Adenovirus E1A Activation Domain Regulates H3 Acetylation Affecting Varied Steps in Transcription at Different Viral Promoters.

How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated lacO array of fusions of LacI-mCherry to E1A fragments with YFP-p300. Viruses with mutations in E1A subdomains were constructed and analyzed for kinetics of early viral RNA expression and association of acetylated H3K9, K18, K27, TBP, and RNA polymerase II (Pol II) across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously (P. Pelka, J. N. G. Ablack, J. Torchia, A. S. Turnell, R. J. A. Grand, J. S. Mymryk, Nucleic Acids Res 37:1095-1106, 2009, https://doi.org/10.1093/nar/gkn1057), and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters

Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?

The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system

The human adenovirus 5 L4 promoter is activated by cellular stress response protein p53

During adenovirus infection, the emphasis of gene expression switches from early genes to late genes in a highly regulated manner. Two gene products, L4-22K and L4-33K, contribute to this switch by activating the Major Late Transcription Unit (MLTU) and regulating the splicing of its transcript. L4-22K and L4-33K expression is driven initially by a recently described L4 promoter (L4P) embedded within the MLTU that is activated by early and intermediate viral factors: E1A, E4 Orf3 and IVa2. Here we show that this promoter is also significantly activated by the cellular stress response regulator, p53. Exogenous expression of p53 activated L4P in reporter assays whilst depletion of endogenous p53 inhibited the induction of L4P by viral activators. Chromatin immunoprecipitation studies showed that p53 associates with L4P and that during adenovirus type 5 (Ad5) infection this association peaks at 12 h.p.i., coinciding with the phase of the infectious cycle when L4P is active, and is then lost as MLP activation commences. P53 activation of L4P is significant during Ad5 infection since depletion of p53 prior to infection of either immortalised or normal cells led to severely reduced late gene expression. The association of p53 with L4P is transient due to the action of products of L4P activity (L4-22K/33K), which establish a negative feedback loop that ensures the transient activity of L4P at the start of the late phase and contributes to an efficient switch from early to late phase virus gene expression

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling

Rational engineering of microRNA-regulated viruses for cancer gene therapy

MicroRNAs (miRNAs) are small noncoding RNA molecules that have important regulatory roles in a wide range of biological processes. miRNAs are often expressed in a tissue- and/or differentiation state-specific patterns, and it is estimated that miRNAs can regulate the expression of more than 50% of all human genes. We have exploited these tissue-specific miRNA expression patterns in the modification of viral replicative tropism. In order to engineer the replicative tropism of oncolytic adenoviruses, we developed a recombinant adenovirus that in the 3 UTR of the critical E1A gene contains sequences complementary to the liver-specific miRNA miR122. This allowed us to generate a novel recombinant adenovirus that was severely attenuated in human liver, but replicated to high titres in colorectal cancer. Systemic injection of miR122-targeted adenovirus into mice did not induce liver toxicity. In a human lung cancer xenograft mouse model this miR122-targeted adenovirus showed potent antitumour activity. We also studied the possibility to exploit neuron-specific miRNA expression patterns in the modification of tissue tropism of an alphavirus Semliki Forest virus (SFV). We engineered SFV genome to contain sequences complementary to the neuron-specific miRNA miR124. In vitro characterization of this novel virus showed that the modification of the SFV genome per se did not affect polyprotein processing or oncolytic potency. Intraperitoneally administered miR124-targeted SFV displayed an attenuated spread into the central nervous system (CNS) and increased survival of infected mice. Also, mice pre-infected with miR124-targeted SFV elicited strong protective immunity against otherwise lethal challenge with a highly virulent wild-type SFV strain. In conclusion, these results show that miRNA-targeting is a potent new strategy to engineer viral tropism in development of safer and more efficient reagents for virotherapy applications.MikroRNA:t (miRNA) ovat pieniä ei-koodaavia RNA molekyylejä joilla on tärkeä tehtävä useiden erilaisten biologisten prosessien säätelyssä. MiRNA:t ekpressoituvat usein kudos- ja/tai kehitysvaihespesifisesti sekä säätelevät jopa yli 50 prosenttia kaikista ihmisen geeneistä. Tässä väitöskirjatutkimuksessa pyrimme käyttämään hyväksi miRNA:iden kudosspesifistä ekpressiota virusten kudostropismin muokkaamisessa vähentääksemme virusvektoreiden haitallista kudostoksisuutta. Muokataksemme adenovirusvektoreiden kudostropismia, kehitimme uudentyyppisen adenoviruksen jonka E1A-geenin 3 ei-koodaavalle alueelle lisäsimme ihmisen maksaspesifisen miRNA miR122:n tunnistussekvenssejä. Tunnistussekvenssien lisäyksellä saimme aikaan adenoviruksen (miR122-targetoitu adenovirus) jonka replikaatiokyky oli huomattavasti heikentynyt ihmisen maksassa, mutta pystyi replikoitumaan voimakkaasti perä- ja paksusuolisyöpäkudoksessa. Hiireen systeemisesti injisoitu miR122-targetoitu adenovirus ei aiheuttanut maksatoksisuutta. Ihmisen keuhkosyöpähiirimallissa miR122-targetoitu virus tappoi tehokkaasti syöpäsoluja. Tässä väitöskirjatutkimuksessa tutkimme myös hermosoluspesifisen miRNA miR124:n hyväksikäyttöä Semliki Forest-viruksen (SFV) kudostropismin muokkauksessa. Kehitimme SFV:n jonka genomiin oli sisällytetty miR124:n tunnistussekvenssejä. In vitro-kokeilla osoitimme tämän miR124-targetoidun SFV:n proteiinien prosessoituvan normaalisti sekä onkolyyttisen tehon säilyneen villityypin viruksen kaltaisena. Vatsaonteloon injisoitu miR124-targetoitu SFV levisi hyvin heikosti keskushermostossa joka johti vähentyneeseen neurotoksisuuteen. Osoitimme myös miR124-targetoidun viruksen toimivan tehokkaana rokotteena erittäin patogeeniselle L10 SFV-kannalle. Tässä väitöskirjatutkimuksessa pystyimme osoittamaan miRNA-targetoinnin olevan tehokas uusi tapa muokata virusten kudostropismia ja parantaa virusvektoreiden turvallisuutta

Adenovirus-Mediated Sensitization to the Cytotoxic Drugs Docetaxel and Mitoxantrone Is Dependent on Regulatory Domains in the E1ACR1 Gene-Region

Oncolytic adenoviruses have shown promising efficacy in clinical trials targeting prostate cancers that frequently develop resistance to all current therapies. The replication-selective mutants AdΔΔ and dl922–947, defective in pRb-binding, have been demonstrated to synergise with the current standard of care, mitoxantrone and docetaxel, in prostate cancer models. While expression of the early viral E1A gene is essential for the enhanced cell killing, the specific E1A-regions required for the effects are unknown. Here, we demonstrate that replicating mutants deleted in small E1A-domains, binding pRb (dl1108), p300/CBP (dl1104) and p400/TRRAP or p21 (dl1102) sensitize human prostate cancer cells (PC-3, DU145, 22Rv1) to mitoxantrone and docetaxel. Through generation of non-replicating mutants, we demonstrate that the small E1A12S protein is sufficient to potently sensitize all prostate cancer cells to the drugs even in the absence of viral replication and the E1A transactivating domain, conserved region (CR) 3. Furthermore, the p300/CBP-binding domain in E1ACR1 is essential for drug-sensitisation in the absence (AdE1A1104) but not in the presence of the E1ACR3 (dl1104) domain. AdE1A1104 also failed to increase apoptosis and accumulation of cells in G2/M. All E1AΔCR2 mutants (AdE1A1108, dl922–947) and AdE1A1102 or dl1102 enhance cell killing to the same degree as wild type virus. In PC-3 xenografts in vivo the dl1102 mutant significantly prolongs time to tumor progression that is further enhanced in combination with docetaxel. Neither dl1102 nor dl1104 replicates in normal human epithelial cells (NHBE). These findings suggest that additional E1A-deletions might be included when developing more potent replication-selective oncolytic viruses, such as the AdΔCR2-mutants, to further enhance potency through synergistic cell killing in combination with current chemotherapeutics

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection

Prion expression is activated by Adenovirus 5 infection and affects the adenoviral cycle in human cells

The prion protein is a cell surface glycoprotein whose physiological role remains elusive, while its implication in transmissible spongiform encephalopathies (TSEs) has been demonstrated. Multiple interactions between the prion protein and viruses have been described: viruses can act as co-factors in TSEs and life cycles of different viruses have been found to be controlled by prion modulation. We present data showing that human Adenovirus 5 induces prion expression. Inactivated Adenovirus did not alter prion transcription, while variants encoding for early products did, suggesting that the prion is stimulated by an early adenoviral function. Down-regulation of the prion through RNA interference showed that the prion controls adenovirus replication and expression. These data suggest that the prion protein could play a role in the defense strategy mounted by the host during viral infection, in a cell autonomous manner. These results have implications for the study of the prion protein and of associated TSEs

Screening for the optimal siRNA targeting a novel gene (HA117) and construction and evaluation of a derivative recombinant adenovirus

We found a novel gene named as HA117 in our previous research. At this study, we screened for an optimal siRNA targeting the novel gene HA117 using the pSOS-HUS method, verified the results of pSOS-HUS siRNA screening for optimal affinity for the target gene, and constructed and evaluated a recombinant adenovirus carrying the DNA template for transcription of the optimal HA117 siRNA. The pSOS-HUS vector method was successfully utilized as a rapid and effective screen for an optimal siRNA for a target gene. Among five pairs of DNA templates, siRNA transcribed from HAi5 gave the strongest interference with the novel gene HA117; a HAi5-carrying recombinant adenovirus (Ad-HAi5) was successfully constructed and evaluated, laying a foundation for the further study of HA117 gene function with RNAi technology