Effect of cleavage mutants on syncytium formation directed by the wild-type fusion protein of Newcastle disease virus.
The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.
Li
Z
Z
Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester 01655, USA.
Sergel
T
T
Razvi
E
E
Morrison
T
T
D000818:Animals; D019556:COS Cells; D015870:Gene Expression; D015726:Giant Cells; D008561:Membrane Fusion; D016297:Mutagenesis, Site-Directed; D009522:Newcastle disease virus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014357:Trypsin / Q000378:metabolism; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism*
D014760:Viral Fusion Proteins; D014357:Trypsin