E4 open reading frame 3 (E4orf3) is a viral oncogene that interferes with the ability of cells to respond to DNA damage by disrupting MRN complex formation. Our study reveals that E4orf3 is required for the enhanced fraction of ΔE1B Ad-infected cells with DNA > 4n. For that reason, we explored signaling events mediated by E4orf3. We found that in ΔE1B Ad-infected cells, E4orf3, as reported by others, isolates NBS1 in nuclear dots and tracks. This allows for elevated levels of phosphorylated ATM that is linked to transcriptionally active NF-κB. Pharmacological inhibition of NF-κB reduced the fraction of ΔE1B Ad-infected cells with DNA > 4n while pharmacological inhibition of ATM reduced the levels of nuclear NF-κB and the fraction of ΔE1B Ad-infected cells with DNA > 4n and increased the fraction of dead or dying cells with fragmented DNA. This ability of E4orf3 to disrupt MRN complex formation that allows cells to bypass the cell cycle, evade death, and accumulate DNA > 4n, may be linked to its oncogenic potential.IMPORTANCE Genome instability, a hallmark of cancer, exists as part of a cycle that leads to DNA damage and DNA > 4n that further enhances genome instability. Ad E4orf3 is a viral oncogene. Here, we describe E4orf3 mediated signaling events that support DNA > 4n in ΔE1B Ad-infected cells. These signaling events may be linked to the oncogenic potential of E4orf3 and may provide a basis for how some cells survive with DNA > 4n.

Abstract 192: Persistence of First Generation Adenovirus in the Myocardium: Refuting Old Dogma

Introduction: Adenovirus (Ad) vectors do not integrate into host genomes and thus offer clinical benefits as non-mutagenic, acute-term gene delivery vehicles. One limitation to Ad vector use is the transient duration of transgene expression, supposedly resulting from immune-mediated cytolysis of Ad-infected cells. Our recent observation of Ad-induced transdifferentiation of infected cardiac fibroblasts into induced cardiomyocytes over 28 days in vivo called this premise into question. Therefore, we hypothesized that Ad remains in the myocardium for over 28 days and, to our knowledge, are the first to suggest alternate mechanisms for this persistence. Methods and Results: Ad encoding green fluorescent protein (AdGFP) or mock was administered via intramyocardial injection into Sprague-Dawley rats, and Ad genome and GFP expression were analyzed 3 or 28 days later (n=6 per group, 3 euthanized at each time point). Polymerase chain reaction typing and amplicon sequencing confirmed the presence of Ad DNA in all 6 treated animals. Quantitative real time polymerase chain reaction assays further demonstrated more than a three-fold increase in Ad DNA levels at both days 3 and 28 compared to untreated control animals (3.41±1.40 and 3.37±1.23, respectively). Thus, at 28 days Ad DNA persisted at a high level comparable to that which was present at 3 days. Next, AdGFP expression and host immune response were characterized by fluorescence microscopy and histology. We found that GFP-expressing cells decreased five-fold by 28 days (49±9 v. 3±0 cells). In addition, hematoxylin and eosin staining showed minimal infiltrating mononuclear inflammatory cells in AdGFP and mock-treated animals at both time points. Conclusions: Ad DNA and infected host cell persistence suggests that host-mediated cytolysis may not be responsible for the downregulation of transgene expression, indicating that Ad vectors may consequently be useful for therapies like cellular reprogramming wherein host cell persistence is required.

Active and separate secretion of fiber and penton base during the early phase of Ad2 or Ad5 infection

Fiber and penton base overproduced in adenovirus (Ad) infected cells can be secreted prior to progeny release and thereby regulate progeny spread. We aimed to investigate the mechanisms of fiber and penton base secretion in Ad2- or Ad5-infected A549 cells. Our flow cytometry analyses detected abundant surface fiber molecules, but little penton base molecules at 12h post infection. Immunogold staining combined with transmission electron microscopic analyses revealed separate, non-co-localized release of fiber and penton base in the proximity of the plasma membrane. Depolymerization of microtubule and actin cytoskeletons, and inhibition of Rock kinase and myosin II activity together demonstrated cytoskeletal network-dependent fiber secretion. Inhibition of intracellular calcium [Ca2+]i signaling caused diminished fiber secretion, which was associated with diminished progeny production. Thus, fiber and penton base are actively and separately secreted during the early stages of Ad2 or Ad5 infection, their secretion may play important role in Ad life cycle.

Automatic detection and measurement of viral replication compartments by ellipse adjustment.

Viruses employ a variety of strategies to hijack cellular activities through the orchestrated recruitment of macromolecules to specific virus-induced cellular micro-environments. Adenoviruses (Ad) and other DNA viruses induce extensive reorganization of the cell nucleus and formation of nuclear Replication Compartments (RCs), where the viral genome is replicated and expressed. In this work an automatic algorithm designed for detection and segmentation of RCs using ellipses is presented. Unlike algorithms available in the literature, this approach is deterministic, automatic, and can adjust multiple RCs using ellipses. The proposed algorithm is non iterative, computationally efficient and is invariant to affine transformations. The method was validated over both synthetic images and more than 400 real images of Ad-infected cells at various timepoints of the viral replication cycle obtaining relevant information about the biogenesis of adenoviral RCs. As proof of concept the algorithm was then used to quantitatively compare RCs in cells infected with the adenovirus wild type or an adenovirus mutant that is null for expression of a viral protein that is known to affect activities associated with RCs that result in deficient viral progeny production.

The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation.

ABSTRACTModulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection.

Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells.

Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that the synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle.

Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased proinflammatory cytokine response of human alveolar macrophages and more severe inflammatory lung disease in infected hamsters. This is the first evidence of a clinical relevance of differential expression of the small Ad E1B gene product. The results suggest that there is a low, critical threshold of E1B 19/20K expression that is needed for viral replication and infection transmission but that a higher level of E1B 19/20K expression is required for the usual repression and control of the Ad-triggered host innate immune response.

Antitumor effects of Newcastle disease virus hemagglutinin-neuraminidase used as a molecular adjuvant

Gene therapy is a potentially powerful tool used in cancer therapy. The strength of immune responses induced by some strategies is usually low, therefore, the development of agents capable of enhancing these responses is highlighted. The authors investigated the potential of an approach based on the hemagglutinin-neuraminidase(HN) of Newcastle disease virus(NDV) as a potential immune adjuvant. It was found that recombinant adenovirus(Ad) infected SGC7901 cells expressing HN exhibited both hemagglutinin(HA) and neuraminidase(NA) activities. It was demonstrated that administration of HN induced higher levels of the effector cytokines TNF-α, IFN-α and IFN-γ and increased natural killer(NK) cell activity. Based on the therapeutic tumor model, the results show that the administration of HN with Apoptin led to improved survival and tumor suppression. In conclusion, this study indicates that HN stimulates innate immune responses to make the activity of NK cells increased, which highlights the potential adjuvant activity of HN in cancer gene therapy.

Molecular Basis for Viral Selective Replication in Cancer Cells: Activation of CDK2 by Adenovirus-Induced Cyclin E

Adenoviruses (Ads) with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL), promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

Model System Using Controlled Receptor Expression for Evaluating Targeted Ultrasound Contrast Agents

This report details a model system for evaluating targeted ultrasound (US) contrast agents using adenoviral (Ad) vectors to regulate target receptor expression. Receptor density in vitro was modulated in breast cancer cells by varying the multiplicity of infection (MOI) from 0 to 100. Target receptors were induced using a green fluorescent protein (GFP) reporter Ad vector for gene transfer and expression of the hemagglutinin (HA) tag. These reporter genes were under the control of the ubiquitous cytomegalovirus (CMV) promoter. Subsequently, receptor expression and anti-HA antibody (Ab) binding was examined with flow cytometry. Targeted US contrast agents, or microbubbles (MB), were created by conjugating either biotinylated anti-HA or isotype control Ab to the surface of biotin coated MBs via a streptavidin bridge. Targeted MBs were incubated with Ad infected 2LMP cells to evaluate in vitro MB binding. Experimental results found GFP expression to be directly correlated with Ad MOI ( r 2 = 0.96). Increasing the Ad MOI produced a corresponding increase in binding and accumulation of anti-HA Ab on the cell surface ( p < 0.01). However, no difference was found between Cy5-labeled anti-HA Ab exposed cell groups at an MOI of 0 ( p > 0.29). Additionally, no difference was found between the isotype control Ab group ( p > 0.44) indicating minimal nonspecific binding. No difference was found between cell groups incubated with isotype-targeted MBs ( p > 0.42) regardless of receptor density. However, cells exposed to HA-targeted MBs showed increased levels of cell binding proportional to induced receptor expression levels ( p < 0.02).

The E3 Ubiquitin Ligase Activity Associated with the Adenoviral E1B-55K–E4orf6 Complex Does Not Require CRM1-Dependent Export

The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase.

Co‐chaperone BAG3 and adenovirus penton base protein partnership

The BAG family of Hsp70/Hsc70 co‐chaperones is characterised by the presence of a conserved BAG domain at the carboxyl‐terminus. BAG3 protein is the only member of this family containing also the N‐terminally located WW domain. We describe here the identification of adenovirus (Ad) penton base protein as the first BAG3 partner recognising BAG3 WW domain. Ad penton base is the viral capsid constituent responsible for virus internalisation. It contains in the N‐terminal part two conserved PPxY motifs, known ligands of WW domains. In cells producing Ad penton base protein, cytoplasmic endogenous BAG3 interacts with it and co‐migrates to the nucleus. Preincubation of BAG3 with Ad base protein results in only slight modulation of BAG3 co‐chaperone activity, suggesting that this interaction is not related to the classical BAG3 co‐chaperone function. However, depletion of BAG3 impairs the cell entry of the virus and viral progeny production in Ad‐infected cells, suggesting that the interaction between virus penton base protein and cellular co‐chaperone BAG3 positively influences virus life cycle. These results thus demonstrate a novel host–pathogen interaction, which contributes to the successful infectious life cycle of adenoviruses. In addition, these data enrich our knowledge about the multifunctionality of the BAG3 co‐chaperone. J. Cell. Biochem. 111: 699–708, 2010. © 2010 Wiley‐Liss, Inc.

Nectin-like Molecule 1 Inhibits the Migration and Invasion of U251 Glioma Cells by Regulating the Expression of An Extracellular Matrix Protein Osteopontin

To investigate the molecular mechanism of nectin-like molecule 1 (NECL1) inhibiting the migration and invasion of U251 glioma cells. We infected U251 glioma cells with adeno-nectin-like molecule 1 (Ad-NECL1) or empty adenovirus (Ad). Transwell and wound healing assays were performed to observe the migration of U251 cells incubated with the cell supernatant from Ad-NECL1 or Ad infected U251 cells. DNA microarray was applied to screen the gene expression profile after the restoration of NECL1 in U251 glioma cell lines. The differential expression of osteopontin (OPN), a gene related to migration and invasion, was further analyzed with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunohistochemistry. The restoration of NECL1 inhibited migration of U251 cells significantly (P<0.05). Altogether 195 genes were found differentially expressed by microarray, in which 175 were up-regulated and 20 down-regulated, including 9 extracellular matrix proteins involved in the migration of cells. Both mRNA and protein expressions of OPN, the most markedly reduced extracellular matrix protein, were found decreased in U251 cells after restoration of NECL1. Immunohistochemical assay also detected an increase of OPN in glioma tissues, related with the progressing of malignant grade. A link might exist between NECL1 and the extracellular matrix protein OPN in inhibiting the migration and invasion of U251 glioma cells.

Cytosolic calcium regulates liver regeneration in the rat†

Liver regeneration is regulated by growth factors, cytokines, and other endocrine and metabolic factors. Calcium is important for cell division, but its role in liver regeneration is not known. The purpose of this study was to understand the effects of cytosolic calcium signals in liver growth after partial hepatectomy (PH). The gene encoding the calcium-binding protein parvalbumin (PV) targeted to the cytosol using a nuclear export sequence (NES), and using a discosoma red fluorescent protein (DsR) marker, was transfected into rat livers by injecting it, in recombinant adenovirus (Ad), into the portal vein. We performed two-thirds PH 4 days after Ad-PV-NES-DsR or Ad-DsR injection, and liver regeneration was analyzed. Calcium signals were analyzed with fura-2-acetoxymethyl ester in hepatocytes isolated from Ad-infected rats and in Ad-infected Hela cells. Also, isolated hepatocytes were infected with Ad-DsR or Ad-PV-NES-DsR and assayed for bromodeoxyuridine incorporation. Ad-PV-NES-DsR injection resulted in PV expression in the hepatocyte cytosol. Agonist-induced cytosolic calcium oscillations were attenuated in both PV-NES-expressing Hela cells and hepatocytes, as compared to DsR-expressing cells. Bromodeoxyuridine incorporation (S phase), phosphorylated histone 3 immunostaining (mitosis), and liver mass restoration after PH were all significantly delayed in PV-NES rats. Reduced cyclin expression and retinoblastoma protein phosphorylation confirmed this observation. PV-NES rats exhibited reduced c-fos induction and delayed extracellular signal-regulated kinase 1/2 phosphorylation after PH. Finally, primary PV-NES-expressing hepatocytes exhibited less proliferation and agonist-induced cyclic adenosine monophosphate responsive element binding and extracellular signal-regulated kinase 1/2 phosphorylation, as compared with control cells. Cytosolic calcium signals promote liver regeneration by enhancing progression of hepatocytes through the cell cycle.

Arginine Methylation of Human Adenovirus Type 5 L4 100-Kilodalton Protein Is Required for Efficient Virus Production

The adenovirus type 5 (Ad5) late region 4 (L4) 100-kDa nonstructural protein (L4-100K) mediates inhibition of cellular protein synthesis and selective translation of tripartite leader (TL)-containing viral late mRNAs via ribosome shunting. In addition, L4-100K has been implicated in the trimerization and nuclear localization of hexon protein. We previously proved that L4-100K is a substrate of the protein arginine methylation machinery, an emergent posttranslational modification system involved in a growing list of cellular processes, including transcriptional regulation, cell signaling, RNA processing, and DNA repair. As understood at present, L4-100K arginine methylation involves protein arginine methyltransferase 1 (PRMT1), which asymmetrically dimethylates arginines embedded in arginine-glycine-glycine (RGG) or glycine-arginine-rich (GAR) domains. To identify the methylated arginine residues and assess the role of L4-100K arginine methylation, we generated amino acid substitution mutations in the RGG and GAR motifs to examine their effects in Ad-infected and plasmid-transfected cells. Arginine-to-glycine exchanges in the RGG boxes significantly diminished L4-100K methylation in the course of an infection and substantially reduced virus growth, demonstrating that L4-100K methylation in RGG motifs is an important host cell function required for efficient Ad replication. Our data further indicate that PRMT1-catalyzed arginine methylation in the RGG boxes regulates the binding of L4-100K to hexon and promotes the capsid assembly of the structural protein as well as modulating TL-mRNA interaction. Furthermore, substitutions in GAR, but not RGG, regions affected L4-100K nuclear import, implying that the nuclear localization signal of L4-100K is located within the GAR sequence.

Adenovirus DNA polymerase is recognized by human CD8+ T cells

Donor lymphocytes have potential as a treatment for adenovirus (Ad) disease in haematopoietic stem cell transplant (SCT) recipients, but better understanding of Ad-specific T-cell responses is required. Most healthy adults exhibit memory T-cell responses to hexon, a capsid protein synthesized late after infection. However, since the Ad E3-19k downregulates major histocompatibility complex (MHC) class I molecules, cytotoxic T cells (CTLs) targeted to early viral proteins may be more effective in eliminating Ad-infected cells in vivo. Here we show that Ad-specific CTLs recognize the early region 2 proteins DNA polymerase (Pol) and DNA-binding protein (DBP). Firstly, memory Ad-specific CD8(+) T cells were amplified from healthy donors by in vitro stimulation with Ad-infected dendritic cells and found to exhibit MHC-restricted cytotoxicity to targets expressing Pol and DBP. Secondly, gamma interferon responses to HLA A2-binding motif peptides from Pol and DBP were directly detected in peripheral blood mononuclear cells (PBMCs) from a recently infected normal donor. Peptide-specific CTLs generated to Pol and DBP epitopes were confirmed to exhibit HLA A2-restricted killing of targets expressing Pol or DBP. Lastly, Pol-epitope-specific T cells were detected at similar or higher frequencies than hexon and DBP in three of three SCT recipients recovering from invasive Ad disease. Pol epitopes were well conserved among different Ad serotypes. Therefore, Pol is a promising target for immunotherapy of Ad disease.

Construction and identification of recombinant adenoviral vector encoding B7-H4 gene

To construct the recombinant adenovirus containing B7-H4 gene with AdEasy XL system and to identify its biological activities. The full-length mouse B7-H4 gene was amplified by RT-PCR from C57 mouse lung and put into T vector, then verified by sequencing. Digested with Xhol I and EcoR V the B7-H4 gene was inserted into pshuttle-CMW(PSC). Pme I linearized shuttle plasmid was transformed into E.coli BJ5183-AD-1 to obtain the recombinant adenoviral plasmid pAd-mB7-H4 by efficient homologous recombination. Then the recombinant adenovirus-mB7-H4/Ad was obtained by packaging Pac I linearized in D-293 cells. The mRNA and protein expression of B7-H4 in mB7-H4/Ad infected AD-293 cells were detected by RT-PCR and Western blot, respectively. mB7-H4/Ad was used to infect L929 cells, the bioactivity of expressed B7-H4 in stimulation of T lymphocytes proliferation and cytokine production were tested. The full-length of mB7-H4 was cloned from mouse lung tissue cDNA and verified by sequencing. The recombinant plasmid pAd-m B7-H4 was successfully generated by homologous recombination, and the primary mB7-H4/Ad was obtained by packaging pAd-B7-H4 in AD-293 cells. Compared with the negative control, L929 cells infected with mB7-H4/Ad effectively inhibited the proliferation of T lymphocytes and cytokines production. The bioactive recombinant adenovirus mB7-H4/Ad has been successfully constructed.

A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.

Adenovirus E3/19K Promotes Evasion of NK Cell Recognition by Intracellular Sequestration of the NKG2D Ligands Major Histocompatibility Complex Class I Chain-Related Proteins A and B

The adenovirus (Ad) early transcription unit 3 (E3) encodes multiple immunosubversive functions that are presumed to facilitate the establishment and persistence of infection. Indeed, the capacity of E3/19K to inhibit transport of HLA class I (HLA-I) to the cell surface, thereby preventing peptide presentation to CD8(+) T cells, has long been recognized as a paradigm for viral immune evasion. However, HLA-I downregulation has the potential to render Ad-infected cells vulnerable to natural killer (NK) cell recognition. Furthermore, expression of the immediate-early Ad gene E1A is associated with efficient induction of ligands for the key NK cell-activating receptor NKG2D. Here we show that while infection with wild-type Ad enhances synthesis of the NKG2D ligands, major histocompatibility complex class I chain-related proteins A and B (MICA and MICB), their expression on the cell surface is actively suppressed. Both MICA and MICB are retained within the endoplasmic reticulum as immature endoglycosidase H-sensitive forms. By analyzing a range of cell lines and viruses carrying mutated versions of the E3 gene region, E3/19K was identified as the gene responsible for this activity. The structural requirements within E3/19K necessary to sequester MICA/B and HLA-I are similar. In functional assays, deletion of E3/19K rendered Ad-infected cells more sensitive to NK cell recognition. We report the first NK evasion function in the Adenoviridae and describe a novel function for E3/19K. Thus, E3/19K has a dual function: inhibition of T-cell recognition and NK cell activation.

Evidence for Involvement of BH3-Only Proapoptotic Members in Adenovirus-Induced Apoptosis

Mammalian cells infected with human adenoviruses (Ads) undergo an apoptotic response as a result of expression of the viral E1A proteins, and this process is suppressed by the viral E1B-19K protein. The intermediary steps in the Ad-induced apoptosis pathway are not fully resolved. The apical step in the canonical mammalian apoptosis pathway involves functional activation of one or more of the BH3-only BCL-2 family proapoptotic proteins. Previous reports have suggested that Ad-induced apoptosis may be initiated at checkpoints downstream of the BH3-only proteins. Here, we undertook genetic and biochemical studies to determine the roles of BH3-only proteins in Ad-induced apoptosis. We examined the activities of the cellular antiapoptosis protein BCL-xL and its mutants expressed from the E1B region of the Ad5 genome. Our results showed efficient suppression of Ad-induced apoptosis by a BCL-xL mutant (mt1) deficient in interaction with multidomain proapoptotic proteins BAX and BAK but proficient in interaction with BH3-only proteins, suggesting a role for BH3-only proteins in the initiation of Ad-induced apoptosis. Further, the antiapoptotic activity of BCL-xL mt1 in Ad-infected cells was observed in spite of BAK activation as a consequence of MCL-1 degradation. Analysis of the mRNA levels of various BH3-only members by reverse transcription-PCR revealed prominent activation of the Bik gene. Further, the BIK protein was also modified into an apoptotically enhanced phosphorylated form during the viral infection. In addition to BIK, enhanced level of BIM was observed in Ad-infected cells. Between the two major E1A proteins coded by the 12S and 13S mRNAs, the 13S product appeared to contribute to the activation of these BH3-only members and apoptosis during viral infection. Depletion of BIK by the use of small interfering RNA reduced the level of Ad-induced apoptosis. Our results are consistent with a model that activation of the BH3-only members may initiate Ad-induced apoptosis.