Activation of fusion by the SER virus F protein: a low-pH-dependent paramyxovirus entry process

Abstract

SER virus, a paramyxovirus closely related to simian virus 5, induces no syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT), and truncation or mutations of the CT result in enhanced syncytium formation (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77:167-178, 2003; S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). We hypothesized that the presence of the long CT serves to stabilize the metastable conformation of the F protein. We observed that the hemifusion, cytoplasmic content mixing, and syncytium formation ability of the wild-type SER virus F coexpressed with the SER virus hemagglutinin-neuraminidase (HN) protein was enhanced, both qualitatively and quantitatively, at elevated temperatures. We also observed enhanced hemifusion, content mixing, and syncytium formation in SER virus F- and HN-expressing cells at reduced pH conditions ranging between 4.8 and 6.2. We have obtained evidence that in contrast to other paramyxoviruses, entry of SER virus into cells occurs by a low-pH-dependent process, indicating that the conversion to the fusion-active state for SER virus F is triggered by exposure to reduced pH.

FIG. 1.

Effect of elevated temperature on hemifusion and content mixing induced by SER virus F/HN. BHK21 cells expressing SER virus F and HN were incubated with guinea pig RBCs labeled with either R-18 or calcein AM at 4°C for 1 h and were then washed to remove unbound RBCs. Cells were further incubated at a temperature of 22, 30, 37, 41, 45, 47, 55, or 65°C for 2 min in a water bath, shifted back to 37°C for 30 min, and observed for dye transfer under an inverted fluorescence microscope (magnification, ×200). Vaccinia virus-infected, mock-transfected cells served as negative controls. (A) Left panels, R-18 transfer (hemifusion); right panels, calcein transfer (content mixing). The incubation temperature for each panel is shown on the panel. (B and C) Extent of fusion expressed as number of lipid-mixing or content-mixing events per microscopic field (averaged over 10 fields) in SER virus F/HN-expressing cells (B) and in SER virus F-L539,548A/HN-expressing cells (C). Differently sized syncytia were scored the same, as we were unable to determine the number of nuclei in the labeled syncytia.

FIG. 2.

(A) Effect of temperature on syncytium formation. At 16 h posttransfection, BHK21 cells expressing SER virus F and HN were incubated at a temperature of 37, 41, 45, 47, 55, or 65°C for 2 min, shifted back to 37°C, and observed under an inverted light microscope. Vaccinia virus-infected, mock-transfected cells served as negative controls, as shown on the top left panel. Magnification, ×200. (B) Effect of pH on SER virus F/HN-induced syncytium formation. At 16 h posttransfection, BHK21 cells expressing SER virus F and HN were incubated for 2 min at 37°C with various pH buffers as indicated, neutralized with neutral pH buffer, and observed under an inverted light microscope for syncytium formation. Vaccinia virus-infected, mock-transfected cells served as negative controls (top left panel). Magnification, ×200.

FIG.3.

Effect of pH on hemifusion and content mixing. BHK21 cells expressing SER virus F and HN were incubated with guinea pig RBCs labeled with either R-18 or calcein AM at 4°C for 1 h and then washed with fusion buffer (pH 7.4) (HEPES-saline buffered with sodium citrate and citric acid) to remove unbound RBCs as described by Steinhauer et al. (38), washed, and incubated for 2 min at 37°C with the buffer adjusted to various pH conditions as shown, followed by a wash with neutral pH buffer and further incubation for 30 min. Cells were observed for dye transfer under a fluorescence microscope (magnification, ×200). (A) Top two panels, vaccinia virus-infected, mock-transfected cells with R18- and calcein-labeled RBCs; other panels, SER virus F/HN-expressing cells with R18- and calcein-labeled RBCs treated under the indicated pH conditions. (B and C) Extent of fusion expressed as number of lipid-mixing (B) and content-mixing (C) events per microscopic field, as averaged over 10 fields. Each error bar represents the standard deviation from the mean.

FIG. 4.

Effect of BFLA1 and ammonium chloride on SER virus infection. MDBK cells were mock infected or infected with SER virus or SV5 at a multiplicity of infection of 10 PFU/cell for 2 h at 37°C in the presence of different concentrations of BFLA1 (A) or ammonium chloride (B). Cells were washed to remove excess virus and inhibitor, followed by incubation with fresh medium. Results for HA titers at 48 h postinfection on culture medium from cells infected or treated with BFLA1 or ammonium chloride at different concentrations or from untreated infected cells are shown as percentages of control (HA titer of untreated SER virus-infected cells, 256 hemagglutinating units; HA titer of SV5-infected cells, 4,096 hemagglutinating units).