Amphiphilic Polyphenylene Dendron Conjugates for Surface Remodeling of Adenovirus 5

Abstract Early pregnancy features complex signaling between fetal trophoblast cells and maternal endometrium directing major peri-implantation events including localized inflammation and remodeling to establish proper placental development. Proinflammatory mediators are important for conceptus attachment, but a more precise understanding of molecular pathways regulating this process is needed to understand how the endometrium becomes receptive to implantation. Both chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal tissues. We identified this pair as a critical driver of placental angiogenesis, but their additional importance to inflammation and trophoblast cell survival, proliferation, and invasion imply a role in syncytia formation at the fetal–maternal microenvironment. We hypothesized that CXCL12 encourages both endometrial inflammation and conceptus attachment during implantation. We employed separate ovine studies to (1) characterize endometrial inflammation during early gestation in the ewe, and (2) establish functional implications of CXCL12 at the fetal–maternal interface through targeted intrauterine infusion of the CXCR4 inhibitor AMD3100. Endometrial tissues were evaluated for inflammatory mediators, intracellular signaling events, endometrial modifications, and trophoblast syncytialization using western blotting and immunohistochemistry. Endometrial tissue from ewes receiving CXCR4 inhibitor demonstrated dysregulated inflammation and reduced AKT and NFKB, paired with elevated autophagic activity compared to control. Immunohistochemical observation revealed an impairment in endometrial surface remodeling and diminished trophoblast syncytialization following localized CXCR4 inhibition. These data suggest CXCL12–CXCR4 regulates endometrial inflammation and remodeling for embryonic implantation, and provide insight regarding mechanisms that, when dysregulated, lead to pregnancy pathologies such as intrauterine growth restriction and preeclampsia.

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Disruption of the coordinate expression of muscle genes in a transfected BC3H1 myoblast cell line producing a low level of the adenovirus E1A transforming protein

Biochemistry and Cell Biology ◽

10.1139/o92-173 ◽

1992 ◽

Vol 70 (10-11) ◽

pp. 1268-1276 ◽

Cited By ~ 2

Author(s):

Joe S. Mymryk ◽

Janice D. Oakes ◽

Senthil K. Muthuswamy ◽

Pietro D'Amico ◽

Stanley T. Bayley ◽

...

Keyword(s):

Myogenic Differentiation ◽

Calf Serum ◽

Adenovirus E1a ◽

Myoblast Cell Line ◽

Muscle Genes ◽

E1a Gene ◽

H1 Cells ◽

Myoblast Cell ◽

Muscle Gene ◽

Adenovirus 5

Mouse BC3H1 myoblasts were stably transfected with the adenovirus 5 E1A gene. One clonal line, BC3E7, was found to differ in some important respects from those previously reported for E1A-transformed myoblasts. In contrast to BC3H1 cells which differentiate when confluent in medium containing 0.5% fetal calf serum (FCS), BC3E7 cells failed to elongate and align, to express acetylcholine receptor and creatine kinase, and to down-regulate expression of β- and γ-actins and tropomyosin isoform (TM) 1. However, increased synthesis of TMs 2, 3, and 4, and myosin light chain 1 associated with differentiation in BC3H1 still occurred in BC3E7 cells, and most surprisingly, α-actin was produced at a significant level in both proliferating and confluent BC3E7 cells. Interestingly, myogenin was expressed in confluent BC3E7 cells in 0.5% FCS, but not in 20%. The level of E1A expression in BC3E7 cells was found to be very low by analysis of mRNA, by immunoprecipitation of E1A protein, and by the ability of BC3E7 cells to complement the E1A-deficient adenovirus mutant dl312. These results suggest that different levels of E1A may be needed to repress different promoters and that E1A does not block myogenic differentiation by repressing myogenin expression, but represses each muscle gene independently.Key words: actin, adenovirus 5 E1A, BC3H1 myoblasts, myogenin.

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Identification of RISC-Associated Adenoviral MicroRNAs, a Subset of Their Direct Targets, and Global Changes in the Targetome upon Lytic Adenovirus 5 Infection

Journal of Virology ◽

10.1128/jvi.02336-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1608-1627 ◽

Cited By ~ 20

Author(s):

Florian Bellutti ◽

Maximilian Kauer ◽

Doris Kneidinger ◽

Thomas Lion ◽

Reinhard Klein

Keyword(s):

Gene Expression ◽

Relative Abundance ◽

A549 Cells ◽

Global Changes ◽

Cellular Mirnas ◽

Total Rna ◽

Cellular Mrnas ◽

Comprehensive Picture ◽

Infected Cells ◽

Adenovirus 5

ABSTRACTAdenoviruses encode a set of highly abundant microRNAs (mivaRNAs), which are generated by Dicer-mediated cleavage of the larger noncoding virus-associated RNAs (VA RNAs) I and II. We performed deep RNA sequencing to thoroughly investigate the relative abundance of individual single strands of mivaRNA isoforms in human A549 cells lytically infected with human adenovirus 5 (Ad5) at physiologically relevant multiplicities of infection (MOIs). In addition, we investigated their relative abundance in the endogenous RNA-induced silencing complexes (RISCs). The occupation of endogenous RISCs by mivaRNAs turned out to be pronounced but not as dominant as previously inferred from experiments with AGO2-overexpressing cells infected at high MOIs. In parallel, levels of RISC-incorporated mRNAs were investigated as well. Analysis of mRNAs enriched in RISCs in Ad5-infected cells revealed that only mRNAs with complementarity to the seed sequences of mivaRNAs derived from VA RNAI but not VA RNAII were overrepresented among them, indicating that only mivaRNAs derived from VA RNAI are likely to contribute substantially to the posttranscriptional downregulation of host gene expression. Furthermore, to generate a comprehensive picture of the entire transcriptome/targetome in lytically infected cells, we determined changes in cellular miRNA levels in both total RNA and RISC RNA as well, and bioinformatical analysis of mRNAs of total RNA/RISC fractions revealed a general, genome-wide trend toward detargeting of cellular mRNAs upon infection. Lastly, we identified the direct targets of both single strands of a VA RNAI-derived mivaRNA that constituted one of the two most abundant isoforms in RISCs of lytically infected A549 cells.IMPORTANCEViral and cellular miRNAs have been recognized as important players in virus-host interactions. This work provides the currently most comprehensive picture of the entire mRNA/miRNA transcriptome and of the complete RISC targetome during lytic adenovirus infection and thus represents the basis for a deeper understanding of the interplay between the virus and the cellular RNA interference machinery. Our data suggest that, at least in the model system that was employed, lytic infection by Ad5 is accompanied by a measurable global net detargeting effect on cellular mRNAs, and analysis of RISC-associated viral small RNAs revealed that the VA RNAs are the only source of virus-encoded miRNAs. Moreover, this work allows to assess the power of individual viral miRNAs to regulate cellular gene expression and provides a list of proven and putative direct targets of these miRNAs, which is of importance, given the fact that information about validated targets of adenovirus-encoded miRNAs is scarce.

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Ability of adenovirus 5 E1A proteins to suppress differentiation of BC3H1 myoblasts correlates with their binding to a 300 kDa cellular protein.

Molecular Biology of the Cell ◽

10.1091/mbc.3.10.1107 ◽

1992 ◽

Vol 3 (10) ◽

pp. 1107-1115 ◽

Cited By ~ 40

Author(s):

J S Mymryk ◽

R W Lee ◽

S T Bayley

Keyword(s):

Creatine Kinase ◽

Kinase Activity ◽

Cellular Protein ◽

Creatine Kinase Activity ◽

Cellular Proteins ◽

Deletion Mutants ◽

Transcriptional Enhancers ◽

Actin Mrna ◽

Alpha Actin ◽

Adenovirus 5

We have used deletion mutants to define the regions in Ad5 E1A proteins necessary to suppress differentiation of mouse BC3H1 myoblasts. We examined the differentiation of cells infected at a low multiplicity with viruses containing the E1A deletions and constructed so as to produce only the smaller of the two major E1A proteins. Only four of the mutant viruses containing deletions within the N-terminal 69 residues failed to suppress differentiation as judged by changes in morphology and in levels of muscle-specific alpha-actin mRNA and creatine kinase activity. The results were confirmed by analyses of lines of cells stably transfected with representative E1A mutants. The mouse cellular proteins to which mutant E1A proteins bound were identified by immunoprecipitating E1A proteins specifically from infected BC3H1 cells and by analyzing the precipitates on denaturing gels. Bands of proteins of 300, 130, 107, 105 (the retinoblastoma product), and 60 kDa (cyclin A) were distinguished. Failure to suppress differentiation correlated with loss of binding to the 300-kDa protein but not to any of the others. The regions of E1A defined in this way have been shown to be required for several other activities, including enhancer repression and transformation. One function of the 300-kDa protein appears to be to facilitate the action of transcriptional enhancers of differentiation-specific genes.

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Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.875-882.1984 ◽

1984 ◽

Vol 4 (5) ◽

pp. 875-882

Author(s):

M J Imperiale ◽

J R Nevins

Keyword(s):

Transcription Initiation ◽

Essential Element ◽

Inducible Promoter ◽

Initiation Site ◽

Transcription Unit ◽

Upstream Sequence ◽

Wild Type ◽

Promoter Sequences ◽

Adenovirus 5 ◽

Wild Type Promoter

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.

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