E1B 19K Research Articles

The deletion of adenovirus E1B-19KD gene enhanced its cell killing ability against lung cancer cell line A549

Oncolytic Viruses immunotherapy is one of the most frequently used anti-cancer agents because it can significantly reverse local immune suppression and amplify antitumor immunity. Although drugs were already applied in cancer treatment, a new agent may open a new pathway for developing oncolytic therapy. This study intends to improve the adenoviral vector to increase its killing effect on tumor cells for effective and safe cancer gene therapy. We modified the pDC316 plasmid by adding TERT promoter, E1A, and E1B viral genes through homologous recombination and transfection. Then the virus was reassured of its stability through PCR analysis, Western blot, viral titer assays, and CPE assay. In the result, Ad-E1B 55k virus especially showed strong cytotoxicity of more than 50% of killing efficacy in vivo A549 cells. Cell viability in HepG2 is around 80% while in MRC5 cells, the viability is approximately 100%, which showed a tumor-specificity of the viruses. Moreover, only on day 4 has 20% of the efficacy of E1B 55k virus and the viability quickly returned to normal on the next day, which indicates the relative safety of the modified in MRC5 normal cells. In conclusion, the modified virus may serve as another starting point to design a potentially safer, better efficacy, and specificity in cancer immunotherapy. However, a further test of the vector in animal trials is recommended.

Bloodletting Acupuncture at Jing-Well Points Alleviates Myocardial Injury in Acute Altitude Hypoxic Rats by Activating HIF-1α/BNIP3 Signaling-Mediated Mitochondrial Autophagy and Decreasing Oxidative Stress.

To explore the protective effect and possible mechanisms of bloodletting acupuncture at Jing-well points (BAJP) pre-treatment on acute hypobaric hypoxia (AHH)-induced myocardium injury rat. Seventy-five rats were randomly divided into 5 groups by a random number table: a control group (n=15), a model group (n=15), a BAJP group (n=15), a BAJP+3-methyladenine (3-MA) group (n=15), and a BANA (bloodletting at nonacupoint; tail bleeding, n=15) group. Except for the control group, the AHH rat model was established in the other groups, and the corresponding treatment methods were adopted. Enzyme-linked immunosorbent assay (ELISA) was used to detect creatine kinase isoenzyme MB (CK-MB) and cardiac troponins I (CTnI) levels in serum and superoxide dismutase (SOD) and malondialdehyde (MDA) levels in myocardial tissue. Hematoxylin-eosin (HE) staining was used to observe myocardial injury, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to observe cell apoptosis. Transmission electron microscopy detection was used to observe mitochondrial damage and autophagosomes in the myocardium. The mitochondrial membrane potential of the myocardium was analyzed with the fluorescent dye JC-1. Mitochondrial respiratory chain complex (complex I, III, and IV) activities and ATPase in the myocardium were detected by mitochondrial respiratory chain complex assay kits. Western blot analysis was used to detect the autophagy index and hypoxia inducible factor-1α (HIF-1α)/Bcl-2 and adenovirus E1B 19k Da-interacting protein 3 (BNIP3) signaling. BAJP reduced myocardial injury and inhibited myocardial cell apoptosis in AHH rats. BAJP pretreatment decreased MDA levels and increased SOD levels in AHH rats (all P<0.01). Moreover, BAJP pretreatment increased the mitochondrial membrane potential (P<0.01), mitochondrial respiratory chain complex (complexes I, III, and IV) activities (P<0.01), and mitochondrial ATPase activity in AHH rats (P<0.05). The results from electron microscopy demonstrated that BAJP pretreatment improved mitochondrial swelling and increased the autophagosome number in the myocardium of AHH rats. In addition, BAJP pretreatment activated the HIF-1α/BNIP3 pathway and autophagy. Finally, the results of using 3-MA to inhibit autophagy in BAJP-treated AHH rats showed that suppression of autophagy attenuated the treatment effects of BAJP in AHH rats, further proving that autophagy constitutes a potential target for BAJP treatment of AHH. BAJP is an effective treatment for AHH-induced myocardial injury, and the mechanism might involve increasing HIF-1α/BNIP3 signaling-mediated autophagy and decreasing oxidative stress.

Mouse Adenovirus Type 1 E4orf6 Induces PKR Degradation.

Protein kinase R (PKR) is a cellular kinase involved in the antiviral response. The inactivation or inhibition of this protein is a conserved activity in DNA and RNA virus infections. In contrast to human adenovirus type 5, mouse adenovirus type 1 (MAV-1) inhibits PKR activity through proteasome-dependent degradation. However, the molecular mechanism by which this process takes place is not fully understood. We investigated whether ubiquitination, MAV-1 early region 1B 55k (E1B 55k), and early region 4 orf6 (E4orf6) play a role in PKR degradation in MAV-1 infection, because the enzyme 3 (E3) ubiquitin ligase activity with these viral proteins is conserved among the Adenoviridae family. We provide evidence that E4orf6 is sufficient to induce mouse PKR degradation and that proteasome pathway inhibition blocks PKR degradation. Inhibition of neddylation of cullin, a component of E3 ubiquitin ligase complex, blocked efficient PKR degradation in MAV-1-infected cells. Finally, we demonstrated that MAV-1 degradation of PKR is specific for mouse PKR. These results indicate that counteracting PKR is mechanistically different in two species of adenoviruses. IMPORTANCE Viruses have evolved to counteract the immune system to successfully replicate in the host. Downregulation of several antiviral proteins is important for productive viral infection. Protein kinase R (PKR) is an antiviral protein that belongs to the first line of defense of the host. Because PKR senses dsRNA and blocks the cellular translation process during viral infections, it is not surprising that many viruses counteract this antiviral activity. We previously reported PKR degradation during mouse adenovirus type 1 (MAV-1) infection; however, the molecular mechanism of this activity was not fully known. This work provides evidence about the MAV-1 protein that induces PKR degradation and expands knowledge about involvement of the proteasome pathway.

Adenovirus-Mediated Cytotoxicity of Chronic Lymphocytic Leukemia Cells

We have studied adenovirus-mediated cytotoxicity after infection of malignant cells obtained from patients with chronic lymphocytic leukemia (CLL). Our studies indicate that adenoviruses can infect primary CLL cells and that infection of CLL cells with a replication-competent strain of human adenovirus 5 (Ad5dl309) results in cytotoxicity. Adenovirus-mediated cytotoxicity was also seen after infection of CLL cells with a variety of viruses attenuated by mutations in the adenovirus early region 1 (E1) or early region 2 (E2). Even viruses attenuated by deletion of the entire E1 region resulted in cytotoxicity after infection of the CLL cells obtained from some patients. Although there was variability in the degree of cytotoxicity induced by different viruses in different patients cells, a virus with a mutation in the E1B 19K gene resulted in the greatest degree of cytotoxicity in most of the CLL samples tested. These studies demonstrate that infection of CLL cells by attenuated adenoviruses with specific mutations in the E1 or E2 region results in cell death. Attenuated adenoviruses should be developed further as therapeutic agents for patients with CLL.

The biology of the adenovirus E1B 55K protein.

The adenovirus E1B 55K (E1B) protein plays major roles in productive adenoviral infection and cellular transformation. Interest in E1B increased because of the potential of adenoviruses as therapeutic vectors, and the E1B gene is commonly deleted from adenovirus vectors for anticancer therapy. E1B activities are spatiotemporally regulated through SUMOylation and phosphorylation, and through interactions with multiple partners that occur presumably at different intracellular sites and times postinfection. E1B is implicated in the formation of viral replication compartments and regulates viral genome replication and transcription, transcriptional repression, degradation of cellular proteins, and several intranuclear steps of viral late mRNA biogenesis. Here, we review advances in our understanding of E1B during productive adenovirus replication and discuss fundamental aspects that remain unresolved.

Promyelocytic leukemia protein isoform II inhibits infection by human adenovirus type 5 through effects on HSP70 and the interferon response.

Promyelocytic leukemia (PML) proteins have been implicated in antiviral responses but PML and associated proteins are also suggested to support virus replication. One isoform, PML-II, is required for efficient transcription of interferon and interferon-responsive genes. We therefore investigated the PML-II contribution to human adenovirus 5 (Ad5) infection, using shRNA-mediated knockdown. HelaΔII cells showed a 2-3-fold elevation in Ad5 yield, reflecting an increase in late gene expression. This increase was found to be due in part to the reduced innate immune response consequent upon PML-II depletion. However, the effect was minor because the viral E4 Orf3 protein targets and inactivates this PML-II function. The major benefit to Ad5 in HelaΔII cells was exerted via an increase in HSP70; depletion of HSP70 completely reversed this replicative advantage. Increased Ad5 late gene expression was not due either to the previously described inhibition of inflammatory responses by HSP70 or to effects of HSP70 on major late promoter or L4 promoter activity, but might be linked to an observed increase in E1B 55K, as this protein is known to be required for efficient late gene expression. The induction of HSP70 by PML-II removal was specific for the HSPA1B gene among the HSP70 gene family and thus was not the consequence of a general stress response. Taken together, these data show that PML-II, through its various actions, has an overall negative effect on the Ad5 lifecycle.

A novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expression.

Many human (different serotypes) and nonhuman adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a porcine adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

Clinical Trials with Oncolytic Viruses: Current and Future Prospects

Reviewing the research in the field of oncolytic virus therapy (OVT) of the past two decades, it is inspiring to see the enormous amount of success accomplished by the scholars of this innovative therapeutic technique. Though the experimental trials have been ongoing from 1990s, however, it took a leap forward with approval of the clinical trials in China, 2005. The world's first oncolytic virus to be approved by their government was adenovirus (with E1B 55K gene deletion) for head and neck cancer therapy along with chemotherapy.

Abstract A38: Novel oncolytic adenovirus (ILV-19) induces apoptosis in BRAF inhibitor resistant (V600E) mutant melanoma cell lines 451lu-R and Mel1617-R

Abstract Over 50% percent of melanomas carry activating BRAF mutation with a vast majority of these mutations (&amp;gt;90%) resulting from a single nucleotide switch at amino acid 600 resulting in a glutamic acid substitution for valine (V600E). As a result of this mutation BRAF Kinase is constitutively expressed and results in disregulation of the RAS-RAF-MEK-ERK protein kinase pathway that controls cell proliferation and apoptosis. Although, Vemurafenib, a specific inhibitor of the V600E BRAF mutant, has shown to have initial positive clinical response in clinical trials, drug resistant develops after several months of continuous exposure resulting in the recurrence of the treated cancer. We have developed a modified adenovirus vector (AdILV-19) that effectively kills vemurafenib resistant cells. We demonstrate that AdILV-19 suppresses expression of c-Myc, survivin, cyclin D1, B-catenin, and Mcl-1 in BRAF V600E mutated cells that are resistant to vemurafenib. This novel vector, AdILV-19 is derived from our previously published adenoviral vector TAV-255 through an additional modification of the adenoviral genome. In order to facilitate and maximize recombinant adenoviral induced apoptosis by this new vector, we removed the first 200 base pair of the adenoviral E1b 19k protein, a human BCL-2 homolog. Using this newly modified virus in two highly Vemurafenib resistant melanoma cell lines (451lu-R, Mel1617-R) we were able to not only infect and express viral proteins i.e. E1a, but also induce a significant cell killing in these resistant BRAF mutant cell lines by MTT as well as crystal violet assays. MTT assay showed 35% to 45% cell killing at an MOI as low as 15 to 80 to 90% killing at an MOI of 50. Crystal violet assay on the other hand showed a significant cytotoxicity in these resistant cells at a low MOI of 15. Additionally, we were able to show using Immunoblot analysis a postinfection reduction in the expression of major pro-survival proteins in time dependent manner. We observed as early as 24hrs postinfection c-Myc (an oncogenic transcription factor) and survivin (Pro-survival protein) expression were turned off while expression of cyclin D1 (a regulatory subunit of holoenzyme) which shown to be amplified in BRAF inhibitor resistant melanoma cells and Mcl-1 (a pro-survival protein) were turned off as early as 48hs postinfection in both 451lu-R and Mel1617-R melanoma cells. Furthermore, colony formation assays showed a substantial reduction in the ability of single cells to form colonies after infection at MOI of 20 with AdILV-19 in both of these resistant melanoma cell lines. As an extension of these studies we also studied the activity of AdILV-19 to treat a xenograft nude mouse model with subcutaneous injection of 451lu-R melanoma cells. When the tumor volume reached 100 mm3 the mice received 3 intratumoral injections at an MOI of 20. Following treatment, we observed a significant inhibition of tumor growth (p≤0.02) in treated mice (n=4) when compared with PBS treated mice controls. Taken together these data we show this newly modified adenovirus vector AdILV-19 alters and silences cellular oncogenic protein synthesis in vemurafenib resistant BRAF mutant melanomas and has a great potential as therapeutic for the treatment of these human resistant melanomas. Citation Format: Farah Hedjran, Lingzhi Zhang, Jason Sicklick, Tony Reid. Novel oncolytic adenovirus (ILV-19) induces apoptosis in BRAF inhibitor resistant (V600E) mutant melanoma cell lines 451lu-R and Mel1617-R. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A38.

Adenovirus E1B 19-kilodalton protein modulates innate immunity through apoptotic mimicry.

Cells that undergo apoptosis in response to chemical or physical stimuli repress inflammatory reactions, but cells that undergo nonapoptotic death in response to such stimuli lack this activity. Whether cells dying from viral infection exhibit a cell death-type modulatory effect on inflammatory reactions is unknown. We compared the effects on macrophage inflammatory responses of cells dying an apoptotic or a nonapoptotic death as a result of adenoviral infection. The results were exactly opposite to the predictions from the conventional paradigm. Cells dying by apoptosis induced by infection with an adenovirus type 5 (Ad5) E1B 19-kilodalton (E1B 19K) gene deletion mutant did not repress macrophage NF-κB activation or cytokine responses to proinflammatory stimuli, whereas cells dying a nonapoptotic death from infection with E1B 19K-competent, wild-type Ad5 repressed these macrophage inflammatory responses as well as cells undergoing classical apoptosis in response to chemical injury. The immunorepressive, E1B 19K-related cell death activity depended upon direct contact of the virally infected corpses with responder macrophages. Replacement of the viral E1B 19K gene with the mammalian Bcl-2 gene in cis restored the nonapoptotic, immunorepressive cell death activity of virally infected cells. These results define a novel function of the antiapoptotic, adenoviral E1B 19K protein that may limit local host innate immune inflammation during accumulation of virally infected cells at sites of infection and suggest that E1B 19K-deleted, replicating adenoviral vectors might induce greater inflammatory responses to virally infected cells than E1B 19K-positive vectors, because of the net effect of their loss-of-function mutation. We observed that cells dying a nonapoptotic cell death induced by adenovirus infection repressed macrophage proinflammatory responses while cells dying by apoptosis induced by infection with an E1B 19K deletion mutant virus did not repress macrophage proinflammatory responses and enhanced some cytokine responses. Our results define a new function of the antiapoptotic, adenoviral protein E1B 19K, which we have termed "apoptotic mimicry." Our studies suggest the possibility that the presence or absence of this E1B 19K function could alter the immunological outcome of both natural and therapeutic adenoviral infections. For example, emerging, highly immunopathogenic adenovirus serotypes might induce increased host inflammatory responses as a result of altered E1B 19K function or expression. It is also possible that engineered variations in E1B 19K expression/function could be created during adenovirus vector design that would increase the therapeutic efficacy of replicating adenovirus vectors for vaccines or oncolytic viral targeting of neoplastic cells.

Molecular analysis of a fragment of gene E1B 19K of canine adenovirus 2 (CAV-2) isolated from dogs with symptoms of cough

The aim of this study was to perform molecular analysis of canine adenovirus 2 (CAV-2) E1B 19K gene fragment isolated from 20 dogs of various breeds (12 males and 8 females aged 1-9 years), with clinical symptoms of upper respiratory tract infections, from the Lubelszczyzna region. Nasal swabs were taken from dogs. DNA of CAV-2 was detected using the PCR method in 16 swabs. All PCR products were sequenced, and the obtained sequences were compared with each other and with the sequence of the E1B 19K gene of the CAV-2 strain from an online database of NCBI GenBank: AC 000003. Based on analysis of the obtained sequences, three polymorphic variants of CAV-2 (No. 1-3) with homology of 78 - 100% were distinguished. The nucleotide and amino acid sequences of the most frequently represented polymorphic variant, No. 1, differed from the sequences of polymorphic variant No. 2 with one substitution. The nucleotide and amino acid sequence of the E1B 19K gene of CAV-2 AC 000003 differed from the analogous sequences of representatives of variant No. 1 with 44 nucleotide and 19 amino acid substitutions. The small number of nucleotide differences in the E1B 19K CAV-2 gene among the examined own isolates, compared with AC 000003, suggest that the infections in dogs were caused by a relatively genetically stable virus which occurs in eastern

The α2 helix in the DNA ligase IV BRCT-1 domain is required for targeted degradation of ligase IV during adenovirus infection

In adenovirus E4 mutant infections, viral DNAs form concatemers through a process that requires host Non-homologous End Joining (NHEJ) proteins including DNA Ligase IV (LigIV). Adenovirus proteins E4 34k and E1b 55k form the substrate-selection component of an E3 ubiquitin ligase and prevent concatenation by targeting LigIV for proteasomal degradation. The mechanisms and sites involved in targeting this and other E3 ligase substrates generally are poorly-understood. Through genetic analysis, we identified the α2 helix of one LigIV BRCT domain (BRCT-1) as essential for adenovirus-mediated degradation. Replacement of the BRCT domain of DNA ligase III (LigIII), which is resistant to degradation, with LigIV BRCT-1 does not promote degradation. A humanized mouse LigIV that possesses a BRCT-1 α2 helix identical to the human protein, like its parent, is also resistant to adenovirus-mediated degradation. Thus, both the BRCT-1 α2 helix and an element outside BRCT-1 are required for adenovirus-mediated degradation of LigIV.

The WTX Tumor Suppressor Enhances p53 Acetylation by CBP/p300

WTX encodes a tumor suppressor, frequently inactivated in Wilms tumor, with both plasma membrane and nuclear localization. WTX has been implicated in β-catenin turnover, but its effect on nuclear proteins is unknown. We report an interaction between WTX and p53, derived from the unexpected observation of WTX, p53, and E1B 55K colocalization within the characteristic cytoplasmic body of adenovirus-transformed kidney cells. In other cells without adenovirus expression, the C-terminal domain of WTX binds to the DNA-binding domain of p53, enhances its binding to CBP, and increases CBP/p300-mediated acetylation of p53 at Lys 373/382. WTX knockdown accelerates CBP/p300 protein turnover and attenuates this modification of p53. In p53-reconstitution experiments, cell-cycle arrest, apoptosis, and p53 target-gene expression are suppressed by depletion of WTX. Together, these results suggest that WTX modulates p53 function, in part through regulation of its activator CBP/p300.

Construction of gene transfer vectors based on simian adenovirus 7

The complete nucleotide sequence of an isolate of simian adenovirus 7 (SAdV-7) was determined. The genome organization of this isolate was found to be similar to that of other primate adenoviruses with two principal notable points: severe truncation of the E1A and E1B 19K proteins and an E3 region encoding only the 12.5K homologue. The viral gene products of SAdV-7 are most closely related to simian adenovirus 1 (SAdV-1), and like SAdV-1, are related to the human adenovirus species HAdV-F, such as the enteric adenoviruses HAdV-40 and HAdV-41 and the recently defined HAdV-G (HAdV-52). Two kinds of gene transfer vectors were made: a replication-competent SAdV-7-based vector with no genomic deletion, and a standard replication-incompetent vector deleted for E1. Importantly, the E1-deleted vector could be propagated to high titre by trans-complementation in human HEK 293 cells.

Abstract 613: Depletion of Mdm2 and Mdm4 by adenovirus: Molecular mechanism and anticancer therapeutic implication

Abstract Mdm2 and Mdm4 are two paralogous proteins. They contain a conserved p53-binding domain in the N-terminal region and a RING-finger domain in the C-terminal part. Both proteins physically bind p53 and prevent p53 activation. Biochemically, Mdm2 acts as an E3 ubiquitin ligase of p53 and promotes ubiquitin-dependent p53 degradation. Mdm4 does not appear to have intrinsic E3 ubiquitin ligase activity and may negatively control p53 function through suppressing p53-mediated transactivation of gene expression. Mdm2 and Mdm4 also form heterodimer, which suggests that Mdm2 and Mdm4 might cooperatively control p53 activity. In vivo, both Mdm2 and Mdm4 have an essential but non-redundant function in suppressing p53 activity to enable embryonic development. Mdm2 and Mdm4 are overexpressed in several types of human cancer such as breast cancer and osteosarcoma. In transgenic mouse models, overexpression of Mdm2 or Mdm4 leads to tumorigenesis. Thus, both Mdm2 and Mdm4 are bona fide oncogenes. Several small molecules that disrupt the interaction between p53 and Mdm2 and Mdm4 have been shown to induce p53 activation, cell cycle arrest and apoptosis. These compounds also cause tumor repression in preclinical animal models. Thus, they have clinical potentials for treating various types of human cancer. Biological agents that specifically target Mdm2 and Mdm4 have not been described. We found that adenovirus infection of diverse type of cancer cells results in dramatic downregulation of both Mdm2 and Mdm4. Adenovirus E1B-55K binds specifically both Mdm2 and Mdm4 in cells and in vitro, demonstrating specific interactions between E1B-55K and Mdm2 or Mdm4. Cotransfection experiments indicated that E1B 55K alone was sufficient to deplete Mdm2 and Mdm4. E1B-55K is known to form a functional E3 ubiquitin ligase consisting of Cul2 or Cul5. Thus, it is likely that E1B-55K targets Mdm2 and Mdm4 for ubiquitin-mediated proteosomal degradation. Based on these observations, we hypothesize that adenovirus is effective for treating cancers with overexpression of Mdm2 or Mdm4. We are currently testing whether adenoviral therapy is efficacious on treating such cancers in cell culture and animal models. (This work was supported by Bankhead-Coley Cancer Research Program of the Florida Department of Health.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 613. doi:10.1158/1538-7445.AM2011-613

Mitochondrially targeted ceramides preferentially promote autophagy, retard cell growth, and induce apoptosis

C(6)-pyridinium (D-erythro-2-N-[6'-(1''-pyridinium)-hexanoyl]sphingosine bromide [LCL29]) is a cationic mitochondrion-targeting ceramide analog that promotes mitochondrial permeabilization and cancer cell death. In this study, we compared the biological effects of that compound with those of D-erythro-C(6)-ceramide, its non-mitochondrion-targeting analog. In MCF7 cells it was found that C(6)-pyridinium ceramide preferentially promoted autophagosome formation and retarded cell growth more extensively than its uncharged analog. This preferential inhibition of cell growth was also observed in breast epithelial cells and other breast cancer cells. In addition, this compound could promote Bax translocation to mitochondria. This redistribution of Bax in MCF7 cells could be blocked by the pan-caspase inhibitor zVAD-fmk but via a Bid-independent signaling pathway. Moreover, C(6)-pyridinium ceramide-induced translocation of Bax to mitochondria led to mitochondrial permeabilization and cell death. Overall, we show that mitochondrial targeting of C(6)-pyridinium ceramide significantly enhances cellular response to this compound.

E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC complex. These observations suggest that dissociation of the ligase IV/XRCC4 complex occurs at an early stage in E4 34k-mediated degradation of ligase IV and indicate a role for E4 34k in dissociation of the ligase IV/XRCCC4 complex. Expression of E4 34k alone was not sufficient to dissociate the ligase IV/XRCC4 complex, which indicates a requirement for an additional, as yet unidentified, factor in E1B 55k-independent dissociation of the ligase IV/XRCC4 complex.

Loss of DNA ligase IV prevents recognition of DNA by double-strand break repair proteins XRCC4 and XLF

The repair of DNA double-strand breaks by nonhomologous end-joining (NHEJ) is essential for maintenance of genomic integrity and cell viability. Central to the molecular mechanism of NHEJ is DNA ligase IV/XRCC4/XLF complex, which rejoins the DNA. During adenovirus (Ad5) infection, ligase IV is targeted for degradation in a process that requires expression of the viral E1B 55k and E4 34k proteins while XRCC4 and XLF protein levels remain unchanged. We show that in Ad5-infected cells, loss of ligase IV is accompanied by loss of DNA binding by XRCC4. Expression of E1B 55k and E4 34k was sufficient to cause loss of ligase IV and loss of XRCC4 DNA binding. Using ligase IV mutant human cell lines, we determined that the absence of ligase IV, and not expression of viral proteins, coincided with inhibition of DNA binding by XRCC4. In ligase IV mutant human cell lines, DNA binding by XLF was also inhibited. Expression of both wild-type and adenylation-mutant ligase IV in ligase IV-deficient cells restored DNA binding by XRCC4. These data suggest that the intrinsic DNA-binding activities of XRCC4 and XLF may be subject to regulation and are down regulated in human cells that lack ligase IV.

Adenovirus E4 34k and E1b 55k Oncoproteins Target Host DNA Ligase IV for Proteasomal Degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.

Adenovirus Type 37 Keratitis in the C57BL/6J Mouse

To develop a mouse model of adenoviral keratitis that will allow further study of viral and host pathogenic mechanisms. Corneas of C57BL/6J mice were injected with adenovirus type 37 (Ad37) or virus-free dialysis buffer by a gas-powered microinjection system coupled to a glass micropipette needle. Mouse corneas were examined for signs of inflammation, by clinical examination, immunohistochemistry, and confocal microscopy; assayed for viral and chemokine mRNA expression by real-time PCR; titered to assess viral replication; and subjected to ELISA for chemokine and myeloperoxidase (MPO) protein expression. C57BL/6J mice corneas injected with 10(5) TCID (tissue culture infective dose) Ad37 showed stromal opacification and inflammation beginning from 1 day after injection and continuing for several months, while buffer-injected corneas showed no signs of inflammation. Ad37-injected corneas expressed adenoviral E1A 10S and E1B 19k mRNA but not IIIa, and viral titers had fallen two logs by day 4 after injection. When compared to untouched and buffer-injected corneas, Ad37-injected corneas expressed significantly higher levels of IL-6, KC, and MCP-1 mRNA at 4 hours after injection (P < 0.05). By ELISA, KC protein was significantly elevated in Ad37-injected corneas at 8 and 16 hours, and MCP-1 protein at 16 hours after injection (P < 0.05). Ad37-injected corneas showed elevated levels of MPO (P = 0.0024) at 4 days after injection consistent with immunohistochemical evidence for a predominance of neutrophils in the corneal stroma. Ad37 induces an acute immunopathologic response in the C57BL/6J mouse cornea, despite an absence of viral replication. This new animal model of Ad37 keratitis will facilitate studies of the molecular pathogenesis of the disorder.