Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement - PubMed (original) (raw)

Deletion of the cytoplasmic tail of the fusion protein of the paramyxovirus simian virus 5 affects fusion pore enlargement

R E Dutch et al. J Virol. 2001 Jun.

Abstract

The fusion (F) protein of the paramxyovirus simian parainfluenza virus 5 (SV5) promotes virus-cell and cell-cell membrane fusion. Previous work had indicated that removal of the SV5 F protein cytoplasmic tail (F Tail- or FDelta19) caused a block in fusion promotion at the hemifusion stage. Further examination has shown that although the F Tail- mutant is severely debilitated in promotion of fusion as measured by using two reporter gene assays and is debilitated in the formation of syncytia relative to the wild-type F protein, the F Tail- mutant is capable of promoting the transfer of small aqueous dyes. These data indicate that F Tail- is fully capable of promoting formation of small fusion pores. However, enlargement of fusion pores is debilitated, suggesting that either the cytoplasmic tail of the F protein plays a direct role in pore expansion or that it interacts with other components which control pore growth.

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Figures

FIG. 1

FIG. 1

Transfer of lipid and aqueous dyes by wt F and F Tail−. SV5 HN and either wt F or F Tail− were coexpressed in CV-1 cells by using the recombinant vac-T7 polymerase expression system. At 24 h p.t., human RBCs double labeled with the lipid probe R18 and aqueous probe CF were bound to CV-1 cells at 4°C. Fusion was initiated by replacement of cold PBS with PBS prewarmed to 37°C, and cells were incubated at 37°C for 10 min. Fusion was stopped by replacement with ice-cold PBS. Cells were examined using a confocal microscope (Zeiss LSM 410; Carl Zeiss, Inc., Thornwood, N.Y.), with dual images recorded on both fluorescein and rhodamine channels.

FIG. 2

FIG. 2

Time course of transfer of the aqueous probe CF. Human RBCs labeled with the aqueous probe CF were bound to CV-1 cells coexpressing HN and either wt F or F Tail−, as described in the legend to Fig. 1. The cells were incubated at 37°C for various times, and the results were analyzed by confocal microscopy.

FIG. 3

FIG. 3

Reporter gene assays of wt F and F Tail−. (A) β-Galactosidase assay. CV-1 cells infected with the vac-T7 polymerase recombinant and coexpressing HN and either wt F or F Tail− were incubated with a second population of CV-1 cells infected with wt vaccinia virus and transfected with the plasmid pINTT7 β-gal, which encodes β-galactosidase under the control of the T7 polymerase promoter. After incubation at 37°C for 4 h, samples were analyzed by a colorimetric lysate assay. Results shown are the average of triplicate samples and are representative of two separate experiments. (B) CAT assay. Vero cells were cotransfected with pCAGGS expressing HN and either wt F or F Tail−. In addition, these cells were transfected with the plasmid encoding CAT under control of the T7 polymerase promoter. A second set of Vero cells was transfected with pCAGGS expressing T7 polymerase. After overnight incubation, the T7 polymerase-expressing cells were overlaid on the cells expressing the F and HN proteins. Membrane fusion between the cell populations allows the T7 polymerase to transcribe the CAT gene, and subsequent CAT activity was assayed as described previously (14). Samples are an average of duplicates and are representative of three separate experiments.

FIG. 4

FIG. 4

Syncytium formation assay. BHK 21F cells or Vero cells were transfected using a total of 2 μg of pCAGGS wt F, F Tail−, or HN plasmid. At 18 to 24 h p.t. (BHK) or 36 h p.t. (Vero), monolayers were examined using a Nikon Diaphot inverted phase-contrast microscope (Nikon Inc., Garden City, N.Y.). Photographs were taken using a Kodak DCS 420 digital camera (Eastman Kodak Company, Rochester, N.Y.).

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