Membrane fusion promoted by increasing surface densities of the paramyxovirus F and HN proteins: comparison of fusion reactions mediated by simian virus 5 F, human parainfluenza virus type 3 F, and influenza virus HA

Abstract

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins.

FIG. 1

Expression of varying surface densities of the SV5 F protein. (A) Duplicate plates of vTF7-3-infected CV-1 cells were transfected (as described in Materials and Methods) with varying quantities of SV5 F DNA and pGEM3X DNA to give a final DNA amount of 7.5 μg per 6-cm-diameter plate. Samples were processed for flow cytometric analysis using MAb F1a as described in Materials and Methods. The MFI was compared to that observed in SV5-infected cells at 18 h p.i., with the MFI of mock-transfected samples subtracted. (B) Example of raw data from flow cytometric analysis, showing data for mock-infected cells or cells transfected with 0.1 μg of SV5 F DNA and 2.5 μg of SV5 F DNA.

FIG. 2

Effect of increasing amounts of SV5 F protein on extent of membrane fusion. vTF7-3-infected CV-1 cells were transfected with 2.5 μg of SV5 HN DNA and various amounts of SV5 F DNA and pGEM3X DNA to give a final amount of 7.5 μg of DNA per 6-cm-diameter plate. Flow cytometry using MAb F1a was performed in duplicate, and MFI was compared to that observed in SV5-infected cell at 18 h p.i. The β-galactosidase fusion assay was performed in triplicate as described in Materials and Methods.

FIG. 3

Effect of SV5 HN protein surface density on fusion promoted by the SV5 F protein. vTF7-3 infected CV-1 cells were transfected with 2.5 μg of SV5 F DNA and varying amounts of SV5 HN DNA and pGEM3X DNA to give a final amount of 7.5 μg of DNA per 6-cm-diameter plate. Flow cytometry was performed in duplicate with either MAb F1a or MAb HN4b. The β-galactosidase fusion assay was performed in triplicate.

FIG. 4

Effect of HPIV-3 HN protein surface density on fusion promoted by the HPIV-3 F protein. vTF7-3-infected CV-1 cells were transfected with 0.5 μg of HPIV-3 F DNA (the amount required to yield approximately 100% of F protein expression observed in an HPIV-3-infected cell) and varying amounts of HPIV-3 HN DNA and pGEM3X DNA to give a final amount of 7.5 μg of DNA per 6-cm-diameter plate. Flow cytometric analysis was performed in duplicate with either MAb 145/50 specific for HPIV-3 F or MAb 66/4 specific for HPIV-3 HN. The β-galactosidase fusion assay was performed in triplicate.

FIG. 5

Effect of increasing surface densities of SV5 F protein on rates of fusion. (A) vTF7-3-infected CV-1 cells were transfected with 2.5 μg of pTF7.5 HA DNA and various amounts of SV5 F DNA and pGEM3X DNA to yield final amounts of 7.5 μg of DNA per 6-cm-diameter plate. Flow cytometric analysis with MAb F1a was performed with duplicate samples. Spectrofluorometry assays were performed as described in Materials and Methods. Individual curves shown are representative of three independent experiments. (B) Spectrofluorimetry and flow cytometric analysis were performed as for panel A except that SV5 HN DNA was substituted for pTF7.5 HA DNA. (C) vTF7-3-infected CV-1 cells were transfected with varying amounts of HA (A/Udorn/72 [H3 subtype]) DNA and pGEM3X DNA to yield a final amount of 7.5 μg per 6-cm-diameter plate. Flow cytometric analysis with MAb D6/1 was performed on duplicate samples; results are shown as a percentage of the value for the highest-expressing sample.

FIG. 6

Examination of fusion by confocal microscopy. Six-centimeter-diameter dishes of vTF7-3-infected CV-1 cells, either with or without coverslips, were transfected with 2.5 μg of SV5 HN DNA and increasing amounts of SV5 F DNA and pGEM3X DNA to yield a final amount of 7.5 μg of DNA. Flow cytometric analysis using MAb F1a was performed in duplicate on plates lacking coverslips. Confocal microscopy analysis was performed as described in Materials and Methods. (A) Representative portions (one-quarter of complete field) of confocal images at various time points (minutes) for cells expressing either 60 or 100% of the SV5 F-protein surface density observed in an SV5-infected cell at 18 h p.i. (B) Representative confocal images from cells expressing HN alone (no SV5 F) or cells expressing 20% of the SV5 F-protein surface density observed in SV5-infected cells at 18 h p.i.

FIG. 7

Quantitation of fusion by confocal microscopy. The number of fusion events was determined by analysis of images made with a confocal microscope as shown in Fig. 6. The data from the rhodamine channel were used during the counting of fusion events so that the cases where even small amounts of dye spread could be properly quantified. At least three complete fields were counted per time point.