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, 85 (6), 2771-80

Recombinant Sendai Viruses Expressing Fusion Proteins With Two Furin Cleavage Sites Mimic the Syncytial and Receptor-Independent Infection Properties of Respiratory Syncytial Virus

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Recombinant Sendai Viruses Expressing Fusion Proteins With Two Furin Cleavage Sites Mimic the Syncytial and Receptor-Independent Infection Properties of Respiratory Syncytial Virus

Joanna Rawling et al. J Virol.

Abstract

Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.

Figures

FIG. 1.
FIG. 1.
Rescue of recombinant Sendai viruses (rSeV). (A) Alignment of SeV fusion protein (FSeV) (amino acids 99 to 129; blue) and RSV fusion protein (FRSV) (amino acids 100 to 144; red). Cleavage sites of FRSV (site I, RARR109, and site II, KKRKRR136) and FSeV (R116) are shown in bold and indicated by arrows. Residues from FRSV that were inserted into the FSeV backbone to produce cleavage mutants are shown in red. The numbering assigned to FSeV mutants produced by mutation of Fc refers to the last residue of FRSV inserted, excluding cleavage site II. Recombinant Sendai virus expressing the F protein mutants was rescued by cotransfection of BSR-T7/5 cells with the FL5 plasmid containing the complete SeV cDNA and pTM1 plasmids encoding SeV N, P, and L genes. Rescued virus was subsequently passaged three times in embryonated chicken eggs. Titration of the allantoic fluid revealed that double-cleavage-site viruses were obtained at lower titers (FFU/ml) than single-cleavage-site viruses (SeV-FH titer, 5 × 1011; SeV-Fc, 9 × 1011; SeV-F117, 9 × 1010; SeV-F130, 5 × 1010). Allantoic fluid from the third passage in eggs was clarified and centrifuged through a 25% glycerol cushion, and equivalent titers of SeV-FH, SeV-Fc, SeV-F117, and SeV-F130 viruses were analyzed on a 10% SDS-PAGE gel stained with Coomassie blue (B) or by Western blotting (C) using the polyclonal rabbit antiserum that recognizes the cytoplasmic tail of FSeV. P, phosphoprotein; HN, hemagglutinin-neuraminidase; N, nucleoprotein; M, matrix protein.
FIG. 2.
FIG. 2.
Multistep growth curve. LLC-MK2 cells were infected at an MOI of 0.05 and incubated in the absence (A) or presence (B) of 0.3 μg/ml trypsin for 96 h. At 24-h intervals, aliquots of the cell culture supernatant were collected and replaced with fresh medium with or without trypsin. Titration of the culture supernatant was performed by immunohistochemical staining of infected LLC-MK2 cell foci in 96-well plates. Results represent the mean of two independent infections, which were titrated in duplicate. FFU, focus forming units.
FIG. 3.
FIG. 3.
Proteolytic cleavage of rSeV. Western blotting was employed to analyze cell culture supernatant from infected LLC-MK2 cells (MOI of 0.05) incubated for 96 h in the absence (−) or presence (+) of 0.3 μg/ml trypsin. Western blots were developed using polyclonal rabbit serum αF_SeV_CT, which recognizes the CT of SeV F protein, or αF_104-117, which recognizes amino acids 104 to 117 of the RSV F protein. Also indicated are bands representing uncleaved F protein precursor (F0), the fully cleaved F1 chain (F1), and partially cleaved intermediate (F1+), which results from cleavage at site I in the absence of cleavage at site II. The percentage of F protein cleavage to F1 in the absence of trypsin (upper left panel) was determined by measuring the densitometry of bands, as detailed in Materials and Methods (Fc, 66%; F117, 64%; F130, 66%).
FIG. 4.
FIG. 4.
Thermostability of recombinant Sendai viruses. (A) Purified SeV was preincubated at 37°C for 2, 4, or 6 h and subsequently incubated at 33°C for 45 min with 0.5 ml of washed guinea pig erythrocytes (1%, vol/vol, in PBS). Hemolysis was measured by determining the optical density of the erythrocyte supernatant at 520 nm. Results represent the mean of two independent experiments performed in duplicate. (B) Supernatant from infected LLC-MK2 cells was preincubated at 37°C for 2, 4, or 6 h and subsequently titrated on LLC-MK2 cells in 96-well plates by immunohistochemical staining. Results represent the mean of three independent experiments.
FIG. 5.
FIG. 5.
Syncytium formation assay. (A) LLC-MK2 cells growing in microchamber wells were infected with SeV-FH, SeV-Fc, SeV-F117, or SeV-F130 virus at an MOI of 0.5 in the absence or presence of 0.3 μg/ml trypsin. Infected cells were processed for syncytium formation and immunostaining at 30 h postinfection. (B) The number of nuclei in 10 syncytia (chosen at random) in duplicate infections were counted using an AxioCam HRc digital camera (×20 magnification). Results are representative of three independent experiments.
FIG. 6.
FIG. 6.
Quantitative cell-cell fusion assay. BSR-T7/5 cells were infected with SeV-FH, SeV-Fc, SeV-F117, or SeV-F130 virus at an MOI of 5 and incubated for 14 h in the absence or presence of 0.25 μg/ml trypsin. LLC-MK2 cells, which had previously been transfected with pTM1-Luc, were overlaid onto infected BSR-T7/5 cells at 14 h postinfection and incubated in the absence or presence of 0.25 μg/ml trypsin for 3 h at 37°C (A) or 33°C (B) to allow fusion. Cells were subsequently lysed and analyzed for luciferase activity. Results (as relative light units) are expressed as a percentage of wild-type (wt) fusion (SeV-FH with trypsin), with mean values from at least two independent experiments shown.
FIG. 7.
FIG. 7.
Effect of neuraminidase treatment on rSeV infection. (A) LLC-MK2 cells growing in microchamber wells were pretreated with 120 mU of neuraminidase (NA) for 90 min at 37°C and subsequently infected with SeV-FH, SeV-Fc, SeV-F117, or SeV-F130 virus at an MOI of 1. Cells were processed for immunostaining at 20 h postinfection. (B) The number of infected cells in five fields (chosen at random) in duplicate infections were counted using an AxioCam HRc digital camera (×20 magnification). Also indicated is the percent infection in NA-treated (+) cells compared to untreated (−) cells. Results are representative of three independent experiments.
FIG. 8.
FIG. 8.
Infection of sialic acid-deficient cells. (A) Pro-5 or sialic acid-deficient Lec2 cells growing in microchamber wells were infected with SeV-FH, SeV-Fc, SeV-F117, or SeV-F130 virus at an MOI of 1. Cells were processed for immunostaining at 20 h postinfection. (B) Pro-5 or Lec2 cells growing in 96-well plates were infected with SeV-FH, SeV-Fc, SeV-F117, or SeV-F130 virus at an MOI of 1 and fixed at 20 h postinfection. Cells were subsequently stained for expression of SeV F protein with MAb GB5, and the optical density (OD) at 490 nm was measured in order to quantitate infection. The percent infection in Lec2 cells relative to Pro-5 cells is indicated. Results represent the mean of three independent experiments performed in duplicate.

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