Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence.
A full-length cDNA clone of Newcastle disease virus (NDV) vaccine strain LaSota was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Transfection of this plasmid into cells that were infected with a recombinant fowlpoxvirus that expressed T7 RNA polymerase, resulted in the synthesis of antigenomic NDV RNA. This RNA was replicated and transcribed by the viral NP, P, and L proteins, which were expressed from cotransfected plasmids. After inoculation of the transfection supernatant into embryonated specific-pathogen-free eggs, infectious virus derived from the cloned cDNA was recovered. By introducing three nucleotide changes in the cDNA, we generated a genetically tagged derivative of the LaSota strain in which the amino acid sequence of the protease cleavage site (GGRQGR downward arrowL) of the fusion protein F0 was changed to the consensus cleavage site of virulent NDV strains (GRRQRR downward arrowF). Pathogenicity tests in day-old chickens showed that the strain derived from the unmodified cDNA was completely nonvirulent (intracerebral pathogenicity index [ICPI] = 0.00). However, the strain derived from the cDNA in which the protease cleavage site was modified showed a dramatic increase in virulence (ICPI = 1.28 out of a possible maximum of 2.0). Pulse-chase labeling of cells infected with the different strains followed by radioimmunoprecipitation of the F protein showed that the efficiency of cleavage of the F0 protein was greatly enhanced by the amino acid replacements. These results demonstrate that genetically modified NDV can be recovered from cloned cDNA and confirm the supposition that cleavage of the F0 protein is a key determinant in virulence of NDV.
Peeters
B P
BP
Department of Avian Virology, Institute for Animal Science and Health, 8200 AB Lelystad, The Netherlands. b.p.h.peeters@id.dlo.nl
de Leeuw
O S
OS
Koch
G
G
Gielkens
A L
AL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D018076:DNA, Complementary / Q000235:genetics*; D010450:Endopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D009522:Newcastle disease virus / Q000235:genetics / Q000276:immunology / Q000472:pathogenicity*; D013329:Structure-Activity Relationship; D014162:Transfection; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D014765:Viral Vaccines / Q000276:immunology; D014774:Virulence
D018076:DNA, Complementary; D014760:Viral Fusion Proteins; D014765:Viral Vaccines; D010450:Endopeptidases