The attachment function of the Newcastle disease virus hemagglutinin-neuraminidase protein can be separated from fusion promotion by mutation.
Using Cos-7 cells and an SV40-based vector, quantitative assays for fusion directed by the Newcastle disease virus hemagglutinin-neuraminidase (HN) and fusion glycoproteins were developed. Data from these assays showed that after cotransfection of the HN protein and the fusion protein DNAs, there was a progressive increase in syncytia size over background levels while transfection with HN protein or F protein DNA alone resulted in no changes over background. Using immunofluorescence, the efficiency of syncytia formation directed by the glycoproteins was assessed. Virtually all cells expressing the fusion protein alone or the HN protein alone existed as single nucleated cells. However, all cells coexpressing the two genes existed in syncytia with 4 to 50 nuclei. Using these assays, the fusion promotion activity of two deletion mutants of the HN protein was quantitated. One mutation deleted amino acids 4 through 26, effectively removing the cytoplasmic domain of the protein. This mutant protein was found at the cell surface, although in very reduced amounts. The mutant protein could bind red blood cells and could promote fusion, although the syncytia formed were smaller (4 to 9 nuclei) than those promoted by the wild-type protein. A second mutation deleted nine amino acids (91-99) located 37 amino acids from the transmembrane region in the ectodomain. This mutant was detected at the cell surface at 33% the level of wild type and it retained near normal ability to bind red blood cells. However, this mutation completely eliminated the fusion promotion activity of the HN protein. This mutation effectively separates the attachment function of the HN protein from fusion promotion.
Sergel
T
T
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655.
McGinnes
L W
LW
Peeples
M E
ME
Morrison
T G
TG
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D015726:Giant Cells / Q000502:physiology; D015707:HN Protein / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D051379:Mice; D016296:Mutagenesis; D009522:Newcastle disease virus / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D013539:Simian virus 40; D014162:Transfection; D014760:Viral Fusion Proteins / Q000235:genetics / Q000502:physiology*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004279:DNA, Viral; D015707:HN Protein; D014760:Viral Fusion Proteins