Conditional gene vectors regulated in cis.

Non-integrating gene vectors, which are stably and extrachromosomally maintained in transduced cells would be perfect tools to support long-term expression of therapeutic genes but preserve the genomic integrity of the cellular host. Small extrachromosomal plasmids share some of these ideal characteristics but are primarily based on virus blueprints. These plasmids are dependent on viral trans-acting factors but they can replicate their DNA molecules in synchrony with the chromosome of the cellular host and segregate to daughter cells in an autonomous fashion.

IL-12-induced T-bet expression and IFNgamma release in lymphocytes from asthmatics--role of MAPkinases ERK-1/-2, p38(MAPK) and effect of dexamethasone.

The transcription factor T-box-expressed-in-T-cells (T-bet) is required for T(H)1 lymphocyte differentiation, regulates the IL-12-induced expression of the T(H)1-specific cytokine IFNgamma and may be dysregulated in asthmatics. The modulatory role of extracellular signal-regulated kinase (ERK)-1/-2, p38mitogen-activated protein kinase (MAPK) and dexamethasone on IL-12 induced T-bet and IFNgamma expression was assessed in peripheral blood lymphocytes of 10 atopic asthmatics and 10 nonatopic normals. IFNgamma production was dependent on phosphorylation of ERK-1/-2 and p38MAPK, as examined by PD098059, an inhibitor of the upstream activator of MAPKkinase (MKK-1), and SB203580, an inhibitor of p38MAPK.

Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism.

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype.

CD8 T cell recognition of endogenously expressed epstein-barr virus nuclear antigen 1.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I-restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon gamma release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter-dependent pathway.

B cell receptor signal strength determines B cell fate.

B cell receptor (BCR)-mediated antigen recognition is thought to regulate B cell differentiation. BCR signal strength may also influence B cell fate decisions. Here, we used the Epstein-Barr virus protein LMP2A as a constitutively active BCR surrogate to study the contribution of BCR signal strength in B cell differentiation.

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus.

The sequential binding of the origin recognition complex (ORC), Cdc6p and the minichromosome maintenance proteins (MCM2-7) mediates replication competence at eukaryotic origins of DNA replication. The latent origin of Epstein-Barr virus, oriP, is a viral origin known to recruit ORC. OriP also binds EBNA1, a virally encoded protein that lacks any activity predicted to be required for replication initiation.

The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold.

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the earliest viral proteins expressed after infection and is the only latent protein consistently expressed in viral-associated tumors. EBNA1's crucial role in viral DNA replication, episomal maintenance, and partitioning is well examined whereas its importance for the immortalization process and the tumorgenicity of EBV is unclear. To address these open questions, we generated, based on the maxi-EBV system, an EBNA1-deficient EBV mutant and used this strain to infect primary human B cells.