Open Access

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), has recently emerged as a promising target for antiviral intervention. Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp. This mAb, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyprotein or subgenomic replicons. Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected. mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level. The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells. The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV. More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents.

The natural cytotoxicity receptor (NCR) NKp30 (CD337) is a key player for NK cell immunosurveillance of infections and cancer. The molecular details of ligand recognition and its connection to CD3ζ signaling remain unsolved. Here, we show that the stalk domain (129KEHPQLGAGTVLLLR143) of NKp30 is very sensitive to sequence alterations, as mutations lead to impaired ligand binding and/or signaling capacity. Surprisingly, the stalk domains of NKp30 and NKp46, another NCR employing CD3ζ for signaling, were not exchangeable without drastic deficiencies in folding, plasma membrane targeting, and/or ligand-induced receptor signaling. Further mutational studies, N-glycosylation mapping, and plasma membrane targeting studies in the absence and presence of CD3ζ suggest two interconvertible types of NCR-CD3ζ assemblies: 1) a signaling incompetent structural NKp30-CD3ζ complex and 2) a ligand-induced signaling competent NKp30-CD3ζ complex. Moreover, we propose that ligand binding triggers translocation of Arg-143 from the membrane interface into the membrane to enable alignment with oppositely charged aspartate residues within CD3ζ and activation of CD3ζ-signaling.

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis, only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34(+) cells. Tal1 expression activated UBE2H expression, whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis.

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.