E1A Protein Research Articles

Post-translational modifications on protein VII are important during the early stages of adenovirus infection.

Due to the importance of post-translational modification (PTM) in cellular function, viruses have evolved to both take advantage of and be susceptible to such modification. Adenovirus encodes a multifunctional protein called protein VII, which is packaged with the viral genome in the core of virions and disrupts host chromatin during infection. Protein VII has several PTMs whose addition contributes to the subnuclear localization of protein VII. Here, we used mutant viruses that abrogate or mimic these PTMs on protein VII to interrogate their impact on protein VII function during adenovirus infection. We discovered that acetylation of the lysine in positions 2 or 3 (K2 or K3) is deleterious during early infection as mutation to alanine led to greater intake of protein VII to the nucleus and enhanced early gene expression. Furthermore, we determined that protein VII is acetylated at alternative residues late during infection which may compensate for the mutated sites. Lastly, due to the role of the early viral protein E1A in viral gene activation, we investigated the interaction between protein VII and E1A and demonstrated that protein VII interacts with E1A through a chromatin-mediated interaction. Together, these results emphasize that the complexity of virus-host interactions is intimately tied to post-translational modification.

DIPG-45. TARGETING HYPERACETYLATION IN PEDIATRIC DIFFUSE MIDLINE GLIOMAS USING ONCOLYTIC ADENOVIRUSES

Abstract BACKGROUND H3K27-altered pediatric diffuse midline glioma (DMG) is a highly aggressive type of brain tumor with a 5-year survival rate of less than 2%. Conventional treatments are restricted to palliative radiation therapy. Therefore, effective treatments are urgently needed. Results from a recent phase I clinical trial for Diffuse Intrinsic Pontine Glioma showed that intratumoral administration of Delta-24-RGD oncolytic adenovirus in combination with radiation improves survival without unwanted toxicity. Based on the rationale that, during infection, the early viral protein E1A binds P300, we hypothesize that combining oncolytic adenoviruses with p300 inhibitors will induce a robust modification of the post-translational landscape of DMGs and increase the anti-cancer effect of the oncolytic virus. METHODS Studies on Delta-24-RGD infectivity and replication were performed in a panel of clinically significant DMG cells. Modification of the acetylation levels of acid-extracted histones after Delta-24-RGD was analyzed using Western blot. Cell Titer Blue was utilized to measure cell viability, and ZIP-synergy scores were calculated using SynergyFinder. RESULTS We observed a time-dependent reduction in H3K27ac in human DMG cells following Delta-24-RGD infection with an 89% reduction in H3K27ac at 48h post-infection. Delta-24-RGD treatment of DMG cells resulted in optimum viral infectivity and replication. Combining Delta-24-RGD with the p300 inhibitor C646 resulted in enhanced cell death in vitro. Using a panel of oncolytic viruses that induce differential enzymatic and binding functions of P300, we showed that Delta-24-RGD mediated alteration of P300 activity is required for hypoacetylation in DMGs. We characterized the in vivo dynamics of K27M-H3.1 and K27M-H3.3 DMG-derived tumors in the brain stem of syngeneic animal models. In vivo experiments are in progress to examine the combinatorial anti-glioma effect of Delta-24-RGD and P300 inhibitors. CONCLUSION Collectively, our studies provide a strong mechanistic rationale for combining oncolytic adenoviruses and p300 inhibitors and should propel the eventual clinical testing of this approach in DMG patients.

Inhibition of human adenovirus replication by TRIM35-mediated degradation of E1A.

Human adenovirus (HAdV) is ubiquitous in the human population, constituting a significant burden of global respiratory diseases. Children and individuals with low immunity are at risk of developing severe infections without approved antiviral treatment for HAdV. Our study demonstrated that TRIM35 inhibited HAdV-C5 early gene transcription, early protein expression, genome replication, and infectious virus progeny production. Furthermore, TRIM35 was found to inhibit HAdV replication by attenuating E1A expression. Mechanistically, TRIM35 interacts with and degrades E1A by promoting its K48-linked ubiquitination. Additionally, K253 and K285 are the key sites necessary for TRIM35 degradation. Moreover, an oncolytic adenovirus carrying shTRIM35 was constructed and observed to exhibit improved oncolysis in vivo, providing new ideas for clinical tumor treatment. Our results expand the broad antiviral activity of TRIM35 and mechanically support its application as a HAdV replication inhibitor. IMPORTANCE E1A is an essential human adenovirus (HAdV) protein responsible for the early replication of adenovirus while interacting with multiple host proteins. Understanding the interaction between HAdV E1A and TRIM35 helps identify effective antiviral therapeutic targets. The viral E1A protein is a crucial activator and regulator of viral transcription during the early infection stages. We first reported that TRIM35 interacts with E1A to resist adenovirus infection. Our study demonstrated that TRIM35 targets E1A to resist adenovirus, indicating the applicability of targeting virus-dependent host factors as a suitable antiviral strategy.

Structural basis of CBP/p300 recruitment by the microphthalmia-associated transcription factor

The microphthalmia-associated transcription factor (MITF) is a master regulator of the melanocyte cell lineage. Aberrant MITF activity can lead to multiple malignancies including skin cancer, where it modulates the progression and invasiveness of melanoma. MITF-regulated gene expression requires recruitment of the transcriptional co-regulator CBP/p300, but details of this process are not fully defined. In this study, we investigate the structural and functional interaction between the MITF N-terminal transactivation domain (MITFTAD) and CBP/p300. Using pulldown assays and nuclear magnetic resonance spectroscopy we determined that MITFTAD is intrinsically disordered and binds to the TAZ1 and TAZ2 domains of CBP/p300 with moderate affinity. The solution-state structure of the MITFTAD:TAZ2 complex reveals that MITF interacts with a hydrophobic surface of TAZ2, while remaining somewhat dynamic. Peptide array and mutagenesis experiments determined that an acidic motif is integral to the MITFTAD:TAZ2 interaction and is necessary for transcriptional activity of MITF. Peptides that bind to the same surface of TAZ2 as MITFTAD, such as the adenoviral protein E1A, are capable of displacing MITF from TAZ2 and inhibiting transactivation. These findings provide insight into co-activator recruitment by MITF that are fundamental to our understanding of MITF targeted gene regulation and melanoma biology.

Antiviral effect of natural and semisynthetic diterpenoids against adenovirus infection in vitro.

The emergence and re-emergence of viruses has highlighted the need to develop new broad spectrum antivirals to mitigate human infections. Pursuing our search for new bioactive plant-derived molecules, we studied several diterpene derivatives synthesized from jatropholones A and B and carnosic acid isolated from Jatropha isabellei and Rosmarinus officinalis, respectively. Here, we investigated the antiviral effect of the diterpenes against human adenovirus (HAdV-5) that causes several infections for which there is no approved antiviral therapy yet. Ten compounds were evaluated and none of them presented cytotoxicity in A549 cells. Only compounds 2, 5 and 9 inhibited HAdV-5 replication in a concentration-dependent manner, without virucidal activity, whereas the antiviral action took place after virus internalization. The expression of viral proteins E1A and Hexon was strongly inhibited by compounds 2 and 5 and, in a lesser degree, by compound 9. Since compounds 2, 5 and 9 prevent ERK activation, they might exert their antiviral action by interfering host cell functions required for virus replication. Besides, the compounds had an anti-inflammatory profile since they significantly inhibited the levels of IL-6 and IL-8 produced by THP-1 cells infected with HAdV-5 or with an adenoviral vector. In conclusion, diterpenes 2, 5 and 9 not only exerted antiviral activity against adenovirus but also are able to restrain pro-inflammatory cytokines induced by the virus.

Evolution of SLiM-mediated hijack functions in intrinsically disordered viral proteins.

Viruses and their hosts are involved in an 'arms race' where they continually evolve mechanisms to overcome each other. It has long been proposed that intrinsic disorder provides a substrate for the evolution of viral hijack functions and that short linear motifs (SLiMs) are important players in this process. Here, we review evidence in support of this tenet from two model systems: the papillomavirus E7 protein and the adenovirus E1A protein. Phylogenetic reconstructions reveal that SLiMs appear and disappear multiple times across evolution, providing evidence of convergent evolution within individual viral phylogenies. Multiple functionally related SLiMs show strong coevolution signals that persist across long distances in the primary sequence and occur in unrelated viral proteins. Moreover, changes in SLiMs are associated with changes in phenotypic traits such as host range and tropism. Tracking viral evolutionary events reveals that host switch events are associated with the loss of several SLiMs, suggesting that SLiMs are under functional selection and that changes in SLiMs support viral adaptation. Fine-tuning of viral SLiM sequences can improve affinity, allowing them to outcompete host counterparts. However, viral SLiMs are not always competitive by themselves, and tethering of two suboptimal SLiMs by a disordered linker may instead enable viral hijack. Coevolution between the SLiMs and the linker indicates that the evolution of disordered regions may be more constrained than previously thought. In summary, experimental and computational studies support a role for SLiMs and intrinsic disorder in viral hijack functions and in viral adaptive evolution.

Characterization of the conserved regions of E1A protein from human adenovirus for reinforcement of cytotoxic T lymphocytes responses to the all genogroups causes ocular manifestation through an in silico approach.

Adenovirus species B, C, D, and E are the most common causes of ocular manifestations caused by adenoviruses. FDA-approved treatment agents for adenovirus infections are not available. Cell-mediated immunity is the major protective mechanism versus human adenoviruses (HAdVs) infection and T cells specific for peptide epitopes from nonstructural proteins can prevent adenoviral dissemination. E1A CR2 region of HAdVs Epitopes predicted for reinforcing cytotoxic T lymphocytes (CTLs) in the EKC patients. Among human adenoviruses E1 protein, four distinct E1A regions had a significantly higher level of homology than the rest of E1A protein. E1A protein inhibits IFN signal transduction. Epitope-based vaccines are designed to have flexible and simple methods to synthesize a vaccine, using an adjuvant to trigger fast immune responses. CTL epitopes were applied to create a multiepitope vaccine. Conserve region1 (CR1) and CR3 have less antigenicity compared to CR2. Additionally, CR3 in HAdV-D8 contains three toxic areas. CR4 similar to the two regions CR1 and CR3 do not show acceptable antigenic properties. Bioinformatics' tools were used to predict, refine and validate the 3D structure of the construct. Effective binding was predicted by protein-protein docking of the epitope vaccine with MHC-I molecules and revealed the safety and efficacy of the predicted vaccine construct. In silico analysis show that rising levels of cytotoxic CD8 + T cells, TH1 cells, macrophages, and neutrophils are linked to IFN-dominant TH1-type responses, which are detected in putative immune individuals. Combined with 3D protein modeling, this study predicted the epitopes of E1A CR2 protein in HAdVs.

Tyrosine hydroxylase gene promoter activity is upregulated in female catecholaminergic neuroblastoma cells following activation of a Gαq-coupled designer receptor

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. The tyrosine hydroxylase gene is regulated by extracellular signaling molecules such as epidermal growth factor, nerve growth factor and steroids. Here, we investigated whether the activity of the tyrosine hydroxylase gene promoter is upregulated by activation of G protein-coupled receptors, the largest group of plasma membrane receptors. We used catecholaminergic neuroblastoma cells as a cellular model and chromatin-integrated tyrosine hydroxylase promoter-luciferase reporter genes. The results show that stimulation of Rαq, a Gαq-coupled designer receptor, triggered transcription of a reporter gene driven by the tyrosine hydroxylase promoter. Transcription was attenuated by overexpression of regulator of G-protein signaling-2, which activates the GTPase activity of the G protein α-subunit, and by a truncated, dominant-negative mutant of phospholipase Cβ3. Extracellular signal-regulated protein kinase was identified as the signal transducer. At the transcriptional level, tyrosine hydroxylase promoter activity was found to be controlled by the transcription factor CREB. Expression experiments with the adenoviral regulator protein E1A, an inhibitor of CBP/p300 histone acetyltransferases, showed that transcription of the reporter gene controlled by the tyrosine hydroxylase is under epigenetic control. We identified the protein phosphatases MAP kinase phosphatase-1 and calcineurin as part of a shutdown device of the signaling cascade linking Rαq designer receptor activation to tyrosine hydroxylase gene transcription. We conclude that tyrosine hydroxylase promoter activity is controlled by Gαq-coupled receptors.

Targeting Triple Negative Breast Cancer With Oncolytic Adenoviruses.

Breast cancer (BC) is the most common cancer globally, accounting for 685,000 deaths in 2020. Triple-negative breast cancers (TNBC) lack oestrogen (ER) and progesterone (PR) hormone receptor expression and HER2 overexpression. TNBC represent 10–15% of all BC with high incidence in women under 50-years old that have BRCA mutations, and have a dismal prognosis. African American and Hispanic women are at higher risk partly due to the common occurrence of BRCA mutations. The standard treatment for TNBC includes surgery, radiotherapy, and chemotherapy although, resistance to all standard-of-care therapies eventually develops. It is crucial to identify and develop more efficacious therapeutics with different mechanisms of action to improve on survival in these women. Recent findings with oncolytic adenoviruses (OAds) may generate a new strategy to improve on the outcomes for women afflicted by TNBC and other types of BC. OAds are genetically engineered to selectively lyse, eliminate and recruit the host antitumour immune responses, leaving normal cells unharmed. The most common modifications are deletions in the early gene products including the E1B55 KDa protein, specific regions of the E1A protein, or insertion of tumour-specific promoters. Clinical trials using OAds for various adenocarcinomas have not yet been sufficiently evaluated in BC patients. Preclinical studies demonstrated efficacy in BC cell lines, including TNBC cells, with promising novel adenoviral mutants. Here we review the results reported for the most promising OAds in preclinical studies and clinical trials administered alone and in combination with current standard of care or with novel therapeutics. Combinations of OAds with small molecule drugs targeting the epidermal growth factor receptor (EGFR), androgen receptor (AR), and DNA damage repair by the novel PARP inhibitors are currently under investigation with reported enhanced efficacy. The combination of the PARP-inhibitor Olaparib with OAds showed an impressive anti-tumour effect. The most promising findings to date are with OAds in combination with antibodies towards the immune checkpoints or expression of cytokines from the viral backbone. Although safety and efficacy have been demonstrated in numerous clinical trials and preclinical studies with cancer-selective OAds, further developments are needed to eliminate metastatic lesions, increase immune activation and intratumoural viral spread. We discuss shortcomings of the OAds and potential solutions for improving on patient outcomes.

Abstract 2000: The adenovirus Delta-24-RGD in combination with ONC201 provides a therapeutic benefit and a proinflammatory recruitment in preclinical models of pHGGs and DMGs

Abstract Purpose of the work: Pediatric High Grade Gliomas (pHGGs), including Diffuse Midline Gliomas (DMGs), are very aggressive solid tumors developed during childhood with a poor overall survival underscoring the need for effective treatments. The adenovirus Delta-24RGD and the imipridone ONC201 have demonstrated safety and effectiveness in preclinical models and clinical trials. Thus, we evaluated the therapeutic efficacy of the Delta-24-RGD/ONC201 combination and analyzed possible changes in the tumor microenvironment in preclinical models of pHGGs and DMGs. Experimental procedures: A battery of pHGGs (SF188 and CHLA-03-AA), human DMG (SU-DIPG IV), and murine DMG cell lines (XFM and NP-53) were used. We assessed the viral replication by hexon titration, the protein expression amount (E1A, fiber, and mTORC1 key proteins)by immunoblotting, and oxygen consumption by Seahorse Analyses. Viability was measured by MTS. CHLA-03-AA and XFM were engrafted orthotopically in athymic nude mice and Balb/c mice, respectively. To assess in vivo efficacy, the animals were treated with Delta-24-RGD (107 PFU/animal) or PBS, followed by ONC201administration (125 mg/kg/twice/weekly). The tumor immune microenvironment was evaluated by flow cytometry. Results: First, the potential adverse interactions between both agents were checked in vitro, showing that ONC201 cotreatment did not interfere with the Delta-24-RGD replication capacity. As expected, ONC201 treatment led to a reduction in oxygen consumption, which was also observed in the combination treatment. Evaluation of cytotoxicity in a panel of cell lines showed that the combination was significantly better than either agent alone. In vivo, CHLA-03-AA bearing mice treated with the combination showed a significant increase in the median overall survival (PBS: 48 days; ONC201: 54.5 days; Delta-24-RGD: 62 days; Delta-24-RGD/ONC201: 95 days (P=0.0008)), leading to 20%long-term survivors, free of disease. Evaluation of the combination in an immunocompetent orthotopic DMG model also revealed the superior efficacy of the Delta-24-RGD/ONC201 (PBS: 11 days; ONC201: 12 days; Delta-24-RGD: 12 days; ONC201+Delta-24-RGD: 15 days (P=0.02)). Of importance, the combination treatment resulted in the reshaping of the tumor microenvironment towards a proinflammatory phenotype (T cells, NK cells, macrophages, granulocytes, monocytes, dendritic cells, and microglia). Conclusions: Our data indicate that the combination Delta-24-RGD/ONC201 generates a pro-inflammatory microenvironment that ultimately leads to a superior therapeutic effect in preclinical pHGG and DMGs models. Citation Format: Daniel de la Nava, Javier Marco-Sanz, Virginia Laspidea, Sara Labiano, Iker Ausejo-Mauleon, Reyes Hernandez-Osuna, Sabine Mueller, Javad Nazarian, Joshua E. Allen, Oren Becher, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-Garcia, Marta M. Alonso. The adenovirus Delta-24-RGD in combination with ONC201 provides a therapeutic benefit and a proinflammatory recruitment in preclinical models of pHGGs and DMGs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2000.

Host diversification is concurrent with linear motif evolution in a Mastadenovirus hub protein

Over one hundred Mastadenovirus types infect seven orders of mammals. Virus-host coevolution may involve cospeciation, duplication, host switch and partial extinction events. We reconstruct Mastadenovirus diversification, finding that while cospeciation is dominant, the other three events are also common in Mastadenovirus evolution. Linear motifs are fast-evolving protein functional elements and key mediators of virus-host interactions, thus likely to partake in adaptive viral evolution. We study the evolution of eleven linear motifs in the Mastadenovirus E1A protein, a hub of virus-host protein–protein interactions, in the context of host diversification. The reconstruction of linear motif gain and loss events shows fast linear motif turnover, corresponding a virus-host protein–protein interaction turnover orders of magnitude faster than in model host proteomes. Evolution of E1A linear motifs is coupled, indicating functional coordination at the protein scale, yet presents motif-specific patterns suggestive of convergent evolution. We report a pervasive association between Mastadenovirus host diversification events and the evolution of E1A linear motifs. Eight of 17 host switches associate with the gain of one linear motif and the loss of four different linear motifs, while five of nine partial extinctions associate with the loss of one linear motif. The specific changes in E1A linear motifs during a host switch or a partial extinction suggest that changes in the host molecular environment lead to modulation of the interactions with the retinoblastoma protein and host transcriptional regulators. Altogether, changes in the linear motif repertoire of a viral hub protein are associated with adaptive evolution events during Mastadenovirus evolution.

B-1b Cells Possess Unique bHLH-Driven P62-Dependent Self-Renewal and Atheroprotection.

B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers, leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet, the mechanism underlying this regulation remains unexplored. Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. Through RNA sequencing, P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-κB (nuclear factor kappa B), leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings, P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover, analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects, suggesting P62 as a new immunomodulatory target for treating atherosclerosis.

Ras Participates in the Regulation of the Stability of Adenoviral Protein E1A via MAP-kinase ERK.

The E1A adenoviral protein required for the initiation of the viral life cycle is being actively studied as a sensitizing agent in the combination therapy of cancer, and tumors with activated Ras in particular. We investigated the role played by the Ras signaling pathway in the regulation of E1A protein stability and showed that overexpression of activated Ras increases the basal level of E1A, but enhances the degradation of the E1A protein under treatment with histone deacetylase inhibitors (HDIs). It has been found that the MAP kinase ERK is the key factor in E1A stabilization, and ERK inactivation upon HDI treatment reduces the E1A protein level. Our results indicate that the combination treatment of tumors with activated Ras using adenoviral E1A and HDI has limitations attributed to intense HDI-dependent degradation of E1A. Nevertheless, the established contribution of ERK kinase to the regulation of E1A stability can be used to search for new effective drug combinations based on the adenoviral E1A protein.

Nonhuman Primate Adenoviruses of the Human Adenovirus B Species Are Potent and Broadly Acting Oncolytic Vector Candidates.

The use of human adenoviruses (hAds) as oncolytic agents has demonstrated considerable potential. However, their efficacy in clinical studies is generally moderate and often varies between patients. This may, in part, be attributable to variable pre-existing neutralizing immunity in patients, which can impact the antitumor efficacy and lead to response heterogeneity. Our aim was to isolate new Ads for the development of oncolytic vectors with low prevalence of neutralizing immunity in the human population. To this end, we isolated a collection of new nonhuman primate (nhp) Ads from stool samples of four great ape species held captive. We elected 12 isolates comprising the broadest genetic variability for further characterization. For three new nhpAds, all classified as the human adenovirus B (HAdV-B) species, no neutralizing activity could be detected when exposed to a preparation of immunoglobulins isolated from a pool of >1,000 donors as a surrogate of population immunity. In addition, the nhpAds of the HAdV-B species showed enhanced oncolytic potency compared to nhpAds of the HAdV-C species as well as to human adenovirus type 5 (HAdV-C5) in vitro when tested in a panel of 29 human cancer cell lines. Next-generation sequencing of the viral genomes revealed higher sequence similarity between hAds and nhpAds of HAdV-B compared to HAdV-C, which might underlie the differences in oncolytic ability. As a proof-of-concept, the Rb-binding domain of the E1A protein of the gorilla-derived HAdV-B nhpAd-lumc007 was deleted, thereby creating a new oncolytic derivative, which demonstrated increased oncolytic potential compared to HAdV-C5. Collectively, our data demonstrate that nhpAds of the HAdV-B species can serve as an alternative for the development of potent oncolytic Ad vectors with limited pre-existing neutralizing immunity in humans.

The Adenoviral E1B-55k Protein Present in HEK293 Cells Mediates Abnormal Accumulation of Key WNT Signaling Proteins in Large Cytoplasmic Aggregates.

HEK293 cells are one of the most widely used cell lines in research, and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA, and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here, we show that HEK293 cells display an unexpected distribution of key components of the WNT/β-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. The reduction of E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/β-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/β-catenin signaling components and on transcriptional regulation of WNT/β-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line.

Multiomic Mapping of Copy Number and Structural Variation on Chromosome 1 (Chr1) Highlights Multiple Recurrent Disease Drivers

Multiomic Mapping of Copy Number and Structural Variation on Chromosome 1 (Chr1) Highlights Multiple Recurrent Disease Drivers

A viral histone-like protein exploits antagonism between linker histones and HMGB proteins to obstruct the cell cycle

Virus infection necessarily requires redirecting cellular resources toward viral progeny production. Adenovirus encodes the histone-like protein VII, which causes catastrophic global reorganization of host chromatin to promote virus infection. Protein VII recruits the family of high mobility group box (HMGB) proteins to chromatin along with the histone chaperone SET. As a consequence of this recruitment, we find that protein VII causes chromatin depletion of several linker histone H1 isoforms. The relationship between linker histone H1 and the functionally opposite HMGB proteins is critical for higher-order chromatin structure. However, the physiological consequences of perturbing this relationship are largely unknown. Here, we employ complementary systems in Saccharomyces cerevisiae and human cells to demonstrate that adenovirus protein VII disrupts the H1-HMGB balance to obstruct the cell cycle. We find that protein VII causes an accumulation of G2/M cells both in yeast and human systems, underscoring the high conservation of this chromatin vulnerability. In contrast, adenovirus E1A and E1B proteins are well established to override cell cycle regulation and promote transformation of human cells. Strikingly, we find that protein VII obstructs the cell cycle, even in the presence of E1A and E1B. We further show that, in a protein-VII-deleted infection, several cell cycle markers are regulated differently compared to wild-type infection, supporting our model that protein VII plays an integral role in hijacking cell cycle regulation during infection. Together, our results demonstrate that protein VII targets H1-HMGB1 antagonism to obstruct cell cycle progression, revealing an unexpected chromatin vulnerability exploited for viral benefit.

Human Adenovirus Serotypes Efficiently Transducing HEK293 Cells: An In Vitro Propagation of HAdv

Generally, a gene which is inserted directly into a cell does not operate independently. Instead, the transmission of the gene is genetically modified by a biological messenger called a vector, consists of a transgene and a large DNA sequence as a backbone. Since they can deliver the new gene by infecting the cell, such viruses are also used as vectors. The adenovirus is a non-enveloped virus that can be tailored to transfer DNA to target cells, and it has sparked a lot of interest in the field, particularly in clinical trial therapy techniques. For the new age production of COVID-19 vaccine, development of different mammalian cell lines like HEK293 (most reliable growth and prosperity for transfection) and recombinant adenoviral vectors have become the first priority for biopharmaceutical giants and globally approved vaccine manufacturers to scale up their vaccine production. Adenoviruses have an icosahedral shape, with a protein coat encasing the viral double-stranded DNA genome. Because the adenovirus genome is relatively small, it's a good candidate for insertion of foreign DNA. The adenovirus E1A gene is deleted, and the virus loses its capacity to replicate. This ability can be restored during cell culture propagation by employing cells that produce the E1A protein, for example. Hence, in this mini research, I have shared an overview of the propagation of adenoviral vectors, i.e. recombinant adenovirus SARS CoV-2 vector in HEK-293 cell suspension culture.

Immediate-early transcriptional response to insulin receptor stimulation

Insulin binding to the insulin receptor triggers intracellular signaling cascades involving the activation of protein and lipid kinases. As a result, multiple biological functions of the cells are changed. Here, we analyzed the regulation and signaling cascades leading to insulin-induced activation of the stimulus-responsive transcription factors. For the analyses, we used chromatin-embedded reporter genes having a cellular nucleosomal organisation, and fibroblasts expressing human insulin receptors (HIRcB cells). The results show that stimulation of the insulin receptor induced the expression of the transcription factor Egr-1. Attenuation of Egr-1 promoter activation was observed following expression of a dominant-negative mutant of the ternary complex factor Elk-1. These data were corroborated by experiments showing that insulin receptor stimulation increased the transcriptional activation potential of Elk-1. In addition, the transcriptional activity of AP-1 was significantly elevated in insulin-stimulated HIRcB cells. Expression of the dominant-negative mutant of Elk-1 reduced insulin-induced activation of AP-1, indicating that Elk-1 controls both serum response element and AP-1-regulated transcription. Moreover, we show that stimulation of the insulin receptor activates cyclic AMP response element (CRE)-controlled transcription, involving the transcription factor CREB. Insulin-induced transcription of Elk-1 and CREB-controlled reporter genes was attenuated by overexpression of MAP kinase phosphatase-1 or a constitutively active mutant of calcineurin A, indicating that both phosphatases are part of a negative feedback loop for reducing insulin-mediated gene transcription. Finally, we show that expression of the adenoviral protein E1A selectively reduced CRE-mediated transcription following stimulation of the insulin receptor. These data indicate that insulin-regulated transcription of CRE-containing genes is under epigenetic control.

E2A Modulates Stemness, Metastasis, and Therapeutic Resistance of Breast Cancer.

Cancer stem cells (CSC) are considered responsible for tumor initiation, therapeutic resistance, and metastasis. A comprehensive knowledge of the mechanisms governing the acquisition and maintenance of cancer stemness is crucial for the development of new therapeutic approaches in oncology. E2A basic helix-loop-helix (bHLH) transcription factors are associated with epithelial-mesenchymal transition (EMT) and tumor progression, but knowledge of their functional contributions to cancer biology is still limited. Using a combination of in vivo and in vitro analyses in a novel PyMT-E2A conditional knockout mouse model and derived primary tumor cell lines, we report here an essential role of E2A in stemness, metastasis, and therapeutic resistance in breast cancer. Targeted deletion of E2A in the mammary gland impaired tumor-initiating ability and dedifferentiation potential and severely compromised metastatic competence of PyMT-driven mammary tumors. Mechanistic studies in PyMT-derived cell lines indicated that E2A actions are mediated by the upregulation of Snai1 transcription. Importantly, high E2A and SNAIL1 expression occurred in aggressive human basal-like breast carcinomas, highlighting the relevance of the E2A-Snail1 axis in metastatic breast cancer. In addition, E2A factors contributed to the maintenance of genomic integrity and resistance to PARP inhibitors in PyMT and human triple-negative breast cancer cells. Collectively, these results support the potential for E2A transcription factors as novel targets worthy of translational consideration in breast cancer. SIGNIFICANCE: These findings identify key functions of E2A factors in breast cancer cell stemness, metastasis, and drug resistance, supporting a therapeutic vulnerability to targeting E2A proteins in breast cancer.