Characterization of the trans-activation-responsive element of the parvovirus H-1 P38 promoter

The parvovirus early protein NS1 positively regulates the expression of the P38 promoter for the viral capsid protein gene. We have examined the trans-activation of P38 by NS1 by using fusions of P38 to the reporter gene, chloramphenicol acetyltransferase (cat). Maximal trans-activation requires a small 5' cis element (tar) between -137 and -116. The tar element has activity in both orientations when 5' to the P38 promoter, but no activity has been detected 3' to the promoter. The wild-type P38 has a biphasic response to NS1 depending on the dosage of the NS1-expressing plasmid. Promoters lacking the tar also have a biphasic response that is reduced about 10-fold, and they can be inhibited by larger doses of the NS1 plasmid. Heterologous promoters from other viruses and the Harvey-ras oncogene promoter are inhibited by NS1. Truncated and internally deleted versions of NS1 lose the trans-activation, but some of them retain the inhibitory properties. Thus transactivation can be uncoupled from inhibition. The tar element has shown no activity with the heterologous simian virus 40 early promoter. In contrast, the P38 promoter responds to a heterologous enhancer, but the enhanced promoter loses activity to trans-activation by NS1. In summary, the P38 tar element has some of the properties of an enhancer with a high preference for a 5' position and a stringent requirement for the P38 promoter.     

Tumor necrosis factor-alpha activation by adenovirus E1A 13S CR3 occurs in a cell-dependent and cell-independent manner

The adenovirus (Ad) early gene product 13S transactivates the tumor necrosis factor (TNF)-alpha promoter in inflammatory cells. We examined both the subdomains of E1A and the upstream TNF promoter elements involved. In both Jurkat and U-937 cells, zinc finger or carboxyl region mutation of Ad E1A 13S conserved region 3 resulted in a significant loss of transactivation of the TNF promoter (> or =69%). For both cell types there was a TNF-negative regulatory element in the -242 to -199 region and a positive regulatory element between -199 and -118. In contrast, an upstream positive regulatory element was detected in different regions in both cell types. In U-937 cells the positive regulatory unit was between -600 and -576, whereas in Jurkat cells it was between -576 and -242. The U-937 upstream element was dependent on a site previously designated epsilon in cooperation with an adjacent nuclear factor-kappaB-2a site. Therefore, transactivation of the TNF promoter by Ad 13S in lymphocyte and monocyte cell types involves similar subdomains of the E1A protein, but cell-specific TNF promoter elements.     

Mutations in DNA Binding and Transactivation Domains Affect the Dynamics of Parvovirus NS1 Protein

The multifunctional replication protein of autonomous parvoviruses, NS1, is vital for viral genome replication and for the control of viral protein production. Two DNA-interacting domains of NS1, the N-terminal and helicase domains, are necessary for these functions. In addition, the N and C termini of NS1 are required for activation of viral promoter P38. By comparison with the structural and biochemical data from other parvoviruses, we identified potential DNA-interacting amino acid residues from canine parvovirus NS1. The role of the identified amino acids in NS1 binding dynamics was studied by mutagenesis, fluorescence recovery after photobleaching, and computer simulations. Mutations in the predicted DNA-interacting amino acids of the N-terminal and helicase domains increased the intranuclear binding dynamics of NS1 dramatically. A substantial increase in binding dynamics was also observed for NS1 mutants that targeted the metal ion coordination site in the N terminus. Interestingly, contrary to other mutants, deletion of the C terminus resulted in slower binding dynamics of NS1. P38 transactivation was severely reduced in both N-terminal DNA recognition and in C-terminal deletion mutants. These data suggest that the intranuclear dynamics of NS1 are largely characterized by its sequence-specific and -nonspecific binding to double-stranded DNA. Moreover, binding of NS1 is equally dependent on the N-terminal domain and conserved β-loop of the helicase domain.     

Transactivation of a cellular promoter by the NS1 protein of the parvovirus minute virus of mice through a putative hormone-responsive element.

The promoter of the thyroid hormone receptor alpha gene (c-erbA-1) is activated by the nonstructural protein 1 (NS1) of parvovirus minute virus of mice (prototype strain [MVMp]) in ras-transformed FREJ4 cells that are permissive for lytic MVMp replication. This stimulation may be related to the sensitivity of host cells to MVMp, as it does not take place in parental FR3T3 cells, which are resistant to the parvovirus killing effect. The analysis of a series of deletion and point mutants of the c-erbA-1 promoter led to the identification of an upstream region that is necessary for NS1-driven transactivation. This sequence harbors a putative hormone-responsive element and is sufficient to render a minimal promoter NS1 inducible in FREJ4 but not in FR3T3 cells, and it is involved in distinct interactions with proteins from the respective cell lines. The NS1-responsive element of the c-erbA-1 promoter bears no homology with sequences that were previously reported to be necessary for NS1 DNA binding and transactivation. Altogether, our data point to a novel, cell-specific mechanism of promoter activation by NS1.     

Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells.

The cotransfection of selectable marker genes and the gene for the nonstructural proteins NS1 and NS2 of the autonomous parvovirus H-1 failed to produce cell lines that constitutively expressed NS1. A plasmid, pP38NS1cat, was constructed that expressed the NS1-NS2 gene from the H-1 P38 coat protein promoter in place of the natural P4 promoter. The P38 promoter is constitutively weak and is trans-activated by NS1. Stable cell lines were isolated that contained pP38NS1cat that was constitutively silent, but inducible with exogenous NS1 by superinfection or by treatment with sodium butyrate. The cells that were induced for this self-stimulatory genetic circuit did not remain in the culture, suggesting that expression of NS1-NS2 is cytotoxic or that the expression is not sustained. The properties of these cell lines and an example of the construction of a cell line inducible for expression of the viral coat protein gene and the bacterial gene for chloramphenicol acetyltransferase (cat) are described.     

The RFA regulatory sequence-binding protein in the promoter of

The prostate-specific antigen (PSA), which plays an important role during the liquefaction process of semen, is a differentiation marker for human prostate. It has become the most sensitive marker for monitoring and detecting prostate cancer. PSA as a serine protease can activate some growth factors that might be related to the advancement of prostate cancer by hydrolyzing growth factor-binding protein. The PSA gene is expressed specifically in prostate epithelial cells. The ex-pression of…     

The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound specifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunctional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding proteins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA promoter.