Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus

Abstract

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.

Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease

“…A similar situation has been reported for NDV, although intracellular cleavage is limited by the presence of a single basic residue at the cleavage site [46,53]. This leads to the F protein being cleaved and fusion triggered only when trypsin and low pH are applied at the cell surface [53,90], as observed in this study ( Fig 4A). This would explain why the same ISAV F proteins coupled with a full length HPR HE did not show any fusion activity when cell monolayers were left untreated ( Fig 4C).…”

Interaction of Peptides with Sequences from the Newcastle Disease Virus Fusion Protein Heptad Repeat Regions

“…Viral genes were inserted into SacI-and XbaI-cut plasmid DNA. Two mutants with mutations of the F gene with altered cleavage sites, mutants F115G and F117L, have been described previously (30,35).…”

“…Transfections. Transfections with dioleoyl-L-␣ phosphatidylethanolamine (DOPE) were done essentially as described previously (8,30). Briefly, Cos-7 cells were plated at 2 ϫ 10 5 per 35-mm plate and transfected 18 h later (the monolayers were 50 to 80% confluent).…”

“…Fusion assays. At 48 h posttransfection, the nuclei in 20 to 40 fusion areas were counted to determine the average size at each time point as previously described (30,40). Values obtained after transfection of the vector alone were subtracted.…”

Analysis of Cathepsin and Furin Proteolytic Enzymes Involved in Viral Fusion Protein Activation in Cells of the Bat Reservoir Host

“…The reason for this complex method of proteolytic activation remains unclear, but the cathepsin activation of henipavirus F proteins cannot be functionally replaced by other proteases, as a Nipah F protein mutant containing trypsin-or furin-cleavable sites displays reduced F processing [55]. Cleavage of the Hendra and Nipah F proteins occurs at a monobasic cleavage site GDV-K/R [56,57]; however, mutagenesis studies demonstrated that mutation of the basic residue at the cleavage site or of amino acids upstream of this site did not eliminate F protein processing [57,58], contradictory to other viral fusion proteins [59][60][61][62].…”