High cell surface expression of Newcastle disease virus proteins via replicon vectors demonstrates syncytia forming activity of F and fusion promotion activity of HN molecules.
For functional studies, the hemagglutinin-neuraminidase (HN) and the fusion protein (F) of Newcastle disease virus (NDV) were expressed in BHK cells using two vectors which are based on the Semliki Forest virus (SFV) replicon. The first system of high protein expression works by transfection of RNA which before has been in vitro transcribed from a vector containing the gene for the SFV self-amplifying replicase (REP) and a foreign gene using the SP6 promoter. A high level of protein (HN or F) expression was detected 18-20 h after transfection. To study the host range of this expression system, a panel of different cell lines were compared for transfections with SFV RNA. A wide range of expression efficiency was observed, the highest being BHK cells. The second system is based on a DNA plasmid in which the SFV-REP and a foreign gene are expressed in cells under the transcriptional and translational control of the cytomegalovirus immediate-early enhancer T7 promoter. DNA-electroporated BHK cells expressed also high levels of the recombinant proteins but at a delayed time point (40-48 h) as compared with the corresponding RNA. Co-expression of the two NDV proteins, HN and F, via this DNA vector in the same cells led to syncytium formation in the cell monolayer, showing that both proteins expressed in this way, were functionally active. F alone, expressed via this vector, displayed residual fusion activity suggesting its proteolytic cleavage and its functional independence from HN.
Zeng
Jinyang
J
Division of Cellular Immunology, German Cancer Research Center, D010, D-69129 Heidelberg, Germany.
Fournier
Philippe
P
Schirrmacher
Volker
V
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D015726:Giant Cells / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D015707:HN Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009439:Neuraminidase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D009522:Newcastle disease virus / Q000235:genetics*; D012093:Replicon / Q000235:genetics*; D012672:Semliki forest virus / Q000235:genetics; D014760:Viral Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D015707:HN Protein; D014760:Viral Fusion Proteins; D009439:Neuraminidase