Nipah virus fusion protein: influence of cleavage site mutations on the cleavability by cathepsin L, trypsin and furin

Abstract

Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae which originated from bats, encodes for a fusion (F) protein which is proteolytically processed within endosomes by cathepsin L. We show here that sequence requirements for NiV F activation differ markedly from other para- or orthomyxoviral fusion proteins. In contrast to other viral fusion proteins with monobasic cleavage sites, processing of NiV F proteins with one single basic amino acid in the cleavage peptide by exogenous trypsin is very inefficient, and introduction of a consensus sequence for furin does not result in cleavage by this ubiquitous protease. In contrast, a multibasic cleavage peptide in the NiV F protein completely impairs proteolytic processing and the generation of biological activity.

Fig. 1

Alignment of NiV and MV F protein cleavage site mutants. Amino acid sequences around the cleavage site of NiV F and MV F_Edm proteins analyzed in this study are presented. Boldfaced letters indicate amino acid residues exchanged in the standard NiV F and MV F_Edm. The N-glycosylation site in the NiV F protein is underlined, N-glycan g3 is attached to asparagine at position 99 (N₉₉). cm, cleavage mutant. Description of the mutants: MutantNiV F_YA has no exchanges at the cleavage site but has two point mutations in the tyrosine-dependent internalization signals in the cytoplasmic tail (described previously in Diederich et al., 2005). NiV F_YA is no longer endocytosed and cleaved by endosomal cathepsins. MutantNiVFcm1 represents a NiV F protein with the monobasic cleavage site of the trypsin-activatable measles virus F protein MV Fcm. MutantNiVFcm2 contains the multibasic furin cleavage site of the measles virus MV F_Edm. MutantNiV Fg3 has a mutation in the N-glycosylation site but no exchanges at the cleavage site. MutantNiV Fg3cm2 lacks g3 due to a mutation in the N-glycosylation site and contains the multibasic furin-cleavage motif of the measles virus MV F_Edm. MutantMVFcm is a MV F protein with a monobasic cleavage site that has been shown to be cleaved by trypsin (Maisner et al., 2000). MutantMVFcm* contains the monobasic cleavage peptide of the NiV F.

Fig. 2

In vitro peptide cleavage assay. Cleavage of a fluorogenic peptide mimicking the NiV F cleavage site (H₂N-Abz-DLVGDVRLAGV-3-nitroYA-CONH₂) by trypsin and cathepsin L (CTSL) was analyzed. Reactions were carried out at 37 °C in 100 μl buffer containing 10 μM fluorogenic peptides and either trypsin (1 μg) or 0.5 μg (0.1 mU) CTSL. Enzymatic activity was measured with a PerkinElmer LS55 luminescence spectrometer (excitation 320 nm, emission 460 nm). Relative fluorescence units (RFU) versus hydrolysis time are plotted.

Fig. 3

Influence of trypsin addition on the cleavage of MV F and NiV F proteins. MDCK cells were transfected in (A) with plasmids carrying the gene encoding for standard MV F_Edm, the MV Fcm protein with a monobasic cleavage site, the wildtype NiV F protein, or the endocytosis-negative mutant NiV F_YA protein. In (B), cells were transfected with pczCFG5 NiV Fg3 carrying a NiV F gene with a mutation in the N-glycosylation site, or with pczCFG5 NiV Fcm1 encoding for a NiV F gene with the multibasic cleavage site of MV F_Edm. At 6 h p.t., the culture medium was replaced by medium without (Ø) or with (+) 0.5 μg of trypsin per ml. After 18 h, cells were surface labeled with biotin and lysed. Following immunoprecipitation, samples were subjected to SDS-PAGE under reducing conditions and blotted to nitrocellulose. Surface-labeled F proteins were visualized with streptavidin-peroxidase and chemiluminescence. F₀* and F₂*: lack of g3 results in faster migration (about 3 kD) of the uncleaved NiV F₀ and the subunit NiV F₂.

Fig. 4

Cleavage and biological activity of mutant MV F proteins with monobasic cleavage sites in the absence and presence of trypsin. (A) MDCK cells were transfected with genes either encoding MV Fcm with a monobasic cleavage site (NHNR) known to be recognized by trypsin, or encoding MV Fcm*_, a MV F protein with the cleavage peptide of NiV F (GDVR). Transfected cells were radiolabeled at 24 h p.t. with [³⁵S]-methionine and -cysteine for 10 min and then incubated for 2 h in the absence (Ø) or presence (+) of 0.5 μg trypsin per ml. F proteins were immunoprecipitated from cell lysates, separated on a 12% SDS gel under reducing conditions, and subjected to autoradiography. (B) Cells were transfected with the MV H gene in combination with either the MV Fcm or the MV Fcm* gene. At 6 h p.t., the culture medium was replaced by medium without (Ø) or with (+) 0.5 μg trypsin per ml. After 18 h, cells were fixed with ethanol and incubated with Giemsa staining solution to visualize syncytium formation. Magnification, 63×.

Fig. 5

Proteolytic processing of NiV F proteins by endogenous host cell proteases. (A) MDCK cells were transfected with genes encoding either for wildtype NiV F, for the g3-deficient mutant NiV Fg3, for mutant NiV Fcm2 containing the multibasic cleavage peptide of the MV F_Edm, or for mutant NiV Fg3cm2, a g3-deficient protein with the multibasic cleavage peptide of the MV F_Edm. At 24 h p.t., cells were metabolically labeled with [³⁵S]-methionine and -cysteine for 10 min and incubated in non-radioactive medium for 3 h. After cell lysis, F proteins were immunoprecipitated and detected by autoradiography after separation on a 12% SDS gel under reducing conditions. NiV F₀*: F₀ of g3 lacking mutants migrates faster (about 3 kD). (B) MDCK cells expressing wildtype or mutant NiV F proteins were subjected to surface biotinylation as described in the legend to Fig. 3B. (C) Syncytium formation in MDCK cells expressing NiV G together with either NiV F, NiV Fg3, NiV Fcm2 or NiV Fg3cm2 was detected by Giemsa staining at 24 h p.t. Magnification, 63×. (D) MDCK cells were transfected with pczCFG5 NiV F, pczCFG5 NiV Fg3cm2, or pCG MV F_Edm. At 24 h p.t., a pulse-chase analysis was carried out as described in the legend to Fig. 4A. Labeling was performed in the absence (control) or presence of either 50 μM CA074ME (CA074ME) or 25 μM Dec-RVKR-CMK (Dec-RVKR-CMK). F proteins were immunoprecipitated, separated by SDS-PAGE under reducing conditions, and subjected to autoradiography.