Patrick Hearing

The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element

The adenovirus type 5 E1A transcriptional control region contains an element with enhancer properties located between -141 and -305 relative to the E1A cap site at +1. The enhancer element is located at or very close to a sequence required in cis for packaging of viral DNA. Deletion of the element reduces both the rate of transcription and steady-state levels of E1A mRNAs in virus-infected cells. Such deletions also cause a modest reduction in activity of the E1B transcription unit, which is located immediately downstream of the E1A unit. The enhancer element includes repeated core sequences. Function is retained when the element is moved to the 3 side of the E1A gene in either possible orientation, and the E1A enhancer functions in cis to enhance transformation by the herpesvirus thymidine kinase gene in both mouse and human cells.

The adenovirus E4-6/7 protein transactivates the E2 promoter by inducing dimerization of a heteromeric E2F complex.

Binding of the mammalian transcription factor E2F to the adenovirus E2a early promoter is modulated through interaction with the viral E4-6/7 protein. E4-6/7 induces the cooperative and stable binding of E2F in vitro to two correctly spaced and inverted E2F binding sites in the E2a promoter (E2F induction) by physical interaction in the protein-DNA complex. The E2a promoter is transactivated in vivo by the E4-6/7 product. The C-terminal 70 amino acids of E4-6/7 are necessary and sufficient for induction of E2F binding and for transactivation. To assess the mechanism(s) of E2a transactivation and the induction of cooperative E2F binding by the E4-6/7 protein, we have analyzed a series of point mutants in the functional C-terminal domain of E4-6/7. Two distinct segments of E4-6/7 are required for interaction with E2F. Additionally, and E4-6/7 mutant with a phenylalanine-to-proline substitution at amino acid 125 (F-125-P) efficiently interacts with E2F but does not induce E2F binding to the E2a promoter and is defective for transactivation. Induction of E2F stable complex formation at the E2a promoter by the F-125-P mutant protein is restored by divalent E4-6/7-specific monoclonal antibodies, but not a monovalent Fab fragment, or by appending a heterologous dimerization domain to the N terminus of the mutant protein. These and other data support the involvement of E4-6/7 dimerization in the induction of cooperative and stable E2F binding and transactivation of the E2a promoter. We present evidence that at least two cellular components are involved in E2F DNA binding activity and that both are required for E2F induction by the E4-6/7 product. The recently cloned E2F-related activities E2F-1 and DP-1 individually bind to an E2F binding site weakly, but when combined generate an activity that is indistinguishable from endogenous cellular E2F. Recombinant E2F-1, DP-1, and E4-6/7 are sufficient to form the induced E2F complex at the E2a promoter.

The adenovirus type 5 E1A enhancer contains two functionally distinct domains: one is specific for E1A and the other modulates all early units in Cis

The adenovirus type 5 genome contains two distinct enhancer elements located at the left end of the viral chromosome. The first element is repeated and specifically regulates region E1A transcription within infected cells. One copy of this element is sufficient to fully activate E1A transcription in vivo. The second element is located between these repeated sequences and regulates transcription in cis of all early regions on the chromosome. These enhancer elements function independently of each other, and neither element is required for efficient viral DNA replication. Since mutations within the two E1A enhancer components generate different physiological responses, transcriptional enhancement can be achieved through multiple mechanisms.

CHARACTERIZATION OF A LYTIC VIRUS INFECTIOUS TO THE BLOOM‐FORMING MICROALGA AUREOCOCCUS ANOPHAGEFFERENS (PELAGOPHYCEAE)

Aureococcus anophagefferens Hargraves and Sieburth has caused recurring monospecific blooms in Long Island embayments since it was first described in 1985. It was termed the “brown tide,” due to the resulting water color, and has had a devastating effect on Long Island’s (New York) marine ecosystem. In 1992, a virus that was capable of causing lysis of A. anophagefferens was isolated and maintained in culture. We report on the further characterization of this virus, Aureococcus anophagefferens virus‐1 (AaV‐1), indicated by a buoyant density of 1.2776 g·mL−1 in a CsCl equilibrium gradient. Electron microscopy revealed a phage with a hexagonal head and tail similar to previously described phages. By using adenovirus for calibration, the virus was found to have a head 50—55 nm wide and a tail 70–75 nm long. The viral band was infectious to A. anophagefferens after dialysis. The virus was composed of at least 16 distinct polypeptides ranging in molecular weight from 20 to 230 kDa. The adsorption coefficient for the virus was 7.2 × 10−9 mL·min−1, and the burst size was calculated to be 9.4 viruses per A. anophagefferens cell at 20° C. Complete lysis of A. anophagefferens occurred with a titer as low as 893 viruses·mL−1, and the lower limit of infectivity was 93 viruses·mL−1. The virus lost its infectivity between 30° and 40° C. These results suggest that AaV‐1 is highly infectious and that the role of the virus in preventing or ending A. anophagefferens blooms needs further investigation.

Chemoselective Attachment of Small Molecule Effector Functionality to Human Adenoviruses Facilitates Gene Delivery to Cancer Cells

We demonstrate here a novel two-step click labeling process in which adenoviral particles are first metabolically labeled during production with unnatural azido sugars. Subsequent chemoselective modification allows access to viruses decorated with a broad array of effector functionality. Adenoviruses modified with folate, a known cancer-targeting motif, demonstrated a marked increase in gene delivery to a murine cancer cell line.

Functional analysis of the nucleotide sequence surrounding the cap site for adenovirus type 5 region E1A messenger RNAs

We have constructed a set of small, dispersed deletion mutations in the sequences surrounding the cap site of the adenovirus type 5 region E1A transcription unit. The effects of the mutations on E1A transcription were studied in vitro using a HeLa cell-free extract, and in vivo by reconstructing the mutations back into intact viral chromosomes and analyzing E1A messenger RNAs synthesized after infection of HeLa cells. The sequence between -35 and +20 (relative to the cap site at +1) was important for efficient E1A transcription and cap site selection in vitro. This region includes the TATA homology, which appeared essential for transcription. Sequences upstream of -35 were dispensable for transcription in vitro. Different results were found upon analysis of the same set of deletions in vivo. None of the mutations affected the steady-state levels of cytoplasmic, E1A-specific mRNAs found in infected HeLa cells by more than twofold. Deletions of the TATA homology, however, generated E1A mRNAs with heterogeneous 5 ends, and deletions downstream of the homology displaced the 5 end of mRNAs by about the size of the deletion.

LEFTY, a Member of the Transforming Growth Factor-β Superfamily, Inhibits Uterine Stromal Cell Differentiation: A Novel Autocrine Role

LEFTY is expressed in normal endometrium in cells that decidualize. To understand the importance of this expression, we have studied the effect of LEFTY on decidualization in vitro and in vivo. Exposure of human uterine fibroblast (HuF) cells to recombinant LEFTY blocked the induction of the decidual differentiation-specific marker genes, IGFBP1 (IGF-binding protein 1) and PRL (prolactin) in response to medroxyprogesterone acetate, estradiol, and prostaglandin E2. The inhibitory effect was associated with decreased induction of the transcription factors ETS1 and FOXO1, both of which are essential for decidualization. Overexpression of LEFTY in decidualized HuF cells with an adenovirus that transduced LEFTY caused a marked decrease in IGFBP1 secretion, and withdrawal of medroxyprogesterone acetate from decidualized cells resulted in a decrease in IGFBP1 secretion and an increase in LEFTY expression. Moreover, overexpression of LEFTY in decidualized cells reprogrammed the cells to a less differentiated state and attenuated expression of decidual markers. Uterine decidualization was markedly attenuated and litter size was significantly reduced by retroviral transduction of LEFTY in the uterine horns of pregnant mice or by induction of LEFTY expression by doxycycline treatment in Tet-On conditional LEFTY transgenic pregnant mice. In addition, administration of the contraceptive agent drospirenone to ovariectomized mice induced a marked increase in LEFTY expression and inhibited decidualization. Taken together, these finding indicate that LEFTY acts as a molecular switch that modulates both the induction of decidual differentiation and the maintenance of a decidualized state. Because decidual cells express abundant amounts of LEFTY, the action of LEFTY on decidualization occurs by an autocrine mechanism.

Methylation-dependent and -independent DNA binding of nuclear factor EF-C

Nuclear factor EF-C binds to important functional sites in the hepatitis B virus and polyomavirus enhancer regions. In this paper, we have characterized new and divergent EF-C binding sites in several viral regulatory regions. We also have demonstrated that EF-C binds to certain DNA sites only when CpG dinucleotide base pairs are methylated (m5C). EF-C binds to other sites in a methylation-independent manner. Based on similar binding properties and identical binding sites, it is very likely that EF-C corresponds to the nuclear protein MDBP previously identified by virtue of binding to methylated DNA.

Tet responsive and adenovirus based constructs for regulated in vivo expression of Lefty.

Regulated expression of Lefty/Ebaf during embryogenesis is required for development of body asymmetry. A tight regulation of Lefty also contributes to the menstrual tissue shedding in humans. In order to replicate this regulated expression, we have developed a tet-on system and an adenovirus driven model. To drive the expression of Lefty, we have placed Lefty under control of a tetracycline-responsive promoter which responds to a sequence variant of the reversed tet transcriptional activator (rtTA), rtTA2S-M2. In this model, Lefty is regulated by the dose of doxycycline. In the adenovirus driven system, Lefty is regulated by the number of adenoviral particles These model systems would be suitable for understanding the dose-dependent biologic role of Lefty in vivo.

Modeling gene expression in the mouse uterine horn by in vivo adenovirus-mediated gene delivery.

Endometrium is a unique tissue that is prepared for implantation of blastocyst during each menstrual cycle by expression of genes during a defined period of endometrial receptivity. Induction of gene over-expression in endometrium allows gaining insight on the role that genes play in endometrial function. Here, we show that induction of a state of gene over-expression in endometrium is feasible by in vivo gene delivery by transduction with adenovirus. Analysis of endometrium following adenoviral transduction of LacZ showed increased beta-galactosidase activity in endometrial glands as early as 24 hours following in vivo gene transfer. By 72 hour, the expression was uniformly strong throughout the uterine horn. Viral transduction was efficient in the range of 0.24-24x10(8) pfu (4.8-480x10(8) particles) in normal, pregnant and decidualized mouse uterine horns. These findings show that induction of a state of gene over-expression can be successfully attained in endometrium by adenoviral gene delivery.