Kerstin Sollerbrant

THE CARBOXY-TERMINAL REGION OF ADENOVIRUS E1A ACTIVATES TRANSCRIPTION THROUGH TARGETING OF A C-TERMINAL BINDING PROTEIN-HISTONE DEACETYLASE COMPLEX

Binding of the C‐terminal binding protein, CtBP, to the adenovirus E1A moiety of a Gal4‐E1A fusion protein abolishes conserved region (CR) 1‐dependent transcription activation. In contrast, a non‐promoter targeted E1A peptide, capable of binding CtBP, can induce transcription from the proliferating cell nuclear antigen (PCNA) promoter. CtBP is shown here to bind the histone deacetylase HDAC1, suggesting that a promoter targeted CtBP‐HDAC1 complex can silence transcription from the PCNA promoter through a deacetylation mechanism. Expression of the CtBP binding domain of E1A is sufficient to alleviate repression, possibly due to the displacement of the CtBP‐HDAC1 complex from the promoter.

The Coxsackievirus and Adenovirus Receptor (CAR) Forms a Complex with the PDZ Domain-containing Protein Ligand-of-Numb Protein-X (LNX)

The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown. A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR. LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb. CAR was able to bind LNX both in vivo and in vitro. Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences. The CAR binding region in LNX was mapped to the second PDZ domain. CAR and LNX were also shown to colocalize in vivo in mammalian cells. We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.

The CtBP Binding Domain in the Adenovirus E1A Protein Controls CR1-Dependent Transactivation

The adenovirus E1A-243R protein has the ability to force a resting cell into uncontrolled proliferation by modulating the activity of key targets in cell cycle control. Most of these regulatory mechanisms are dependent on activities mapping to conserved region 1 (CR1) and the non-conserved N-terminal region of E1A. We have previously shown that CR1 functions as a very patent transactivator when it is tethered to a promoter through a heterologous DNA binding domain. However, artificial DNA binding was not sufficient to convert full-length E1A-243R to a transactivator. Thus, an additional function(s) of the E1A-243R protein modulates the effect of CR1 in transcription regulation. Here we demonstrate that a 44 amino acid region at the extreme C-terminus of ElA inhibited transactivation by a Gal4-CR1 fusion protein. Inhibition correlated with binding of the nuclear 48 kDa C-terminal binding protein (CtBP), which has been implicated in E1A-mediated suppression of the metastazing potential of tumour cells. This might suggest that CtBP binding can regulate E1A-mediated transformation by modulating CR1-dependent control of transcription.

A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors

The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses - a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene - produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.

Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs

BackgroundThe Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown.ResultsUsing antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart.ConclusionThe localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.

Essential Role of the Coxsackie - and Adenovirus Receptor (CAR) in Development of the Lymphatic System in Mice

The coxsackie- and adenovirus receptor (CAR) is a cell adhesion molecule predominantly associated with epithelial tight junctions in adult tissues. CAR is also expressed in cardiomyocytes and essential for heart development up to embryonic day 11.5, but not thereafter. CAR is not expressed in vascular endothelial cells but was recently detected in neonatal lymphatic vessels, suggesting that CAR could play a role in the development of the lymphatic system. To address this, we generated mice carrying a conditional deletion of the CAR gene (Cxadr) and knocked out CAR in the mouse embryo at different time points during post-cardiac development. Deletion of Cxadr from E12.5, but not from E13.5, resulted in subcutaneous edema, hemorrhage and embryonic death. Subcutaneous lymphatic vessels were dilated and structurally abnormal with gaps and holes present at lymphatic endothelial cell-cell junctions. Furthermore, lymphatic vessels were filled with erythrocytes showing a defect in the separation between the blood and lymphatic systems. Regionally, erythrocytes leaked out into the interstitium from leaky lymphatic vessels explaining the hemorrhage detected in CAR-deficient mouse embryos. The results show that CAR plays an essential role in development of the lymphatic vasculature in the mouse embryo by promoting appropriate formation of lymphatic endothelial cell-cell junctions.

Multiple Phenotypes in Adult Mice following Inactivation of the Coxsackievirus and Adenovirus Receptor (Car) Gene

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

Transcription activation by the transforming domain of adenovirus E1A is efficiently repressed by the last 44 amino acids of E1A

REPRESSION OF THE c-JUN trans-ACTIVATION FUNCTION BY THE ADENOVIRUS TYPE 12 E1A 52R PROTEIN CORRELATES WITH THE INHIBITION OF PHOSPHORYLATION OF THE c-JUN trans-ACTIVATION DOMAIN. D. Brocknu an, G. KrOner, C. Bury and H. Esche. The cellular transcription factor complex AP-1 mediates growth factor signals on the level ofgene expression and is considered to be decisive in cell differentiation, proliferation and transformation. AP-1 consists mainly of proteins encoded by the jun gene family (c-jun, junB, junD) and the fos gene family (c-los, fosB) including thefos-related antigensfral andfra2. The early region 1A (E1A) 52R polypeptide, a protein expressed exclusively by the in vivo oncogenic Adenovims subtype 12 (Adl2), represses the trans-activating activity of AP-1 consisting of c-Jun:c-Jun homodimers. Repression is accompanied by a direct physical interaction of the adenovirus protein with the bZIP domain of c-Jun essential for dimerization and DNA-binding. Interestingly this interaction does not lead to the prevention of the promoter bindung of c-Jun/AP-1. Moreover, the association between c-JUN and the TATA-box binding protein TBP is not disturbed by the 52R polypeptide. Down-regulation of c-Jun activity is rather due to the inhibition of the phosphorylatinn of its acidic trans-activation domain located at the amino terminal end. In vivo phosphorylation of the cJun trans-activation domain by JNK kinases enzymes belonging to the mitogen-activated protein (MAP) kinase group is necessary for the interaction of c-Jun with specific co-factors like CBP and therefore a prerequisite for the activation of specific target genes. Due to these results we propose a model in which the 52R protein represses the trans-activating activity of c-Jun by prevemijag its phosphorylation through a JNK kinase(s). (Supported by the Deutsche Forschungsgemeinschaft through SFB 354/TP3 and the Fonds der Chemischen Industrie.)