Adenovirus E4-34kDa requires active proteasomes to promote late gene expression.
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A complex of the Adenovirus (Ad) early region 1b 55-kDa protein (E1b-55kDa) and the early region 4 ORF6 34-kDa protein (E4-34kDa) promotes viral late RNA accumulation in the cytoplasm while inhibiting the transport of most newly synthesized cellular mRNA. The E4 ORF3 11-kDa protein (E4-11kDa) functionally compensates for at least some of the activities of this complex. We find that the same large central region of E4-34kDa that is required for proteasome-mediated degradation of p53 (J. Virol. 75, (2001) 699-709) is also required to promote viral late gene expression in a complementation assay. E4-34kDa does not promote late gene expression in complementation assays performed in the presence of proteasome inhibitors. A proteasome inhibitor also dramatically reduced late gene expression by a virus that lacks the E4-11kDa gene and therefore relies on E4-34kDa for late gene expression. Our results suggest that E4-34kDa activity in promoting late gene expression depends on the proteasome. (+info)
Transcriptional activation of the cyclin A gene by the architectural transcription factor HMGA2.
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The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120(E4F), interfering with p120(E4F) binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation. (+info)
Adenovirus E4 gene promotes selective endothelial cell survival and angiogenesis via activation of the vascular endothelial-cadherin/Akt signaling pathway.
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The early 4 region (E4) of the adenoviral vectors (AdE4(+)) prolongs human endothelial cell (EC) survival and alters the angiogenic response, although the mechanisms for the EC-specific, AdE4(+)-mediated effects remain unknown. We hypothesized that AdE4(+) modulates EC survival through activation of the vascular endothelial (VE)-cadherin/Akt pathway. Here, we showed that AdE4(+), but not AdE4(-) vectors, selectively stimulated phosphorylation of both Akt at Ser(473) and Src kinase in ECs. The phosphatidylinositol 3-kinase (PI3K) inhibitors LY294002 and wortmannin abrogated AdE4(+) induction of both phospho-Akt expression and prolonged EC survival. Regulation of phospho-Akt was found to be under the control of various factors, namely VE-cadherin activation, Src kinase, tyrosine kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Downstream targets of Akt signaling resulted in glycogen synthase kinase-3alpha/beta phosphorylation, beta-catenin up-regulation, and caspase-3 suppression, all of which led to AdE4(+)-mediated EC survival. Furthermore, infection with AdE4(+) vectors increased the angiogenic potential of ECs by promoting EC migration and capillary tube formation in Matrigel plugs. This selective AdE4(+)-mediated enhanced motility of ECs was also blocked by PI3K inhibitors. Taken together, these results suggest that activation of the VE-cadherin/Akt pathway is critical for AdE4(+)-mediated survival of ECs and angiogenic potential. (+info)
Identification of the E1A-regulated transcription factor p120 E4F as an interacting partner of the RASSF1A candidate tumor suppressor gene.
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Epigenetic inactivation of the candidate tumor suppressor gene RASSF1A is a frequent and critical event in the pathogenesis of many human cancers. The RASSF1A protein contains a Ras association domain, suggesting a role in Ras-like signaling pathways, and has also been implicated in cell cycle progression. However, the preliminary data suggests that the RASSF1A gene product is likely to have multiple functions. To identify novel RASSF1A functions, we have sought to identify interacting proteins by yeast two-hybrid analysis in a human brain cDNA library. We identified the E1A-regulated transcription factor p120(E4F) as a RASSF1A interacting partner in yeast and mammalian cells, and demonstrated that RASSF1A protein and p120(E4F) form a complex in vivo. The interaction between RASSF1A and p120(E4F) was confirmed by both in vitro and in vivo pull downs and coimmunoprecipitation assays. In addition, specific inactivation of RASSF1A by short interfering RNA disrupts binding of RASSF1A to p120(E4F) in coimmunoprecipitation assays. In addition, we demonstrated enhanced G(1) cell cycle arrest and S phase inhibition by propidium iodide staining of p120(E4F) in the presence of RASSF1A. As p120(E4F) has been reported previously to interact with p14ARF, retinoblastoma, and p53, these findings provide an important link between the function of RASSF1A and other major human tumor suppressor genes. (+info)
Viral RNAs detected in virions of porcine adenovirus type 3.
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It has been demonstrated that cellular and viral RNAs were packaged in the virions of human cytomegalovirus (CMV) and herpes simplex virus 1 (HSV 1), members of the Herpesviridae family, both of which are enveloped double-stranded DNA viruses. Here, we provide evidence suggesting that RNAs are packaged in the virions of porcine adenovirus type 3 (PAdV-3), which is a member of the Adenoviridae family, a non-enveloped double-stranded DNA virus. The RNAs packaged in PAdV-3 virions were enriched in the size range of 300-1000 bases long. By reverse transcription (RT) of RNAs isolated from purified PAdV-3 virions, PCR amplification, and DNA sequence analysis of PCR products, we determined the identities of some viral RNAs contained in PAdV-3 virions. The results indicated that the RNAs representing transcripts from E1A, E1B, DNA binding protein (DBP), DNA polymerase (POL), E4 and some of the late genes including pIIIA, pIII, pV, Hexon, 33 K, and fiber were detected from purified PAdV-3 virions. In contrast, we could not detect the RNAs representing transcripts of precursor terminal protein (pTP), 52 kDa, pX, or 100-kDa protein genes in purified virions. Because the transcripts of pIX, IVa2, E3, protease, pVI, pVII, and pVIII overlap with those of other genes in PAdV-3, we could not definitely conclude that RNAs representing these transcripts were packaged in virions although the expected DNA fragments were produced by RT-PCR in the RNAs isolated from purified virions. (+info)
Activation of adenovirus type 2 early region 4 ORF4 cytoplasmic death function by direct binding to Src kinase domain.
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Adenovirus type 2 (Ad2) early region 4 ORF4 (E4orf4) triggers a major death pathway that requires its accumulation in cellular membranes and its tyrosine phosphorylation. This program is regulated by Src family kinases and triggers a potent ZVAD (benzyloxycarbonyl-VAD)- and Bcl2-resistant cell death response in human-transformed cells. How E4orf4 deregulates Src-dependent signaling is unknown. Here we provide strong evidence that a physical interaction requiring the kinase domain of Src and the arginine-rich motif of E4orf4 is involved. The Src binding domain of E4orf4 overlaps with, but is distinct from that of the Balpha subunit of protein phosphatase 2A (PP2A-Balpha) and some E4orf4 complexes contain both PP2A and Src. Functional assays using mutant E4orf4 revealed that deregulation of Src signaling, activation of the Jun kinase pathway, and cell blebbing were all critically dependent on Src binding. In contrast, PP2A-Balpha binding per se was not required to engage the Src-dependent death pathway but was more critical for triggering a distinct death activity. Both E4orf4 death activities were manifested within a given cell population, were typified by distinct morphological features, and contributed to overall cell killing, although to different extents in various cell types. We conclude that E4orf4 binding to the Src kinase domain leads to deregulation of Src signaling and plays a crucial role in induction of the cytoplasmic death pathway. Nonetheless, both Src and PP2A enzymes are critical targets of E4orf4 that likely cooperate to trigger E4orf4-induced tumor cell killing and whose relative contributions may vary in function of the cellular background. (+info)
Analyses of melanoma-targeted oncolytic adenoviruses with tyrosinase enhancer/promoter-driven E1A, E4, or both in submerged cells and organotypic cultures.
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We have generated novel conditionally replicative adenoviruses (CRAds) targeted to melanoma cells. In these adenoviruses, the E4 region (AdDelta24TyrE4) or both E1 and E4 regions (Ad2xTyr) were controlled by a synthetic tyrosinase enhancer/promoter (Tyr2E/P) specific for melanocytes. The properties of these CRAds were compared with wild-type adenovirus (Adwt) and our previous CRAd with a targeted E1A CRII mutation (AdTyrDelta24) in submerged cultures of melanoma cells and nonmelanoma control cells. We showed that AdDelta24TyrE4 had a cell type selectivity similar to AdTyrDelta24 but had a distinct block in viral reproduction in nonmelanoma cells and that Ad2xTyr had an augmented selectivity for melanoma cells. These viruses were additionally tested in organotypic cultures of melanoma cell lines, primary human keratinocytes (PHKs), or mixed cell populations. Unexpectedly, the CRAds exhibited somewhat different cell type selectivity profiles in these cultures relative to those observed in submerged cultures, demonstrating the importance of multiple assay systems. Specifically, AdTyrDelta24 and Ad2xTyr were selective for melanoma cells, whereas AdDelta24TyrE4 exhibited no selectivity, similar to Adwt. AdTyrDelta24 and Ad2xTyr were strongly attenuated in their ability to lyse PHKs in organotypic cultures. Furthermore, Ad2xTyr had a superior melanoma selectivity in organotypic cultures of cocultivated melanoma cells and PHKs. The enhanced selectivity for melanoma cells exhibited by Ad2xTyr provides a window of opportunity for therapeutic application. These studies also demonstrate that organotypic cultures derived from mixtures of tumor and normal cells represent a promising new model for analysis of CRAd specificity and toxicity. (+info)
Expression of the adenovirus E4 34k oncoprotein inhibits repair of double strand breaks in the cellular genome of a 293-based inducible cell line.
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The human adenovirus E4 ORF 6 34 kDa oncoprotein (E4 34k), in concert with the 55 kDa product of E1b, prevents concatenation of viral genomes in infected cells, inhibits the repair of double strand breaks (DSBs) in the viral genome, and inhibits V(D)J recombination in a plasmid transfection assay. These activities are consistent with a general inhibition by the E4 34k and E1b 55k proteins of DSB repair by non-homologous end joining (NHEJ) on extrachromosomal substrates. To determine whether inhibition of NHEJ extends to repair of DSBs in the cell chromosome, we have examined the effects of E4 34k on repair of chromosomal DSBs induced by ionizing radiation in a cell line in which E4 34k expression and biological activity is inducible and E1b 55k is produced constitutively. We demonstrate that in this cell line, induction of E4 34k inhibits chromosomal DSB repair. Recently, it has been shown that in infected cells, E4 34k and the adenovirus E1b 55k proteins cooperate to destabilize Mre11 and Rad50, components of mammalian NHEJ systems. Consistent with this, induction of expression of E4 34k in the inducible cell line also reduces the steady state level of Mre11 protein. (+info)