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.
Fournier
Philippe
P
Division of Cellular Immunology, German Cancer Research Center, D-69129 Heidelberg, Germany.
Zeng
Jinyang
J
Von Der Lieth
Claus-W
CW
Washburn
Birgit
B
Ahlert
Thorsten
T
Schirrmacher
Volker
V
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005822:Genetic Vectors; D015707:HN Protein / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism; D016898:Interferon-alpha / Q000378:metabolism; D016899:Interferon-beta / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009522:Newcastle disease virus / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics; D012694:Serine / Q000737:chemistry*; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; D015707:HN Protein; D007370:Interferon Type I; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; C505654:Tnfsf10 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D012694:Serine; D012313:RNA; D016899:Interferon-beta