E1A Region Research Articles

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

Development of Oncolytic Vectors Based on Human Adenovirus Type 6 for Cancer Treatment.

Human Adenovirus type 6 (HAdV-C6) is a promising candidate for the development of oncolytic vectors as it has low seroprevalence and the intrinsic ability to evade tissue macrophages. However, its further development as a therapeutic agent is hampered by the lack of convenient cloning methods. We have developed a novel technology when a shuttle plasmid carrying the distal genome parts with modified E1A and E3 regions is recombined in vitro with the truncated HAdV-C6 genome. Using this approach, we have constructed a novel Ad6-hT-GM vector controlled by the hTERT promoter and expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) instead of 6.7K and gp19K E3 proteins. We have demonstrated that control by the hTERT promoter may result in delayed viral replication, which nevertheless does not significantly change the cytotoxic ability of recombinant viruses. The insertion of the transgene by displacing the E3-6.7K/gp19K region does not drastically change the expression patterns of E3 genes; however, mild changes in expression from major late promoter were observed. Finally, we have demonstrated that the treatment of human breast cancer xenografts in murine models with Ad6-hT-GM significantly decreased the tumor volume and improved survival time compared to mock-treated mice.

Structure–function analyses of the G729R 2-oxoadipate dehydrogenase genetic variant associated with a disorder of l-lysine metabolism

2-Oxoadipate dehydrogenase (E1a, also known as DHTKD1, dehydrogenase E1, and transketolase domain-containing protein 1) is a thiamin diphosphate-dependent enzyme and part of the 2-oxoadipate dehydrogenase complex (OADHc) in l-lysine catabolism. Genetic findings have linked mutations in the DHTKD1 gene to several metabolic disorders. These include α-aminoadipic and α-ketoadipic aciduria (AMOXAD), a rare disorder of l-lysine, l-hydroxylysine, and l-tryptophan catabolism, associated with clinical presentations such as developmental delay, mild-to-severe intellectual disability, ataxia, epilepsy, and behavioral disorders that cannot currently be managed by available treatments. A heterozygous missense mutation, c.2185G→A (p.G729R), in DHTKD1 has been identified in most AMOXAD cases. Here, we report that the G729R E1a variant when assembled into OADHc in vitro displays a 50-fold decrease in catalytic efficiency for NADH production and a significantly reduced rate of glutaryl-CoA production by dihydrolipoamide succinyl-transferase (E2o). However, the G729R E1a substitution did not affect any of the three side-reactions associated solely with G729R E1a, prompting us to determine the structure-function effects of this mutation. A multipronged systematic analysis of the reaction rates in the OADHc pathway, supplemented with results from chemical cross-linking and hydrogen-deuterium exchange MS, revealed that the c.2185G→A DHTKD1 mutation affects E1a-E2o assembly, leading to impaired channeling of OADHc intermediates. Cross-linking between the C-terminal region of both E1a and G729R E1a with the E2o lipoyl and core domains suggested that correct positioning of the C-terminal E1a region is essential for the intermediate channeling. These findings may inform the development of interventions to counter the effects of pathogenic DHTKD1 mutations.

Correction: Novel Oncolytic Adenovirus Selectively Targets Tumor-Associated Polo-Like Kinase 1 and Tumor Cell Viability.

Purpose: Polo-like kinase 1 (plk1) is a serine/threonine protein kinase essential for multiple mitotic processes. Previous observations have validated plk1 as a promising therapeutic target. Despite being conceptually attractive, the potency and specificity of current plk1-based therapies remain limited. We sought to develop a novel plk1-targeting strategy by constructing an oncolytic adenovirus to selectively silence plk1 in tumor cells. Experimental Design: Two artificial features were engineered into one wild-type adenovirus type 5 (wt-Adv5) genome to generate a new oncolytic adenovirus (M1). First, M1 contains a 27-bp deletion in E1A region, which confers potent, oncolytic efficacy. Second, M1 is armed with a fragment of antisense plk1 cDNA that substitutes the E3 region encoding 6.7K and gp19K. In this design, tumor-selective replication of M1 would activate the native adenovirus E3 promoters to express the antisense plk1 cDNA preferentially in tumor cells and silence tumor-associated plk1 protein. Results: By virtue of combining oncolysis with plk1 targeting, M1 exhibited potent antitumoral efficacy in vitro and in vivo . Systemic administration of M1 plus cisplatin induced complete tumor regression in 80% of orthotopic hepatic carcinoma model mice that were otherwise resistant to cisplatin and disseminated metastases. Conclusions: Coupling plk1 targeting with oncolysis had shown superior antitumor efficacy. Present findings would benefit the development of novel oncolytic adenoviruses generally applicable to a wide range of molecule-based therapeutics.

Interplay between sequence, structure and linear motifs in the adenovirus E1A hub protein

E1A is the main transforming protein in mastadenoviruses. This work uses bioinformatics to extrapolate experimental knowledge from Human adenovirus serotype 5 and 12 E1A proteins to all known serotypes. A conserved domain architecture with a high degree of intrinsic disorder acts as a scaffold for multiple linear motifs with variable occurrence mediating the interaction with over fifty host proteins. While linear motifs contribute strongly to sequence conservation within intrinsically disordered E1A regions, motif repertoires can deviate significantly from those found in prototypical serotypes. Close to one hundred predicted residue-residue contacts suggest the presence of stable structure in the CR3 domain and of specific conformational ensembles involving both short- and long-range intramolecular interactions. Our computational results suggest that E1A sequence conservation and co-evolution reflect the evolutionary pressure to maintain a mainly disordered, yet non-random conformation harboring a high number of binding motifs that mediate viral hijacking of the cell machinery.

The Influence of E1A C-Terminus on Adenovirus Replicative Cycle.

Adenovirus Early 1A proteins (E1A) are crucial for initiation of the viral life cycle after infection. The E1A gene is encoded at the left end of the viral genome and consists of two exons, the first encoding 185 amino acids in the 289 residues adenovirus 5 E1A, while the second exon encodes 104 residues. The second exon-encoded region of E1A is conserved across all E1A isoforms except for the 55 residues protein, which has a unique C-terminus due to a frame shift following splicing into the second exon. This region of E1A contributes to a variety of processes including the regulation of viral and cellular gene expression, immortalization and transformation. Here we evaluated the contributions that different regions of the second exon of E1A make to the viral life cycle using deletion mutants. The region of E1A encoded by the second exon was found to be important for overall virus growth, induction of viral and cellular gene expression, viral genome replication and deregulation of the cell cycle. Efficient viral replication was found to require exon 2 and the nuclear localization signal, as loss of either resulted in severe growth deficiency. Induction of cellular DNA synthesis was also deficient with any deletion of E1A within the C-terminus even if these deletions were outside of conserved region 4. Overall, our study provides the first comprehensive insight into the contributions of the C-terminus of E1A to the replicative fitness of human adenovirus 5 in arrested lung fibroblasts.

Exonal switch down-regulates the expression of CD5 on blasts of acute T cell leukaemia.

To date, CD5 expression and its role in acute T cell lymphoblastic leukaemia (T-ALL) have not been studied closely. We observed a significant reduction in surface expression of CD5 (sCD5) on leukaemic T cells compared to autologous non-leukaemic T cells. In this study, we have shown the molecular mechanism regulating the expression and function of CD5 on leukaemic T cells. A total of 250 patients suffering from leukaemia and lymphoma were immunophenotyped. Final diagnosis was based on their clinical presentation, morphological data and flow cytometry-based immunophenotyping. Thirty-nine patients were found to be of ALL-T origin. Amplification of early region of E1A and E1B transcripts of CD5 was correlated with the levels of surface and intracellular expression of CD5 protein. Functional studies were performed to show the effect of CD5 blocking on interleukin IL-2 production and survival of leukaemic and non-leukaemic cells. Lack of expression of sCD5 on T-ALL blasts was correlated closely with predominant transcription of exon E1B and significant loss of exon E1A of the CD5 gene, which is associated with surface expression of CD5 on lymphocytes. High expression of E1B also correlates with increased expression of cytoplasmic CD5 (cCD5) among leukaemic T cells. Interestingly, we observed a significant increase in the production of IL-2 by non-leukaemic T cells upon CD5 blocking, leading possibly to their increased survival at 48 h. Our study provides understanding of the regulation of CD5 expression on leukaemic T cells, and may help in understanding the molecular mechanism of CD5 down-regulation.

The role of alternative GJB2 transcription in screening for neonatal sensorineural deafness in Austria

Conclusion: Alterations within a novel putative Exon 1a within the gap junction beta 2 (GJB2) gene may play a role in the development of genetic hearing impairment in Austria.Objectives: Mutations in the GJB2 gene are the most common cause of hereditary sensorineural deafness. Genome-wide screening for alternative transcriptional start sites in the human genome has revealed the presence of an additional GJB2 exon (E1a). This study tested the hypothesis of whether alternative GJB2 transcription involving E1a may play a role in the development of congenital sensorineural deafness in Austria.Methods: GJB2 E1a and flanking regions were sequenced in randomized normal hearing control subjects and three different patient groups with non-syndromic hearing impairment (NSHI), and bioinformatic analysis was performed. Statistical analysis of disease association was carried out using the Cochran-Armitage test for trend.Results: A single change 2410 bp proximal to the translational start site (c.-2410T > C, rs7994748, NM_004004.5:c.-23 + 792T > C) was found to be significantly associated with the common c.35delG GJB2 mutation (p = .009). c.35delG in combination with c.-2410CC occurred at a 6.9-fold increased frequency compared to the control group. Additionally, one patient with idiopathic congenital hearing loss was found to be homozygous c.-2410CC.

A p53-independent apoptotic mechanism of adenoviral mutant E1A was involved in its selective antitumor activity for human cancer.

The conserved regions (CR) of adenoviral E1A had been shown to be necessary for disruption of pRb-E2F transcription factor complexes and induction of the S phase. Here we constructed a mutant adenoviral E1A with Rb-binding ability absent (E1A 30-60aa and 120-127aa deletion, mE1A) and investigated its antitumor capacities in vitro and in vivo. The mE1A suppressed the viability of tumor cells as efficiently as the wild type E1A, and there was no cytotoxic effect on normal cells. Although the mE1A arrested tumor cell cycle with the same manner as E1A, the former played a different role on cell cycle regulation compared with E1A in normal cells, which might contribute to its selective antitumor activity. E1A and mE1A had accumulated inactive p53, decreased the expression of mdm2, Cdkn1a (also named p21), increased p21's nuclear distribution and induced tumor cell apoptosis in a p53-indenpent manner. Further, E1A or mE1A significantly suppressed tumor growth in subcutaneous hepatocellular carcinoma xenograft models. Especially, tumor-bearing mice treated with mE1A had higher survival rate than those treated with E1A. Our data demonstrated that mutant adenoviral E1A significantly induced tumor cell apoptosis in a p53-indenpednt manner and had selective tumor suppressing ability. The observations of adenoviral E1A mutant had provided a novel mechanism for E1A's complex activities during infection.

Oncolytic adenoviruses targeted to Human Papilloma Virus-positive head and neck squamous cell carcinomas

In recent years, the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has markedly increased. Our aim was to design a novel therapeutic agent through the use of conditionally replicative adenoviruses (CRAds) that are targeted to the HPV E6 and E7 oncoproteins. Each adenovirus included small deletion(s) in the E1a region of the genome (Δ24 or CB016) intended to allow for selective replication in HPV-positive cells. In vitro assays were performed to analyze the transduction efficiency of the vectors and the cell viability following viral infection. Then, the UPCI SCC090 cell line (HPV-positive) was used to establish subcutaneous tumors in the flanks of nude mice. The tumors were then treated with either one dose of the virus or four doses (injected every fourth day). The transduction analysis with luciferase-expressing viruses demonstrated that the 5/3 fiber modification maximized virus infectivity. In vitro, both viruses (5/3Δ24 and 5/3CB016) demonstrated profound oncolytic effects. The 5/3CB016 virus was more selective for HPV-positive HNSCC cells, whereas the 5/3Δ24 virus killed HNSCC cells regardless of HPV status. In vivo, single injections of both viruses demonstrated anti-tumor effects for only a few days following viral inoculation. However, after four viral injections, there was statistically significant reductions in tumor growth when compared to the control group (p<0.05). CRAds targeted to HPV-positive HNSCCs demonstrated excellent in vitro and in vivo therapeutic effects, and they have the potential to be clinically translated as a novel treatment modality for this emerging disease.

The Cellular Protein Complex Associated with a Transforming Region of E1A Contains c-MYC.

The cell-transforming activity of human adenovirus 5 (hAd5) E1A is mediated by the N-terminal half of E1A, which interacts with three different major cellular protein complexes, p300/CBP, TRRAP/p400, and pRb family members. Among these protein interactions, the interaction of pRb family proteins with conserved region 2 (CR2) of E1A is known to promote cell proliferation by deregulating the activities of E2F family transcription factors. The functional consequences of interaction with the other two protein complexes in regulating the transforming activity of E1A are not well defined. Here, we report that the E1A N-terminal region also interacted with the cellular proto-oncoprotein c-MYC and the homolog of enhancer of yellow 2 (ENY2). Our results suggested that these proteins interacted with an essential E1A transforming domain spanning amino acid residues 26 to 35 which also interacted with TRRAP and p400. Small interfering RNA (siRNA)-mediated depletion of TRRAP reduced c-MYC interaction with E1A, while p400 depletion did not. In contrast, depletion of TRRAP enhanced ENY2 interaction with E1A, suggesting that ENY2 and TRRAP may interact with E1A in a competitive manner. The same E1A region additionally interacted with the constituents of a deubiquitinase complex consisting of USP22, ATXN7, and ATXN7L3 via TRRAP. Acute short hairpin RNA (shRNA)-mediated depletion of c-MYC reduced the E1A transforming activity, while depletion of ENY2 and MAX did not. These results suggested that the association of c-MYC with E1A may, at least partially, play a role in the E1A transformation activity, independently of MAX. The transforming region of adenovirus E1A consists of three short modules which complex with different cellular protein complexes. The mechanism by which one of the transforming modules, CR2, promotes cell proliferation, through inactivating the activities of the pRb family proteins, is better understood than the activities of the other domains. Our analysis of the E1A proteome revealed the presence of the proto-oncoprotein c-MYC and of ENY2. We mapped these interactions to a critical transforming module of E1A that was previously known to interact with the scaffolding molecule TRRAP and the E1A-binding protein p400. We showed that c-MYC interacted with E1A through TRRAP, while ENY2 interacted with it independently. The data reported here indicated that depletion of c-MYC in normal human cells reduced the transforming activity of E1A. Our result raises a novel paradigm in oncogenic transformation by a DNA viral oncogene, the E1A gene, that may exploit the activity of a cellular oncogene, the c-MYC gene, in addition to inactivation of the tumor suppressors, such as pRb.

P10.07 Mapping the integration sites e1-e2 of hpv-16 andhpv-18 as a tool to evaluate different stages of cervical disease progression

Introduction Different methodologies have been developed to analyse integration. Most of them are expensive and laborious. Since the most frequent disrupting happen in E1 or E2 genes we believe that amplify fragments covering theses genes could be associated to virus integration status. Methods Hence, in order to evaluate the physical state (episomal or integrated) of HPV 16 and 18 genomes, a PCR combining 10 primers pairs for HPV16 and 11 primers pairs for HPV18 were used, covering the E1-E2 region, adapted as described by Vernon et al. (1997)1 and Collins Constandinou-Williams et al. (2009).2 CASKI and HeLa lineage were used as control. Results Our preliminary results involved 93 samples cervical samples from patients infected by HPV16 and 37 by HPV18, harbouring cervical lesions in different stages of progression, except HPV18, exclusively associated with cancer lesions. Among HPV16, 26 (28%) were presented episomal, 30 (32%) mixed and 37 (40%) integrated forms. The upper region E1a were the most frequently absent (disrupted) (n = 30, 32%), followed by the downstream E2c region, (n = 21, 22,6%). Among HPV 18 cervical cancers, 5 (13,5%) were presented as episomal forms, 8 (21,5%) mixed and 24 (65%) were integrated. The downstream region E2P3 was the most frequently disrupted, (n = 30, 81%). Conclusion It is interesting to observe that literature points out a predominance of disruption in E2 region but our results suggested a highly prevalent E1 inactivation. The approach here described is a very specific methodology that can successfully map the HPV 16 and 18 genome fragile areas. Data are being analysed in order to search for statistical correlation between integration and severity of the lesion but it has been observed that E1-E2 absences were suggestively frequent in HSIL and cancer. In a few cases, episomal forms were observed in cancer samples, suggesting additional biomarkers as responsible for carcinogenesis. Disclosure of interest statement PCR, Genomic integration, episomal, HPV16. Financial support: Bolsa Capes, Faperj APQ1. References Vernon SD, Unger ER, Miller DL, et al. Association of human papillomavirus type 16 integration in the E2 gene with poor disease-free survival from cervical cancer. Int J Cancer 1997;74:50–6 Collins SI, Constandinou-Williams C, Wek K, et al. Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study. Cancer Res. 2009;69:3828–32

632. Oncolytic Adenoviruses Targeted to the HPV E6 and E7 Oncoproteins as a Novel Treatment for Head & Neck Squamous Cell Carcinomas

Introduction:Recent reports have shown that the incidence of Human Papilloma Virus (HPV)-positive head and neck squamous cell carcinomas (HNSCC) has been steadily increasing. HNSCCs are known to have a high recurrence rate and compared to their HPV-negative counterparts, these tumors have a unique biology which necessitates the development of novel treatment modalities. The HPV E6 and E7 oncoproteins are attractive therapeutic targets as they interact with key cell cycle regulatory components, namely p53 and pRb. Our research group has applied conditionally replicating oncolytic adenoviruses (CRAd) with modifications in the E1a region of the genome allowing for selective replication in E6 and E7 expressing HNSCCs.Methods:The in vitro transduction efficiency of E1-deleted luciferase expressing vectors with fiber modifications was assessed in multiple HPV-positive and negative cell lines. The CRAds were designed with a distinct deletion in the E1a region of the adenoviral genome (D24 and CB016) intended to allow for selective replication in HPV-positive HNSCC cells. Additionally, the CRAds have a luciferase transgene in the E3 region that is expressed in a replication dependent manner. By using a luciferase assay, the degree of viral replication was analyzed in numerous HNSCC cell lines. In vitro cell viability following viral infection was analyzed with crystal violet and MTS assays. A HPV-positive cell line (UPCI SCC 090) was used to establish subcutaneous tumors in female nude mice. They were subsequently treated with either one intratumoral viral injection or four injections (given every fourth day).Results:The 5/3 fiber modification significantly increased viral infectivity in all tested HNSCC cell lines. The 5/3 CB016 vector replicated selectively in HPV-positive cell lines, while the 5/3 D24 virus replicated in all cell lines regardless of HPV status. Both of the vectors demonstrated profound cytocidal effects in the crystal violet and MTS assays. For the in vivo experiments, a single intratumoral injection of virus demonstrated an anti-tumor effect for only one week following injection. Given this limited effect, an additional in vivo experiment was performed to analyze the efficacy of multiple intratumoral injections (3.5 x1011 vp/injection). This setup resulted in statistically significant tumor growth suppression at day 26 when compared to the saline control group (p<0.05 for 5/3 CB016, p<0.01 for 5/3 D24).Conclusion:CRAds designed to target HPV-positive HNSCCs demonstrated tremendous, yet selective in vitro cytocidal effects. The profound tumor growth suppression in a mouse model (particularly by multiple injections) shows the excellent potential of our viruses to be clinically translated as a new therapeutic entity for this emerging disease process.

665. Toxicity and Bio-Distribution of a GM-CSF-Expressing, Chimeric Oncolytic Adenovirus ONCOS-102

INTRODUCTION: ONCOS-102 is a serotype 5 adenovirus, comprising a chimeric capsid for enhanced gene delivery to cancer cells and a 24 bp deletion in Rb binding site of E1A region for cancer cell restricted replication. ONCOS-102 is armed with granulocyte-macrophage colony-stimulating factor (GM-CSF) for an enhanced immunostimulatory effect. ONCOS-102 treatment is a promising immunotherapy strategy for advanced cancer by directly recruiting antigen presenting cells (APC) at tumor site leading to an induction of adaptive tumor-specific CD8+ T cell response. It's immunological activity has already been demonstrated in Phase I clinical study. In this phase 1 study, local treatment of pleural mesothelioma with ONCOS-102 induced a systemic antitumor CD8+ T-cell response, prominent infiltration of CD8+ lymphocytes and Th1 type polarization.PURPOSE OF THE STUDY: The aim of this study was to evaluate the toxicity profile and biodistribution of ONCOS-102 after intracardial and repeated intraperitoneal or subcutaneous administration in Syrian hamsters.METHOD: This study was performed in compliance with the OECD principles of GLP. 300 Syrian hamsters were divided into nine groups, each group containing males and females in the same ratio. ONCOS-102 was administered in NaCl solution by intracardial, intraperitoneal or subcutaneous injections. The control animals were administered with NaCl solution without ONCOS-102 in the same volume and the same way. Hamsters were checked daily for mortality and clinical signs. Body weight and food consumption were recorded weekly. For bio-distribution analysis, hamsters were euthanized on days 0, 3, 29, and 183 and tissues and organs were collected for assessment of viral DNA. Gross necropsy and histological examination of tissues and organs were performed on days 29 and 190.RESULTS: Treatment with ONCOS-102 had no effects on body weight and food consumption. No treatment related premature deaths occurred. Apart from some findings that were not related to treatment, none of the hamsters showed signs of intoxication in the course of the first four weeks of the study. No considerable differences in hematology and clinical chemistry parameters were observed in any of the treatment groups when compared to control animals after 26 weeks of the study and recovery period. ONCOS-102 did not cause gross or histopathological changes in liver and kidneys of animals indicative of a toxic effect. The highest accumulation of viral DNA was seen in heart, liver, lungs, spleen, kidneys and bone marrow. An overall decrease of concentration of viral DNA was observed from Day 3 to Day 29 and Day 183. No adverse effects of repeated administration of ONCOS-102 on clinical signs, hematology and clinical chemistry parameters or histopathology were observed in the course of 6-month administration and following 3- month recovery period.CONCLUSIONS: Under the conditions of the study, administration of hamsters with ONCOS 102 was well tolerated, without significant signs indicating toxic effects

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 (258RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP289). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs.

Genome stability of adenovirus types 3 and 7 during a simultaneous outbreak in Greater Manchester, UK.

A total of 96 isolates of species B adenovirus collected in Greater Manchester, UK and typed previously by serum neutralization were analyzed in five genome regions. Of these, 62 isolates were HAdV-B3 and HAdV-B7 collected during a simultaneous 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 and other species B adenovirus types collected in different years following the outbreak. The phylogenetic analysis results of all the isolates in the structural regions hexon L2, penton, and fiber knob were found to be consistent and no mismatches were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable even when different types are circulating in a limited geographical area simultaneously.

Prison and community outbreak of severe respiratory infection due to adenovirus type 14p1 in Tayside, UK

This report describes the investigation and public health management of a community-based outbreak of severe adenovirus serotype 14p1 respiratory infection affecting the Tayside area during 2011. It is the first report of an adenovirus outbreak involving prisons. An outbreak-based/incident management approach was carried out. Alerts were sent out to local doctors, general practitioners, prison healthcare staff and consultants so that cases could be identified prospectively. Sequencing of hexon, fibre and E1A regions of adenovirus were carried out to genotype the viruses. Fifteen cases were identified in total, including 13 confirmed cases and 2 possible cases. There were 3 deaths amongst the 13 confirmed cases, with a case fatality rate of 23%. Eight of the cases had a direct association with one of the two prisons in the area. We advise that surveillance measures for adenovirus infection and guidelines for the management of critically ill patients should be developed in order to identify outbreaks at an early stage and allow patients to receive appropriate treatment. Adenovirus infection should be borne in mind as a cause of severe pneumonia in closed settings such as prisons.

Dissection of the C-Terminal Region of E1A Redefines the Roles of CtBP and Other Cellular Targets in Oncogenic Transformation

Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A.

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells.

The C-terminal region of E1A: a molecular tool for cellular cartography

The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.