Adenovirus Early region 1A(E1A) plays an important role in viral lytic infection and serves as a useful tool in the study of molecular mechanisms of gene regulation and gene expression. In addition to the ability of E1A functions to induce transactivation and transrepression of gene expression, E1A products play a critical role in transformation by adenovirus. A technique employing linker insertion mutagenesis scanning most of the E1A major regions was developed in this laboratory to systematically study the effect of E1A mutations on transcriptional transactivation and trans-repression functions as well as transforming ability (Bautista 1989). Results obtained showed that the unique region was the region primarily for transactivation in agreement with a wealth of other data and that transrepression was sensitive to insertions in CRII (conserved region II) and a region immediately following the unique region at the beginning of Exon II. However, transformation was not affected by repression-sensitive mutants. This thesis describes further studies carried out to create additional insertion mutants within conserved region of E1A to confirm and extend the results obtained by Bautista. A synthetic oligonucleotide of 39 base pair (13 aa) was introduced into two additional restriction sites in this region. As in previous work, the oligonucleotide could be inserted in either of two possible orientations. In one orientation, all three reading frames were open whereas all three reading frames of the other orientation contained stop codons which resulted in truncation of the E1A protein at the site of insertion. In addition, the insertion oligomer was designed with flanking BamHI sites to provide the opportunity of collapsing the full length insertion to a 6 base pair (2 aa) insertion. This allowed comparison to be made between mutations containing 13 aa insertions and those with 2 aa inserts at the same site to see how different oligopeptide segments would influence different functions. Trans-repression assays utilized a novel β-galactosidase assay developed by Bautista for characterizing CRII mutants expressing only the 12S product. The results suggested that CRII was an important region in terms of ability of E1A to transrepress. This negative regulatory function was sensitive to full length 13 aa insertions but not 2 aa inserts for those mutants made to date. Transforming ability of all CRII mutants was more or less impaired. Transactivation activity was not reduced by insertion at CRII except for mutants which were in the reverse orientation and had stop codons in their reading frame thus terminating the translation upstream of the unique region. In an effort to identify functional temperature sensitive mutants, DNA-mediated transformation assays of mutants within E1A functional regions were carried out at different temperatures. Two transformation temperature sensitive mutants were identified which failed to produce transformed colonies at 38.5°C but transformed efficiently at 32°C. No replication temperature sensitive mutants were identified among 61 mutant viruses. / Thesis / Master of Science (MS)

Gene therapy, although a young field, has become an intense area of study for the potential treatment of many inborn or acquired diseases, including cancer. The systemic or intratumoural delivery of cytokines and other immune system modulators has shown that protective, specific immunity to tumours is possible and effective. Adenovirus is an extremely useful vehicle for the transfer of genes, due to the ease of its preparation, its ability to be grown to high concentrations, and its capacity to infect a wide variety of cell types, including nonproliferating cells. Although a great deal of research has been done, there are a number of possible improvements, specifically, the consistency and range of transgene expression. The HCMV IE promoter/enhancer, used in a wide range of vector systems, has proven to drive lower levels of transgene expression in murine cells as compared to human cells. The use of the MCMV IE promoter/enhancer within the context of an adenovirus vector system has been investigated to see if more consistent transgene expression is possible in murine models. Studies with a luciferase reporter gene have demonstrated that in all cell types, the MCMV IE promoter/enhancer drives transgene expression to equivalent or higher levels than the HCMV IE promoter/enhancer. Our lab has investigated adenovirus as an intrtumoural gene delivery vehicle for various cytokines, including IL-12. However, the current IL-12-expressing vector carries the IL-12p35 subunit in E1 and the IL-12p40 subunit in E3. A homologous recombination event between E1 sequences in the viral genome and those that are integrated into 293 cells, would produce a replication-competent adenovirus expressing high levels of the p40 subunit, which is a potential antagonist to IL-12. A bicistronic adenovirus vector using the poliovirus IRES was constructed containing all IL-12 coding sequences in E1 to avoid this potential hazard. This virus expressed between 60 ng to 2.5 μg of IL-12/10⁶ cells, depending on the cell type. These levels of expression, however are 7-30-fold lower than those driven by previous IL-12 vector. Although this new virus is not appropriate for tumour immunotherapy, the shuttle plasmids developed during its construction have many potential uses. / Thesis / Master of Science (MS)

The E1 region of the group C adenoviruses is able to induce oncogenic transformation of rodent cells in culture and is also necessary for the efficient transcription of other viral genes. The proteins of the E1a transcription unit have been shown to play a pivotal role in both of these activities. In order to better understand the regions of the E1a proteins required for transformation and viral growth a program of random insertion mutagenesis was undertaken by D. S. Bautista to help identify important domains. The 39bp linker insertion oligonucleotides were designed to encode a 13 amino acid in frame insertion in one orientation or a closed reading frame insertion in the opposite orientation. As well the insertion mutants could be collapsed by digestion with BamHI to generate a 2 amino acid in frame insertion. Using this method all three types of mutants were generated at 18 different sites within the E1a coding sequences. The purpose of this project was to assay these E1a mutants for the ability to cooperate with EJb in the transformation of primary baby rat kidney cells using DNA-mediated transfection and also to 'rescue' the mutants into infectious virus and study the ability of the mutant virus to replicate on HeLa cells. Results showed that only closed reading frame mutations upstream of the unique region were completely negative for transformation. Conversely, 13aa or 2aa insertions outside of the unique region impaired but did not abolish transformation. However 8 of the 9 insertions in the unique region of the 289R protein of E1a were defective for transformation of BRK cells in E1a plus E1b DNA-mediated transformation assays. To determine whether the unique region played a direct role in the transformation process or if it had an indirect role such as the transactivation of E1b the transformation assay was carried out using selective media that allow~ growth of foci transformed by E1a alone. Results from this assay showed that the unique region mutants combined with E1b were able to transform with about the same efficiency as E1a alone. The transformation assay was also performed using the unique region mutants in an E1a only background cotransfected with the EJ-ras oncogene. Results from these experiments showed that the unique region mutants in an E1a only background could cooperate with ras in transformation as well as wild-type E1a. From these results it was concluded that the unique region does not play a direct role in transformation by E1 but is required for the efficient expression of E1b which results in wild type transforming frequencies. The actual role of E1b in transformation is unknown. The insertion mutants were also 'rescued' back into infectious virus to study their effect on the ability of the viruses to replicate. The results showed that only viruses in which the unique region was either eliminated or altered were defective for growth on HeLa cells. Transactivation assays carried out by D. Bautista showed results which were comparable to results of infectivity assays. Taken together the results suggest that only the unique region is required for transactivation and only the ability of E1a to transactivate is of importance for viral replication in HeLa cells. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

The E1b transforming region of Adenovirus type 5 encodes minor products of 93R and 156R in addition to the more abundant proteins 178R, 496R, and 84R. The goal of this study was to elucidate the function of 93R and 156R to gain a better understanding of their role in oncogenic transformation and productive infection. Mutant viruses were constructed, whose normal splicing pattern was disrupted by point mutations in the 3' acceptor sites for the 1.26 and 1.31Kb mRNAs, which code for the 156R and 93R products, respectively. In the construction of these mutations, it was necessary to ensure that they did not affect the coding region for 496R. These mutants produced transformed foci in primary rat kidney cells with wild type efficiencies in DNA-mediated transformation assays. In the mutant designed to eliminate 156R, although the two wild type 156R species were absent, two new species running slightly faster on SDS-PAGE were detected. These proteins were recognized by sera specific to both the N- and C-termini of 496R, suggesting there utilization of an in-frame cryptic splice acceptor site. Use of this site probably resulted in the production of a mRNA encoding a modified 156R. These mutant proteins also seemed to be produced at the expense of 496R. The mutant designed to eliminate 93R grew with titres equivalent to wild type dl309, yet it was not clear whether a modified protein was produced in this case as well. / Thesis / Master of Science (MS)

Intratumoral injection of oncolytic viruses provides a direct means of tumor cell elimination for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this thesis, I explore the efficacy of conditionally replicating HAdV vectors expressing either the p14 Fusion Associated Small Transmembrane (FAST) protein (CRAdFAST) or p14 FAST protein in combination with the adenovirus death protein (CRAdFAST-ADP). The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect, while ADP aids in cell lysis and HAdV spread at late times in infection. I first explored the efficacy of CRAdFAST in the 4T1 immune competent mouse model of cancer. Treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene in vitro, but did not reduce the tumor growth rate in vivo. The 4T1 model was significantly resistant to HAdV infection and propagation, so I next explored CRAdFAST efficacy in human A549 cell culture and a xenograft mouse model of cancer. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion which led to the formation of large acellular regions within the tumor, and significantly reduced the tumor growth rate compared to control vector. Finally, to assess the use of a newly constructed CRAdFAST vector co-expressing the adenovirus death protein (ADP), a new model was explored comprised of CMT-64.6 mouse lung carcinoma cells which are syngeneic with Balb/C mice. This model was significantly more sensitive to HAdV infection and CRAdFAST induced fusion than the 4T1 cell line. In this model, expression of ADP and p14 FAST from a CRAdFAST-like vector (CRAdFAST-ADP) resulted in significant oncolytic synergy in vitro but not in vivo. My results indicate that expression of p14 FAST protein, and potentially ADP, from an oncolytic HAdV can improve vector efficacy for the treatment of cancer, but improved in vivo models will be required to analyze the full preclinical potential of these oncolytic HAdV vectors.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2014. / Made available in DSpace on 2014-08-06T18:08:08Z (GMT). No. of bitstreams: 1 326940.pdf: 2274596 bytes, checksum: 074ce9d264a989af3e9bdace4790ce98 (MD5) Previous issue date: 2014 / No Brasil, a Portaria MS 2.914/2011 exige a ausência de coliformes totais e E. coli na água tratada, entretanto é fundamental que o tratamento seja eficiente para todos os microrganismos patogênicos. A desinfecção nas Estações de Tratamento de Água (ETA) é comumente realizada com cloro. O adenovírus recombinante (rAdV) expressa proteína verde fluorescente (GFP) quando se replica nas células HEK 293A, e foi escolhido, como um modelo para avaliar a eficiência de desinfecção em amostras de água filtradas de duas ETAs (Lagoa do Peri (pH 6.9) e Morro dos Quadros (pH 6.5)), a 15°C e 20°C e com 0,2 mg/L e 0,5 mg/L de cloro livre. O tampão livre de demanda de cloro (BDF) (pH 6.9 e 8.0) foi utilizado como controle. As amostras foram caracterizadas físico-quimicamente e quanto ao padrão bacteriológico. A partir da curva da expressão da GFP, foi escolhido o período de 24h pós-infecção para os ensaios de citometria de fluxo (FACS) e microscopia de fluorescência (MF). Após padronizadas, as técnicas apresentaram como limite de detecção a diluição viral de 10-4 (FACS) e 10-6 (MF), sendo a MF escolhida para averiguar a inativação de rAdV por cloro. A técnica de qPCR foi escolhida como método molecular. O tempo necessário para inativar 4log10 de rAdV foi menor que 1 min (para 0,2 mg/L e 0,5 mg/L de cloro livre), utilizando MF, com exceção do BDF pH 8.0 (acima de 2,5 min para 4log10). O pH (6,5, 6,9 e 8.0) exerceu grande influência na eficiência de desinfecção. O ensaio de qPCR não forneceu informações a respeito de rAdV inativado. Os dados foram modelados (Chick-Watson) e observado que os valores de Ct para inativação de 4log10 foi menor que 0,25 para todas as condições experimentais, com exceção do BDF pH 8.0, com Ct acima de 1,249. Os valores de Ct são comparáveis aos reportados na literatura e menores do que os recomendados pela EPA. O modelo demonstrou ter se ajustado bem às condições experimentais realizadas e observadas. Também foram coletadas amostras de água tratada das ETAs (na saída da ETA e na rede de distribuição) e avaliado para coliformes totais, E. coli e adenovírus humano (HAdV), que foi detectado na rede de distribuição da ETA Morro dos Quadros (2,75.10³ UFP/L). Finalmente, foi possível comprovar que os adenovírus são rapidamente inativados em águas superficiais tratadas com cloro e que os adenovírus recombinantes mostraram-se bons modelos para esta avaliação.<br> / Abstract : In Brazil, the MS 2.914/2011 ordinance requires the absence of total coliforms and E. coli in treated water, however it is essential that water treatment is effective for all pathogens. Disinfection in Water Treatment Plants (WTP) is commonly performed with chlorine. The recombinant adenovirus (rAdV) expresses green fluorescent protein (GFP) when replicates in HEK 293A cells, and was chosen as a model to evaluate the efficiency of disinfection in filtered water samples from two WTP (Lagoa do Peri (pH 6.9) and Morro dos Quadros (pH 6.5)) under 15 ° C and 20 ° C with 0.2 mg/L and 0.5 mg/L free chlorine. Buffered demand free (BDF) (pH 6.9 and 8.0) was used as control. The samples were characterized physico-chemically as well for bacteriological standards. From curve of GFP expression, the 24-hour period post-infection for flow cytometry assays (FACS) analysis and fluorescence microscopy (FM) was selected. After standardized, techniques showed a detection limit for viral dilution of 10-4 (FACS) and 10-6 (FM) and FM was selected to investigate the decay of rAdV by chlorine. qPCR technique was employed as a molecular method. The time required to inactivate 4log10 of rAdV was less than 1 min (for 0.2 mg/L and 0.5 mg/L) using FM, except for BDF pH 8.0 (up to 2.5 min for 4log10). The pH (6.5, 6.9 and 8.0) had a great influence on the efficiency of disinfection. The qPCR assay was not able to provide information regarding rAdV inactivated. The data were modeled (Chick-Watson) and observed that the Ct values for inactivation of 4log10 was less than 0.25 for all experimental conditions, except for the BDF pH 8.0, with Ct over 1.249. The Ct values are comparable to those reported in the literature and smaller than those recommended by EPA. The model proved to have adjusted well to the experimental conditions used and observed. Samples of treated water from WTP (at the output of WTP and the distribution network) were evaluated for total coliforms, E. coli and human adenovirus (HAdV), which was detected in the distribution network of WTP Morro dos Quadros (2,75.10³ UFP/L). Finally, it was possible to prove that adenoviruses were rapidly inactivated in surface water treated with chlorine and that recombinant adenovirus proved to be good models for this evaluation.

No description available.

p53 is a pivotal tumour suppressor in mammalian cells. It protects the integrity of a number of cellular pathways, preventing the malignant transformation of cells. There is however perhaps nothing more efficient at disrupting cellular pathways than a virus. Viruses infiltrate cells commandeering the normal growth and survival pathways for their narcissistic needs. While the association between viral infections and the induction of p53 has long been recognised, there is controversy surrounding the ultimate role of p53 during a virus infection. The classical model of p53 in an adenovirus infection is that p53 is a formidable obstacle which needs to be overcome. Adenoviruses overcome p53 by degrading the protein and removing its ability to transactivate its target genes. However the degradation is not immediate and there is increasing evidence which would suggest p53 is actually beneficial to an adenovirus infection. In the introductory chapter, I review what is known about p53 and virus infections. What emerges from this review is the sheer number of interactions that occur between viruses and p53, indicating its importance in an infection. Additionally it shows that adenoviruses are not the only virus shown to benefit from the presence of p53. What beneficial role p53 may be fulfilling in an adenovirus infection is unclear. The experiments reported in this thesis investigate the functions of p53 in an adenovirus infection. In Chapter Three, immunoblots on a panel of adenovirus infected cells reveal that p53 levels do not correlate with the level of the classical p53 target proteins. This indicates that p53 is disconnected from its target genes during an infection. Promoter/reporter assays carried out on infected cells show that adenovirus can directly regulate p53 target genes independently of p53. In Chapter Five, I show this regulation is dependent on E1a, with transient transfection of E1a resulting in the marked activation of p53 target promoters. E1a mediated transactivation appears to be reliant on the largest splice variant of E1a (E1a-289R) and the presence of pRB. Electrophoresis mobility shift assays reveal that the transcription factor Sp1 is involved. In Chapter Four, p53 transcription in an adenovirus infection was directly assayed by using an artificial p53 consensus response element. The results show that p53 is unable to activate its consensus response element during an infection. However, I show that p53 is transcriptionally competent in an infection, and is able to transactivate a mutant derivative of the p53 consensus sequence. This shows that p53 is not only transcriptional competent but has a gain-of-function in an infection. This gain-of-function requires E1a, and appears not to be due to a change in the DNA binding affinity of p53. The data in this thesis show that adenoviruses not only appear to inhibit and control the normal transcriptional profile of p53 but appear to modify p53, giving it a new transcriptional profile. This provides a possible mechanism by which p53 could aid an adenovirus infection.

ABSTRACT Adenovirus Death Protein (ADP) expression during late stages of a lytic infection releases mature virions to promote viral spread, thus leading to death of the host cell. We sought to investigate ADP expression patterns in persistently infected human lymphocytes cells. We hypothesized that low expression of ADP allows the virus to persist while high expression would promote lytic infection in lymphocytes. Accordingly, we found ADP expressed in low amount in BJAB and KE37 cells, while lytically infected Jurkat cells demonstrated higher ADP expression in both protein and transcript levels. ADP overexpression in persistently infected lymphocytes did not alter the viability of these cells, or their level of ADP expression. In contrast, Jurkat cells infected with an ADP-deleted virus had increased survival and maintained viral DNA for greater than 1-month, suggesting conversion to a persistent infection. Also manipulating ADP expression had minimal impact on the total virus yield from infected lymphocytes.