Importance of serine 200 for functional activities of the hemagglutinin-neuraminidase protein of Newcastle Disease Virus.

Newcastle disease virus (NDV) is an avian paramyxovirus with replication competence in human tumor cells and interesting anti-neoplastic and immune stimulatory properties. In order to increase tumor selectivity of replication, we prepared mutants from the avirulent strain Ulster with monocyclic replication cycle and adapted them for multicyclic replication in human melanoma cells. Two mutants (M1 and M2) showed interesting functional differences: while M2 showed T cell co-stimulatory effects in a tumor-specific cytotoxic T lymphocyte (CTL) assay, M1 did not. A distinct difference of these 2 virus mutants appeared also when testing their capacity to induce interferon-alpha and -beta as well as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules in human monocytes. Sequence analysis of the hemagglutinin-neuraminidase (HN) molecules of the 2 virus mutants showed 7 non-silent mutational differences. Upon cloning of the HN mutant genes into an expression vector and transfection of cells, only HN derived from M2 (HN-M2) was detected at the cell surface by immunostaining with specific antibodies and showed hemadsorption and neuraminidase activity. In order to define which amino acid was responsible for the loss of functional activity of HN derived from M1 (HN-M1), distinct HN mutants were generated via site-directed mutagenesis and tested. Substitution of serine 200 by a proline abrogated HN expression and its hemadsorption and neuraminidase activities. Molecular modeling revealed that proline 200 in HN influences flexibility of a loop near the entrance to the neuraminidase active site, a function that may be crucial for the functions of this viral protein.

TNF-related apoptosis-inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus.

The Newcastle disease virus (NDV) has antineoplastic and immunostimulatory properties, and it is currently clinically tested in anticancer therapy. However, the tumoricidal mechanisms of NDV tumor therapy are not fully understood. The results presented here demonstrate that NDV-stimulated human monocytes (Mphi) kill various human tumor cell lines and that this tumoricidal activity is mediated by TRAIL. In contrast to soluble TRAIL-R2-Fc, soluble CD95-Fc and TNF-R2-Fc showed only minimal blocking of the antitumor effect. TRAIL expression is induced on human Mphi after stimulation with NDV and UV-inactivated NDV. These results show that TRAIL induction on human Mphi after NDV stimulation is independent from viral replication and that TRAIL mediates the tumoricidal activity of NDV-stimulated human Mphi.

AML-associated translocation products block vitamin D(3)-induced differentiation by sequestering the vitamin D(3) receptor.

Acute myeloid leukemia (AML)-associated chromosomal translocations result in formation of chimeric transcription factors, such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, of which the components are involved in regulation of transcription by chromatin modeling through histone acetylation/deacetylation. The leukemic differentiation block is attributed to deregulated transcription caused by these chimeric fusion proteins, which aberrantly recruit histone-deacetylase (HDAC) activity. One essential differentiation pathway blocked by the leukemic fusion proteins is the vitamin (Vit) D(3) signaling. Here we investigated the mechanisms by which the leukemic fusion proteins interfere with VitD(3)-induced differentiation. The VitD(3)-receptor (VDR) is, like the retinoid receptors RAR, retinoid X receptor, and the thyroid hormone receptor (TR), a ligand-inducible transcription factor. In the absence of ligand, the transcriptional activity of TR and RAR is silenced by recruitment of HDAC activity through binding to corepressors. In the presence of ligand, TR and RAR activate transcription by releasing HDAC activity and by recruiting histone-acetyltransferase activity. Here we report that VDR binds corepressors in a ligand-dependent manner and that inhibition of HDAC activity increases VitD(3) sensitivity of HL-60 cells. Nevertheless, the inhibition of HDAC activity is unable to overcome the block of VitD(3)-induced differentiation caused by PLZF/RARalpha expression. Here we demonstrate that the expression of the translocation products PML/RARalpha and PLZF/RARalpha impairs the localization of VDR in the nucleus by binding to VDR. Furthermore, the overexpression of VDR in U937 cells expressing AML-related translocation products completely abolishes the block of VitD(3)-induced differentiation. Taken together these data indicate that the AML-associated translocation products block differentiation not only by interfering with chromatin-modeling but also by sequestering factors involved in the differentiation signaling pathways, such as VDR in the VitD(3)-induced differentiation.