The adenovirus major late transcription unit (MLTU) encodes for most of the mRNAs that are translated into the structural proteins of the virus capsid. Transcription from the MLTU is directed by the major late promoter (MLP), which is highly activated during the late phase of infection. This thesis discusses how the adenovirus-encoded L4-22K protein regulates the MLP at both the level of transcription and pre-mRNA splicing. The study shed new light on the complex regulation of the early to late shift of adenoviral gene expression. Here we show that the L4-22K protein has opposing effects on MLP transcription, functioning both as an activator and a repressor protein. The stimulatory effect mainly depends on the direct interaction of the L4-22K protein with the downstream element (DE element) located approximately 100 nucleotides downstream of the transcription initiation site. In addition to the DE element we also show that the promoter-proximal upstream element (UPE) acts as an L4-22K responsive enhancer element in the MLP. Preliminary data suggests that the activation of MLP transcription via DE and UPE differs mechanistically. The transactivation domain of the L4-22K protein is localized to the conserved carboxy-terminus of the protein. Our results also defined a novel low affinity L4-22K binding site, the R1 region, which functions as a repressor element in MLP transcription. At high concentrations L4-22K binds to R1 and recruits the cellular transcription factor Sp1 to a DNA segment covering the major late first leader 5´ splice site that is embedded in the R1 region. Sp1 binding to R1 results in a suppression of L4-22K-mediated activation of MLP transcription. This self-limiting effect on MLP transcription might have a function to fine-tune the MLTU gene expression. Interestingly, the L4-22K protein binds with the same sequence specificity to both the R1 double-stranded DNA and R1 single-stranded RNA (ssRNA). L4-22K binds to the R1 ssRNA with the same polarity as the MLTU nascent RNA. This binding results in the recruitment of U1 snRNA to the major late first leader 5´ splice site. This enhanced U1 snRNA recruitment leads to a suppression of MLP transcription and simultaneously an increase of major late first intron splicing.

BACKGROUND:Subspecies B1 human adenoviruses (HAdV-B1) are prevalent respiratory pathogens. Compared to their species C (HAdV-C) counterparts, relatively little work has been devoted to the characterization of their unique molecular biology. The early region 3 (E3) transcription unit is an interesting target for future efforts because of its species-specific diversity in genetic content among adenoviruses. This diversity is particularly significant for the subset of E3-encoded products that are membrane glycoproteins and may account for the distinct pathobiology of the different human adenovirus species. In order to understand the role of HAdV-B-specific genes in viral pathogenesis, we initiated the characterization of unique E3 genes. As a continuation of our efforts to define the function encoded in the highly polymorphic ORF E3-10.9K and testing the hypothesis that the E3-10.9K protein orthologs with a hydrophobic domain contribute to the efficient release of viral progeny, we generated HAdV-3 mutant viruses unable to express E3-10.9K ortholog E3-9K and examined their ability to grow, disseminate, and egress in cell culture.RESULTS:No differences were observed in the kinetics of infected cell death, and virus progeny release or in the plaque size and dissemination phenotypes between cells infected with HAdV-3 E3-9K mutants or the parental virus. The ectopic expression of E3-10.9K orthologs with a hydrophobic domain did not compromise cell viability.CONCLUSIONS:Our data show that despite the remarkable similarities with HAdV-C E3-11.6K, HAdV-B1 ORF E3-10.9K does not encode a product with a "death-like" biological activity.

No description available.

P53 empêche la réplication d'ADN cellulaire pathologique ainsi que viral en activant la transcription d'effecteurs en aval. L'augmentation d'expression de p53 et sa phosphorylation en réponse à des oncogènes ou à des dommages à l'ADN sont considérées comme déterminant l'activation transcriptionnelle de p53. Les mutations tumorales et les protéines virales convergent fonctionnellement en inactivant p53. Par exemple la protéine cellulaire MDM2 et la protéine adénovirale E1 B-55K ciblent toutes les deux p53 pour sa dégradation. Ceci représente la base du raisonnement de la création des molécules antagonistes de MDM2 et de l'adénovirus oncolytique où E1B-55K a été supprimé, ONYX-015 (Oncorine), comme thérapies anticancéreuses ciblant p53. Pourtant ici, nous montrons que E1B-55K est dispensable pour l'inactivation de p53 et nous révélons un mécanisme épigénétique nouveau et dominant qui inactive l'activité de p53, quel que soit le niveau et la phosphorylation de p53. En utilisant une approche génétique, nous dévoilons qu'E4-ORF3, une autre protéine adénovirale, est le point de nucléation de domaines d'hétérochromatine, menant à l'inactivation des promoteurs cibles de p53, en empêchant sa liaison et donc son activation transcriptionnelle. De plus, nous montrons qu'E4-ORF3 forme un échafaudage nucléaire qui dirige deux métyltransferases, SUV39H1 et SUV39H2, vers ces nouveaux domaines répressifs. Notre étude change la définition fondamentale du mécanisme d'inactivation de p53 dans les cellules infectées par un adénovirus, apportant une nouvelle lumière sur un mécanisme critique qui va maintenant permettre le développement de thérapies adénovirale vraiment sélectives du statut p53 des cellules. / P53 guards against pathological cellular and viral DNA replication by activating the transcription of downstream effectors. The induction of p53 levels and its phosphorylation in response to oncogenes and DNA damage is thought to determine p53 transcriptional activation. Tumor mutations and viral proteins functionally converge in inactivating p53. For example the cellular protein MDM2 and the adenoviral protein E1B-55k both target p53 for its degradation. This is the premise for MDM2 antagonists and the E1B-55k deleted oncolytic adenovirus, ONYX-015 (Oncorine) as p53-targeted cancer therapies. However, here we show that E1B-55k is dispensable for p53 inactivation and reveal a novel and dominant epigenetic mechanism that silences p53 activity, irrespective of p53 level and phosphorylation. Using a genetic approach, we reveal that E4-ORF3, another adenoviral protein, nucleates heterochromatin domain, leading to the silencing of p53 target promot er by preventing its binding and subsequent transcriptional activation. Moreover we show that E4-ORF3 forms a novel nuclear scaffold that directs two methyltransferases, SUV39H1 and SUV39H2, to this newly formed repressive domains. Our study changes the fundamental definition of how p53 is inactivated in adenovirus infected cells, and provides a critical mechanistic insight that could now enable the rational development of true p53 tumor selective adenoviral therapies.

RNA interference (RNAi) is a diverse, conserved regulatory mechanism in eukaryotic cells, which silences the target gene expression in a homology-dependent manner. Although it has been well documented that RNAi is an antiviral mechanism in plants and insects, it is still unclear whether RNAi naturally limits viral infections in vertebrates. Viruses are masters of adopting strategies to subvert cellular defense mechanisms. Not only can viruses use elaborate strategies to suppress the effects of defensive RNAi, but they can also redirect or interfere with cellular functions orchestrated by endogenous small RNAs. In our work we have focused on studying the relationship of human adenovirus type 5 (Ad5) infection and the RNAi/miRNA pathways. We show that Ad5 infection inhibits RNAi by blocking the activity of Dicer and the RNA-induced silencing complex (RISC). For Dicer inhibition, the virus-associated RNAs, VA RNAI and VA RNAII bind Dicer through their terminal stems and are cleaved by Dicer into functional small RNAs that are incorporated into active RISC. Furthermore, by cloning small RNAs, we found that approximately 80% of Ago2-containing RISC immunopurified from late infected cells was associated with VA RNA-derived small RNAs (mivaRNAs). Interestingly, the small RNAs derived from VA RNAII, the minor VA RNA species, appear to be the major mivaRNAs occupying RISC and associate with polyribosomes, which indicates their potential roles as miRNAs regulating translation of cellular mRNAs. During our previous work, we observed that the strand bias of VA RNAI derived small RNA (mivaRI) incorporating into active RISC varied in the different viable Ad5 mutant viruses infected cells. It has been reported that Ad5 VA RNAI had two transcription initiation sites, which produced two clusters of VA RNAI with 3 nt difference at their 5’ end. Our data show that this heterogeneity resulted in a dramatic difference in mivaRI guide strand selection. Collectively, our data contributes to understanding the interplay between virus and host. This study would be beneficial in designing optimal adenovirus vectors for therapeutic RNAi application.

Despite the availability of live attenuated measles virus vaccines, a large number of measles-associated deaths occur among infants in developing countries during the "window of susceptibility" (age 4-9 months). During this period declining maternal antibody titers are no longer protective against wild-type measles virus (MV) and impede successful immunization with the live attenuated vaccines. Therefore, the development of a safe vaccine that would induce protective immunity in the presence of maternally derived MV-specific antibodies in young infants and would close the "window of susceptibility" is desirable. Since adenoviruses have been shown as suitable vaccine candidates capable of eliciting potent protection against mucosal infectious diseases, the ability of an adenovirus-vectored anti-measles vaccine to elicit robust immune responses against MV was assessed in this study. Mice immunized intramuscularly or intranasally with a combination of human adenovirus serotype 5 (Ad5) recombinants expressing MV hemagglutinin (H) and fusion (F) glycoproteins developed MV-specific neutralizing antibody titers similar for both routes of immunization. However, intramuscular immunization of mice with Ad5 recombinants resulted in induction of a predominant T helper type (Th1) immune response, whereas intranasal immunization induced a balanced Th1/Th2 immune response. Furthermore, intranasal immunization resulted in increased titers of MV-specific immunoglobulin A (IgA) in lungs in comparison to intramuscularly immunized animals. The ability of the Ad5 recombinants to induce protective immune responses in cotton rats by different routes of administration was also evaluated. Cotton rats that received a single dose of the Ad5 recombinants intramuscularly or intranasally experienced a rise in MV-specific neutralizing antibody titers and reduction of the viral RNA load in the lung tissue after intranasal MV challenge. In addition, the largest reduction in viral replication was observed in the group of cotton rats inoculated with the Ad5 recombinants intranasally. Based on these observations, the Ad5-based vaccine appears to be a suitable candidate against measles. Furthermore, a capability of purified globular head domain of MV H protein produced in a human cell line to induce MV-specific immune responses in mice was tested. Subcutaneous immunization of mice with the recombinant protein alone resulted in both humoral and cell-mediated immunity, characterized by the production of MV-specific serum IgG and neutralizing antibodies, as well as interferon-gamma and interleukin 5 (IL-5) production by in vitro restimulated splenocytes. The former responses were further enhanced by formulation of the protein with aluminium hydroxide. However, very low numbers of INF-gamma secreting cells and low levels of IgG2a in the serum suggested a Th2 immune response. Novel adjuvants (Th1-directing), as well as MV F protein should be considered for the inclusion into the vaccine formulations to induce more balanced Th1/Th2 immune responses against measles.

Inclusion body hepatitis (IBH) has been occurring as an economically important, emerging disease of broiler chickens in several countries. Historically, IBH has been identified as a secondary disease, often associated with common immunosuppressive diseases. However, few studies have identified IBH as a primary disease with no apparent association with immunosuppressive diseases. The objectives of this study were to develop an animal model of IBH in commercial broilers, to demonstrate vertical transmission of fowl adenoviruses (FAdVs) in broiler breeders and to control IBH in broilers by vaccinating their parents with an inactivated FAdV vaccine. In order to develop an animal model of IBH in commercial broilers, fourteen-day old broilers were inoculated intramuscularly with 1x104 1x107 CCID50 of either FAdV x11a-like virus, two strains of FAdV-8a (FAdV-8a strain TR-59 and FAdV-8a strain T8-A) or FAdV-11strain 1047. Four days following FAdV inoculation, 5% - 15% mortality was observed and dead birds showed histologic lesions of hemorrhagic necrotizing hepatitis. This animal model reproduced the clinical disease, and pathological lesions of IBH that have been described in commercial broilers. In order to demonstrate vertical transmission of the FAdV, 35-week-old broiler breeders were inoculated with 1x106 CCID50 of either FAdV x11a-like virus, FAdV-8a strain TR-59, FAdV-8a strain T8-A or FAdV-11 strain 1047. Eggs from infected breeders were collected and hatched seven days post-inoculation. Clinical signs or mortality were not observed in parents; however broiler progeny derived from broiler breeders inoculated with FAdV-8a strain T8-A had 30% IBH mortality by seven days of age. The hexon gene loop 1 sequence of the virus isolated from affected broiler progeny showed 100% identity to FAdV-8a strain T8-A. In order to demonstrate protection of broilers against IBH by vaccination of their parents, four groups of broiler breeders were vaccinated with either FAdV-8a strain T8-A (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion, or FAdV x11a-like virus (2x107 or 2x104 CCID50) formulated with 20% oil-in-water emulsion at the age of 12 and 15 weeks. The control group was inoculated with 20% oil-in-water emulsion. Broiler progeny were challenged with FAdV-8a strain T8-A to study the immunoprotective effect of the vaccine. Although, survival of broilers following FAdV-8a strain T8-A challenge was not significantly different among vaccinated and non-vaccinated groups (P>0.05), immunoprotective effect was enhanced by the increase dose of FAdV antigens (P>0.05). Further studies are necessary to improve the vaccine efficacy to protect broilers against IBH.<p> In conclusion, the results of this study support the hypothesis that IBH in broilers in Canada is a vertically-transmitted primary disease with no known immunosuppressive involvement. The results also demonstrated that inactivated antigens of FAdV are able to partially protect broilers against IBH by vaccinating their parents. Further studies with different formulations, and priming the immune system of broiler breeders with live FAdV prior to vaccination with inactivated FAdV vaccines are necessary to improve the efficacy of inactivated IBH vaccine.

Bovine adenovirus type 3 (BAdV-3) is a non-enveloped icosahedral particle which contains a double stranded DNA genome. The genome of BAdV-3 is organized into early, intermediate and late regions. The late region is organized into seven regions L1-L7 (Reddy et.al., 1998). The L6 region of late transcription unit of BAdV-3 encodes one of the non structural protein named 33K protein. The objective of the present study was to characterize the 33K protein and to identify the viral/cellular proteins involved in the interaction with 33K protein.<p> The RT-PCR analysis revealed the presence of spliced and unsliced mRNAs encoding 33K and 22K proteins respectively in BAdV-3 infected cells. The 33K and 22K proteins share a N-terminus region of 138 amino acids. To determine the specificity of these two proteins, rabbit polyclonal antiserum was raised against peptides representing unique C- terminal regions of the proteins. Anti-33Kp serum detected two major proteins of 42 kDa and 22 kDa and five minor proteins of 39kDa, 35kDa, 29kDa, 25kDa and 19kDa in BAdV-3 infected cells or 33K transfected cells. Similarly, anti-22Kp serum detected three proteins of 41kDa, 39kDa and 37kDa in BAdV-3 infected cells. However, a protein of 39kDa and 37kDa was detected in 22K (having splice sites removed) transfected cells. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells and is involved in stimulating the transcription from major late promoter. Analysis of mutant 33K proteins demonstrated that amino acids 201-240 and amino acid 204-231 are required for nuclear localization and MLP transactivation.<p> The adenovirus 33K protein appears to be a multifunctional protein performing different role in viral infection. Earlier study has shown that the 33K protein plays a role in viral capsid assembly and efficient capsid DNA interaction in BAdV-3 (Kulshreshtha et.al., 2004). The involvement of 33K protein in different steps of adenovirus replication may require protein protein interaction. Using 33K protein as bait in yeast two hybrid system, open reading frames (ORFs) of BAdV-3 were screened for the potential interactions with 33K protein. The 33K protein showed specific interactions with two late viral proteins- 100K and protein V (pV). The yeast two hybrid findings were validated by in vitro binding using <i>in vitro</i> synthesized transcription-translation products. It was demonstrated that the interaction of 33K with 100K and pV takes place during BAdV-3 infection. The stretch of amino acids 81-120 and 161-200 in 33K protein were involved in the interaction with pV and 100K protein.<p> For screening the cellular interactions, the 33K protein was used as a bait to screen bovine retina cDNA library. The yeast two hybrid screening revealed that the 33K protein appears to interact with bovine presenilin-1-associated protein / mitochondrial carrier homolog 1 (BoPSAP / BoMtch1) and bovine microtubule associated protein (BoMAP). However, subsequent analysis by various <i>in vitro</i> and <i>in vivo</i> assays could only confirm the interaction between 33K protein and BoPSAP/BoMtch1. In addition, the 33K protein was also shown to be colocalized with BoPSAP in mitochondria. Based on these observations, it may be possible that 33K protein may play an anti-apoptotic by interacting with BoPSAP since the human homolog of PSAP has been known to induce apoptosis.

Despite the availability of live attenuated measles virus vaccines, a large number of measles-associated deaths occur among infants in developing countries during the "window of susceptibility" (age 4-9 months). During this period declining maternal antibody titers are no longer protective against wild-type measles virus (MV) and impede successful immunization with the live attenuated vaccines. Therefore, the development of a safe vaccine that would induce protective immunity in the presence of maternally derived MV-specific antibodies in young infants and would close the "window of susceptibility" is desirable. Since adenoviruses have been shown as suitable vaccine candidates capable of eliciting potent protection against mucosal infectious diseases, the ability of an adenovirus-vectored anti-measles vaccine to elicit robust immune responses against MV was assessed in this study. Mice immunized intramuscularly or intranasally with a combination of human adenovirus serotype 5 (Ad5) recombinants expressing MV hemagglutinin (H) and fusion (F) glycoproteins developed MV-specific neutralizing antibody titers similar for both routes of immunization. However, intramuscular immunization of mice with Ad5 recombinants resulted in induction of a predominant T helper type (Th1) immune response, whereas intranasal immunization induced a balanced Th1/Th2 immune response. Furthermore, intranasal immunization resulted in increased titers of MV-specific immunoglobulin A (IgA) in lungs in comparison to intramuscularly immunized animals. The ability of the Ad5 recombinants to induce protective immune responses in cotton rats by different routes of administration was also evaluated. Cotton rats that received a single dose of the Ad5 recombinants intramuscularly or intranasally experienced a rise in MV-specific neutralizing antibody titers and reduction of the viral RNA load in the lung tissue after intranasal MV challenge. In addition, the largest reduction in viral replication was observed in the group of cotton rats inoculated with the Ad5 recombinants intranasally. Based on these observations, the Ad5-based vaccine appears to be a suitable candidate against measles. Furthermore, a capability of purified globular head domain of MV H protein produced in a human cell line to induce MV-specific immune responses in mice was tested. Subcutaneous immunization of mice with the recombinant protein alone resulted in both humoral and cell-mediated immunity, characterized by the production of MV-specific serum IgG and neutralizing antibodies, as well as interferon-gamma and interleukin 5 (IL-5) production by in vitro restimulated splenocytes. The former responses were further enhanced by formulation of the protein with aluminium hydroxide. However, very low numbers of INF-gamma secreting cells and low levels of IgG2a in the serum suggested a Th2 immune response. Novel adjuvants (Th1-directing), as well as MV F protein should be considered for the inclusion into the vaccine formulations to induce more balanced Th1/Th2 immune responses against measles.

El càncer de pròstata és el càncer majoritàriament diagnosticat en homes adults i la segona causa de mort, després del càncer de pulmó. Els tractaments convencionals com la prostatectomia radical, radioteràpia i quimioteràpia tan sols funcionen en la malaltia localitzada, essent el desenvolupament de metàstasis la principal causa de mort dels pacients. Per tant, és necessària la cerca de noves teràpies que millorin l'eficàcia dels tractaments del càncer de pròstata, sobretot en presència de focus metastàtics en altres òrgans. En aquest sentit, la teràpia gènica - que consisteix en la transferència de material genètic per a substituir, restablir o potenciar les funcions biològiques de cèl·lules i teixits- s'ha proposat com una alternativa al tractament del càncer de pròstata. La majoria dels protocols de teràpia gènica en càncer de pròstata utilitzen la injecció intratumoral, que limita la teràpia als tumors localitzats, sent necessària l'administració sistèmica del vector terapèutic per tal d'arribar a la malaltia disseminada. Per a poder administrar sistèmicament un vector cal evitar la transferència del transgen als teixits i cèl·lules no diana i, per tant, aquest vector haurà d'estar específicament dirigit a aquests. Els Adenovirus són els vectors més utilitzats tant en teràpia gènica del càncer com en teràpia gènica del càncer de pròstata ja que presenten una elevada transferència del transgen sense introduir el seu genoma al de la cèl·lula hoste, poden transportar fragments grans de DNA i es poden infectar una gran varietat de cèl·lules i teixits. Aquest extens tropisme és alhora que una avantatge una limitació ja que es pot produir la infecció de cèl·lules i teixits no diana degut a l'expressió ubiqua del seu receptor primari: el Coxsackie's and Adenovirus Receptor (CAR). Així doncs, el direccionament específic d'aquests vectors és de gran interès. En aquest treball, mitjançant el cribratge in vitro de la llibreria de Phage Display PhD-C7C sobre cèl·lules de càncer de pròstata metastàtic (PC3MM2) hem seleccionat 3 seqüències peptídiques (CTPQNTTMC, CYPSRSPLC i CPLHQRPMC) que han demostrat una major eficiència d'unió a les variants metastàtiques de càncer de pròstata que a les altres línies cel·lulars analitzades. Per tal de determinar si aquestes seqüències podrien ser funcionals pel direccionament específic de vectors terapèutics, les hem introduït a l'HI loop de la fibra de l'Adenovirus 5. Hem obtingut 3 Adenovirus recombinants: l'AdTLCTPQ (amb la seqüència CTPQNTTMC a l'HI loop), l'AdTLCYPS (amb la seqüència CYPSRSPLC a l'HI loop) i l'AdTLCPLH (amb la seqüència CPLHQRPMC a l'HI loop) i hem comprovat la seva infectivitat tant en sistemes in vitro com in vivo. Tot i que no hem obtingut diferències estadísticament significatives en la infecció de tumors i metàstasis en sistemes in vivo, els resultats obtinguts in vitro demostren que l'AdTLCTPQ i l'AdTLCPLH poden infectar les cèl·lules emprant un receptor diferent a CAR i que, per tant, utilitzant un vector terapèutic adequat, aquestes seqüències podrien ser funcionals pel direccionament de vectors terapèutics. / Prostate cancer is most commonly diagnosed in adult men and is the second cause of death after lung cancer. Radical prostatectomy, radiotherapy and chemotherapy only are effective in localized disease, being metastasis the principal cause of patient's death. Therefore, it's necessary the development of novel therapies to improve prostate cancer treatment. Gene therapy -which consists in the transfer of genetic material to restore, promote or replace biological functions of cells and tissues- has been proposed as an alternative to prostate cancer treatment. The major part of prostate cancer gene therapy studies use intratumoral injection, limiting the therapy to localized disease and being necessary the systemic administration to reach disseminated disease. In order to administrate systemically a vector it is necessary to avoid transgen transfer to non-target cells, and so it will have to be specifically directed to target cells.Adenoviruses are the vectors more used in cancer gene therapy and in prostate cancer gene therapy because present an elevated transgen transfer, can accommodate large DNA fragments and can infect a large variety of cells and tissues. This extended tropism is an advantage but a limitation too because non-specific transgen transfer due to ubiquitous expression of the primary receptor: Coxsackie's and Adenovirus Receptor (CAR). So on, there's a big interest in specific targeting of these vectors.In this work, through the screening in vitro of the Phage Display library PhD-C7C on metastatic prostate cancer cells PC3MM2, we have selected 3 peptide sequences (CTPQNTTMC, CYPSRSPLC and CPLHQRPMC) that have demonstrate a higher binding efficiency to metastatic prostate cancer cells than to the rest of cell lines analyzed. To determine if these peptide sequences could be functional in specific targeting of therapeutic vectors we have introduced them into the HI loop of Ad5 fiber. We have generate 3 recombinant adenoviruses: AdTLCTPQ (CTPQNTTMC sequence into the HI loop), AdTLCYPS (CYPSRSPLC sequence into the HI loop) and AdTLCPLH (CPLHQRPMC sequence into the HI loop) and analyzed its infectivity in vitro and in vivo. Although we have not observed and increase in tumor and metastasis infectivity in vivo, in vitro results demonstrate that AdTLCTPQ and AdTLCPLH can transduce cells using a CAR independent pathway and, so on, using an adequate therapeutic vector these sequences could functions as target specific peptides.