[Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

Blockage by adenovirus E4orf6 of transcriptional activation by the p53 tumor suppressor.

The adenovirus E4orf6 protein is shown here to interact with the cellular tumor suppressor protein p53 and to block p53-mediated transcriptional activation. The adenovirus protein inhibited the ability of p53 to bind to human TAFII31, a component of transcription factor IID (TFIID). Earlier work demonstrated that the interaction of p53 with TAFII31 involves a sequence near the NH2-terminus of p53, whereas the E4orf6-p53 interaction occurs within amino acids 318 to 360 of p53. Thus, the E4orf6 protein interacts at a site on p53 distinct from the domain that binds to TAFII31 but nevertheless inhibits the p53-TAFII31 interaction.

The E2F transcription factor activates a replication-dependent human H2A gene in early S phase of the cell cycle.

Histone gene expression is restricted to the S phase of the cell cycle. Control is mediated by a complex network of sequence-specific DNA-binding factors and protein-protein interactions in response to cell cycle progression. To further investigate the regulatory functions that are associated at the transcriptional level, we analyzed the regulation of a replication-dependent human H2A.1-H2B.2 gene pair. We found that transcription factor E2F binds specifically to an E2F recognition motif in the H2A.1 promoter region. Activation of the H2A.1 promoter by E2F-1 was shown by use of luciferase reporter constructs of the intergenic promoter region. Overexpression of the human retinoblastoma suppressor gene product RB suppressed E2F-1 mediated transcriptional activation, indicating an E2F-dependent regulation of promoter activity during the G1-to-S-phase transition. Furthermore, the activity of the H2A.1 promoter was also downregulated by overexpression of the RB-related p107, a protein that has been detected in S-phase-specific protein complexes of cyclin A, E2F, and cdk2. In synchronized HeLa cells, expression of luciferase activity was induced at the beginning of DNA synthesis and was dependent on the presence of an E2F-binding site in the H2A.1 promoter. Together with the finding that E2F-binding motifs are highly conserved in H2A promoters of other species, our results suggest that E2F plays an important role in the coordinate regulation of S-phase-specific histone gene expression.

p300 acts as a transcriptional coactivator for mammalian Notch-1.

Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-J kappa/CBF1. RBP-J kappa mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-J kappa complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.

Sp100A is a tumor suppressor that activates p53-dependent transcription and counteracts E1A/E1B-55K-mediated transformation.

Human adenoviruses (HAdV) are used as a model system to investigate tumorigenic processes in mammalian cells where the viral oncoproteins E1A and E1B-55K are absolutely required for oncogenic transformation, because they simultaneously accelerate cell cycle progression and inhibit tumor suppressor proteins such as p53, although the underlying mechanism is still not understood in detail. In our present study, we provide evidence that E1B-55K binding to the PML-NB component Sp100A apparently has an essential role in regulating adenovirus-mediated transformation processes. Specifically, when this E1B-55K/Sp100A complex recruits p53, Sp100A-induced activation of p53 transcriptional activity is effectively abolished. Hence, Sp100A exhibits tumor-suppressive activity, not only by stabilizing p53 transactivation but also by depressing E1A/E1B-55K-mediated transformation. E1B-55K counteracts this suppressive activity, inducing Sp100A SUMOylation and sequestering the modified cellular factor into the insoluble matrix of the nucleus or into cytoplasmic inclusions. These observations provide novel insights into how E1B-55K modulates cellular determinants to maintain growth-promoting activity during oncogenic processes and lytic infection.

PML isoforms IV and V contribute to adenovirus-mediated oncogenic transformation by functionally inhibiting the tumor-suppressor p53.

Although modulation of the cellular tumor-suppressor p53 is considered to have the major role in E1A/E1B-55K-mediated tumorigenesis, other promyelocytic leukemia nuclear body (PML-NB)/PML oncogenic domain (POD)-associated factors including SUMO, Mre11, Daxx, as well as the integrity of these nuclear bodies contribute to the transformation process. However, the biochemical consequences and oncogenic alterations of PML-associated E1B-55K by SUMO-dependent PML-IV and PML-V interaction have so far remained elusive. We performed mutational analysis to define a PML interaction motif within the E1B-55K polypeptide. Our results showed that E1B-55K/PML binding is not required for p53, Mre11 and Daxx interaction. We also observed that E1B-55K lacking subnuclear PML localization because of either PML-IV or PML-V-binding deficiency was no longer capable of mediating E1B-55K-dependent SUMOylation of p53, inhibition of p53-mediated transactivation or efficiently transforming primary rodent cells. These results together with the observation that E1B-55K-dependent SUMOylation of p53 is required for efficient cell transformation, provides evidence for the idea that the SUMO ligase activity of the E1B-55K viral oncoprotein is intimately linked to its growth-promoting oncogenic activities.

Adenovirus early E4 genes in viral oncogenesis.

Previous investigations into potential transforming activities of adenovirus (Ad) early genes were largely overshadowed by the more obvious roles of E1A and E1B products. One exception was an Ad9 E4 protein (ORF1) shown to enhance transformation of cultured cells and promote mammary tumors in female rats. Recently, significant advances in understanding Ad E4 gene products at the molecular level have revealed that these proteins possess an unexpectedly diverse collection of functions, which not only orchestrate many viral processes, but overlap with oncogenic transformation of primary mammalian cells. Operating through a complex network of protein interactions with key viral and cellular regulatory components, Ad E4 products are apparently involved in transcription, apoptosis, cell cycle control, DNA repair, cell signaling, posttranslational modifications and the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs). Some of these functions directly relate to known transforming and oncogenic processes, or implicate mechanisms such as modulating the function and subcellular localization of cellular PDZ domain-containing proteins, POD reorganization, targeted proteolytic degradation, inhibition of DNA double-strand break repair and 'hit-and-run' mutagenesis. Here, we summarize the recent data and discuss how E4 gene product interactions may contribute to viral oncogenesis.

The adenovirus E4orf6 protein contributes to malignant transformation by antagonizing E1A-induced accumulation of the tumor suppressor protein p53.

The adenovirus type 5 (Ad5) E4orf6 protein promotes focus formation of primary baby rat kidney (BRK) cells in cooperation with Ad5 E1 proteins. This activity is most likely related to the ability of the E4orf6 protein to bind to p53 and modulate its tumor suppressor functions. In this study we report that transformed BRK cells that stably express E4orf6 in addition to E1A and E1B (ABS cells) displayed multiple additional properties commonly associated with a high grade of oncogenic transformation compared to cells expressing only E1A and E1B (AB cells). These properties included morphological alterations, markedly enhanced growth rates and growth to much higher saturation densities. Following injection into nude mice ABS-derived tumors exhibited accelerated growth and, based on histopathological criteria, proofed to be much more malignant compared to tumors generated by AB cells. Interestingly, these highly transformed properties of ABS cells correlated with a dramatic reduction of p53 steady-state levels which inversely correlated with E4orf6 expression. From these results we conclude that expression of the Ad5 E4orf6 protein (i) confers additional transformed in vitro properties to primary rat cells expressing the Ad5 E1 proteins, and (ii) increases the tumorigenic and malignant potential of these cells in vivo. Our data suggest that the Ad5 E4orf6 protein enhances the intrinsic ability of E1-transformed rat cells to grow in a neoplastic state by completely inactivating p53 tumor suppressor function in combination with the E1A and E1B proteins.

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2.

The E1B-55K and E4orf6 oncoproteins of adenovirus type 5 are involved in the export of viral mRNAs. Previously, it was suggested that a complex composed of E1B-55K and E4orf6 serves as a nucleocytoplasmic transporter for viral mRNAs in which the E4orf6 protein directs both nuclear import and export. We now demonstrate that the E1B-55K protein itself shuttles efficiently in the absence of E4orf6. In addition, E1B-55K trafficking was independent of the defined shuttle proteins Mdm2 or p53, which interacts with E1B-55K. The identified N-terminal E1B-55K leucine-rich nuclear-export signal (NES) conferred rapid nuclear export even in a heterologous system in contrast to the postulated E4orf6NES. Interestingly, although shuttling was blocked by inhibitors of the CRM1 mediated export pathway, E1B-55K inhibited neither the activity nor the trafficking of the retroviral shuttle proteins HIV-1 Rev and HTLV-1 Rex. In contrast, Rev or Rex blocked the nuclear export of E1B-55K, most likely by competing for essential export factors. Our results provide new insights into the regulation of the adenovirus mRNA export system and the processes of adenovirus mediated transformation. Oncogene (2000) 19, 850 - 857.

Transcription factor AP-2alpha triggers apoptosis in cardiac myocytes.

Idiopathic-dilated cardiomyopathy (IDC) is a common primary myocardial disease of unknown etiology associated with apoptosis, cardiac dilatation, progressive heart failure and increased mortality. An elevation of the transcription factor activator protein 2alpha (AP-2alpha) is involved in vertebrate embryonic development and oncogenesis. Here, we show that AP-2alpha protein is expressed in the human heart and increased in human failing myocardium with IDC. Adenovirus-mediated overexpression of human AP-2alpha triggered apoptosis and increased mRNA levels of Bcl-2 family members Bax and Bcl-x in rat cardiomyocytes. Immunohistological analysis of human myocardium revealed an increased percentage of AP-2alpha-positive nuclei in IDC and, interestingly, a colocalization of AP-2alpha-positive but not -negative cells with a caspase-cleaved fragment of poly(ADP-ribose)polymerase. We suggest AP-2alpha as a novel cardiac regulator implicated in the activation of apoptosis in IDC.

Cell transformation by human adenoviruses.

The last 40 years of molecular biological investigations into human adenoviruses have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of their productive infection cycle in permissive host cells. Also, initial observations concerning the carcinogenic potential of human adenoviruses subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer, and established adenoviruses as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human adenoviruses is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in adenovirus-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, detailed studies on the tumorigenic potential of subgroup D adenovirus type 9 (Ad9) E4 have now revealed a new pathway that points to a novel, general mechanism of virus-mediated oncogenesis. In this chapter, we summarize the current state of knowledge about the oncogenes and oncogene products of human adenoviruses, focusing particularly on recent findings concerning the transforming and oncogenic properties of viral proteins encoded in the E1B and E4 transcription units.

Regulation of Human Adenovirus Replication by RNA Interference.

Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

Molecular regulation and biological function of adenovirus early genes: the E4 ORFs.

Over the past few years there have been a number of interesting advances in our understanding of the functions encoded by the adenovirus early transcription unit 4 (Ad E4). A large body of recent data demonstrates that E4 proteins encompass an unexpectedly diverse collection of functions required for efficient viral replication. E4 gene products operate through a complex network of protein interactions with key viral and cellular regulatory components involved in transcription, apoptosis, cell cycle control and DNA repair, as well as host cell factors that regulate cell signaling, posttranslational modifications and the integrity of nuclear multiprotein complexes known as nuclear bodies (NBs) or PML oncogenic domains (PODs). As understood at present, some of the lytic functions overlap with roles in oncogenic transformation of primary mammalian cells. These observations, together with findings that E4 proteins substantially affect cell toxicity and the immune response of the host have profound implications for the development of Ad vectors for gene therapy. In this article we will summarize recent findings regarding the diverse functions of E4 gene products in the context of earlier work. We will emphasize the interaction of E4 proteins with cellular and viral interaction partners, the role of these interactions for lytic virus growth and how these interactions may contribute to viral oncogenesis. Finally, we will discuss their role in Ad vector and adeno-associated virus infections.

Isolation and characterization of the porcine nuclear factor I (NFI) gene.

This study describes the isolation of a major portion of the gene for nuclear factor I (NFI) including its 5'-flanking region with transcriptional start sites. We screened a porcine liver, genomic DNA library in phage EMBL3A with synthetic oligonucleotides derived from tryptic and cyanogen-bromide peptide sequences obtained from purified NFI protein. The NFI gene is present as a single copy in porcine DNA.

A novel divergently transcribed human histone H2A/H2B gene pair.

A genomic clone containing a novel closely linked human histone H2A/H2B gene pair has been isolated and sequenced along with extensive 5' and 3' flanking regions. Both genes are devoid of introns and code for core histone proteins. The nucleotide sequences are 84% and 87% homologous to the coding regions of a human genomic H2A and H2B gene, respectively. A comparison of the nucleotide-derived amino acid sequences shows that the histone H2A protein corresponds to the human H2A.1 subtype, whereas the H2B histone gene predicts an H2B protein sequence which is almost identical to the histone H2B.2 variant from human and bovine obtained by direct protein sequencing. The 3' flanking regions contain previously identified conserved sequence elements thought to be involved in transcription termination and processing of replication-dependent histone gene poly(A)- mRNAs. Primer extension analyses of the histone mRNAs encoded within this clone demonstrate that both genes are divergently transcribed from a 313 bp intergene promoter region. The spatial arrangement and orientation of two TATA-boxes, four CAAT-boxes, and one H2B-box within this region suggests that the linked genes share common promoter elements for transcriptional regulation.

[A new human cellular protein AUP1. I. In vitro interaction of AUP1 with adenoviral proteins E4ORF3 and E1A]. / Novyi kletochnyi belok cheloveka AUP1. I. Vzaimodeistvie s adenovirusnymi belkami E4ORF3 i E1A in vitro.

The 11-kDa product of adenovirus early region 4 (E4) open reading frame (ORF) 3 participates in many processes occurring in infected cell, including post-transcriptional steps in late viral gene expression and viral DNA synthesis. In addition, E4ORF3 from adenovirus type 5 (Ad5) displays the features of a viral oncoprotein. It initiates focal transformation of primary rat cells in cooperation with Ad5 El genes and confers multiple additional transformed properties on E1-expressing cells. Biochemical details of E4ORF3 activities in these processes are not well understood. A large body of evidence indicates that its lytic and transforming functions are mediated by physical interactions with viral and cellular components involved in DNA transcription and repair, as well as by host cell factors that regulate the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs). In this study we have employed the two-hybrid screen in yeast to isolate human cDNAs encoding for E4ORF3-interacting proteins. Among 15 positive clones five cDNAs encode for a cellular protein called AUP1. In vitro-binding assays demonstrated that AUP1 fused to glutathione S-transferase (GST) specifically binds to E4ORF3 from Ad5, Ad9 and Ad40 generated in a coupled transcription-translation system, whereas no interactions was observed with ORF3 from Ad12. Interestingly, GST-AUP1 interacted also specifically with in vitro translated Ad5 E1A proteins. Regions involved in the Ad5 E4ORF3/AUP1 interaction in vitro map to the central part of E4 protein and the carboxy-terminal region of AUP1, while E1A binds to an amino-terminal segment of the cell protein. Taken together, these studies indicate that AUP1 may represent a cellular target of both adenovirus E4ORF3 and E1A proteins. Additional studies are currently under way to confirm the significance of these interactions in living cells in vivo.

[A new human cellular protein AUP1. II. cDNA cloning, genomic organization of Aup1 gene ans preliminary characterization of human AUP1 protein]. / Novyi kletochnyi belok cheloveka AUP1. II. Klonirovanie kDNK gena Aup1, opredelenie ego genomnoi organizatsii i pervichnaia kharakteristika belka AUP1 cheloveka.

We report here the cloning of human cDNA of Aup1 gene (ancient uniquitous protein 1), its genomic organization and preliminary characterization of human AUP1 protein. Genomic length of the human Aup1 gene is composed of 12 exons and 11 introns spanning 3047 bp. A single open reading frame of 1230 bp starts with the first ATG from nucleotide 5630 of the genomic sequence, in accordance with the Kazak rule, and stops with TGA on nucleotide 8500 of the genomic sequence AC005041.2. The 3 noncoding region has a 152 bp length followed by poly(A)tail. All exon-intron junctions conform to 5' donor and 3' acceptor consensus. Exon and intron sizes range from 69 to 187 bp, and from 180 to 262 bp, resp. We have cloned a full-length human Aup1 cDNA. For preliminary characterization of human AUP1 protein, we cloned the full-length human Aup1 cDNA in pcDNA3 vector. In vitro transcription/translation of the cloned cDNA yielded a 45 kDa protein. Human Aup1 coding region demonstrates 90% homology to its rat and mouse homologous at the nucleotide level, and 82% at the amine acid level.

[A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines]. / Novyi kletochnyi belok cheloveka AUP1. III. Lokalizatsiia v razlichnykh kletochnykh liniiakh cheloveka i krysy.

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.

Cross-talk between phosphorylation and SUMOylation regulates transforming activities of an adenoviral oncoprotein.

Since the discovery of post-translational modification (PTM) by the small ubiquitin-related modifiers (SUMOs), a multitude of proteins have been described to be reversibly modified, resulting in the alteration of several cellular pathways. Interestingly, various pathogens gain access to this modification system, although the molecular mechanisms and functional consequences are barely understood. We show here that the adenoviral oncoprotein E1B-55K is a substrate of the SUMO conjugation system, which is directly linked to its C-terminal phosphorylation. This regulative connection is indispensable for modulation of the tumor suppressor p53/chromatin-remodeling factor Daxx by E1B-55K and, consequently, its oncogenic potential in primary mammalian cells. In virus infection, E1B-55K PTMs are necessary for localization to viral transcription/replication sites. Furthermore, we identify the E2 enzyme Ubc9 as an interaction partner of E1B-55K, providing a possible molecular explanation for SUMO-dependent modulation of cellular target proteins. In conclusion, these results for the first time provide evidence how E1B-55K PTMs are regulated and subsequently facilitate exploitation of the host cell SUMOylation machinery.

Expression of the coxsackie adenovirus receptor in neuroendocrine lung cancers and its implications for oncolytic adenoviral infection.

Coxsackie adenovirus receptor (CAR) is the primary receptor to which oncolytic adenoviruses have to bind for internalization and viral replication. A total of 171 neuroendocrine lung tumors in form of multitissue arrays have been analyzed resulting in a positivity of 112 cases (65.5%). Immunostaining correlated statistically significant with histopathology and development of recurrence. The subtype small cell lung cancer (SCLC) showed the highest CAR expression (77.6%), moreover the CAR level was correlated to the disease-free survival. Further, high CAR expression level in SCLC cell lines was found in vitro and in vivo when cell lines had been transplanted into immunodeficient mice. A correlation between CAR expression in the primary tumors and metastases development in the tumor model underlined the clinical relevance. Cell lines with high CAR level showed a high infectivity when infected with a replication-deficient adenovirus. Low levels of CAR expression in SCLC could be upregulated with Trichostatin A, a histone deacetylase inhibitor. As a result of the unaltered poor prognosis of SCLC and its high CAR expression it seems to be the perfect candidate for oncolytic therapy. With our clinically relevant tumor model, we show that xenograft experiments are warrant to test the efficiency of oncolytic adenoviral therapy.