Effect of ERdj5 and PDI overexpression on cell-cell fusion. (A) Syncytia were visualized by the immunofluorescence of cells using anti-NDV antibody. Cells transfected with empty vector or cotransfected with HN and F protein cDNAs with or without ERdj5 or PDI cDNA were processed for immunofluorescence at 48 h posttransfection. (B) Average sizes of syncytia after transfection with HN and F protein cDNAs (HN+F), HN, F, and ERdj5 cDNAs (HN+F+E), or HN, F, and PDI cDNAs (HN+F+P) were determined by counting the number of nuclei per syncytium in 20 different fusion areas at 24, 48, and 72 h posttransfection. The results shown are the averages of three independent experiments, with the error bars indicating standard deviations. (C) Effects of the overexpression of ERdj5 and PDI on content mixing were assayed using a ß-galactosidase reporter assay. Effector cells cotransfected with HN and F protein cDNAs as well as with pTA were mixed with target cells (transfected with pB1-G with or without ERdj5 or PDI). Content mixing between effector and target cells was quantified by measuring ß-galactosidase in cell extracts and is shown as a percentage of the activity detected in cells transfected with HN and F cDNAs (HN+F). The results shown are the averages of three independent experiments, and the error bars indicate the range.

Effect of ERdj5 and PDI overexpression on the production of free thiols in F protein when expressed without (A) or with (B) HN protein. Cells transfected with empty vector, F protein cDNA, or HN and F protein cDNAs with or without ERdj5 or PDI cDNA were labeled with MPB as described in Materials and Methods. Proteins in resulting cell extracts were precipitated with neutravidin-agarose (lanes 1 to 4) and resolved by SDS-PAGE. Total F protein in cell extracts (lanes 5 to 8) was resolved as a control for the expression of the proteins. The quantification of F₁ labeled by MPB is shown at the bottom of each panel and was performed in films with protein bands in the linear range.

Effect of ERdj5 and PDI overexpression on the binding of conformation-sensitive antibodies to F protein on cell surfaces. (A, C, and E) Cells transfected with pSVL vector (top panels) or with pSVL-HN and pSVL-F (bottom panels) were processed for surface immunofluorescence using anti-Fu1a antibody, anti-HR1 antibody, and anti-NDV antibody, respectively (indicated on left). Column 2 shows cells transfected with ERdj5 cDNA along with pSVL or pSVL-HN and pSVL-F, and column 3 shows cells transfected with PDI cDNA along with pSVL or pSVL-HN and pSVL-F. (B, D, and F) Quantification of mean fluorescence intensities of the cells shown in panels A, C, and E, respectively. The results shown are averages of the mean fluorescence intensity of 10 different cells and are normalized for background fluorescence by subtracting the mean fluorescence of cells transfected with empty vector. Error bars represent the range of fluorescence intensity of 10 different cells within one experiment, and the experiment shown is representative of three similar experiments.

Overexpression of ERdj5 and PDI and its effect on HN and F protein expression and cell viability. (A) Proteins in extracts of COS-7 cells, transfected with empty vector or cDNA encoding ERdj5 (top) or PDI (bottom), were resolved by SDS-PAGE and analyzed by Western blotting. The blots, with actin as a loading control, are shown below each panel. Numbers at bottom of each panel represent the expression of the respective proteins in transfected cells as a percentage of expression in untransfected cells. The quantification was performed in films with protein bands in the linear range. (B) Cells, untransfected (UT) or at 48 h after transfection with ERdj5 or PDI cDNA and with or without F and HN protein cDNA, were incubated with MTT for 3 h at 37°C. The blue metabolic product of MTT, measured as the optical density at 570 nm, is represented as a percentage of the value obtained for untransfected cells. The values represented are averages from three different experiments, and error bars represent the ranges. (C) Cells were transfected with empty vector (lanes 2 and 6) or were cotransfected with HN and F protein cDNAs (lanes 3 and 7) or HN and F protein with ERdj5 cDNA (lanes 4 and 8) or with PDI cDNA (lanes 5 and 9). Surface HN protein (top) or F protein (bottom), biotinylated using sulfo-NHS-SS-biotin, was precipitated with neutravidin-agarose (lanes 6 to 9), and total F or HN protein in the extracts (lanes 2 to 5) was resolved by SDS-PAGE and analyzed by Western blotting using anti-F protein antibody (anti-HR2) or anti-HN protein antibody (anti-AS). The amount of the total extract loaded represents one-third of the amount of extract used to precipitate biotinylated surface proteins. Lane 1 shows the infected cell extract that was used as a marker. (D) Surface expression of PDI. Cells were transfected with increasing amounts of cDNAs encoding PDI and, 48 h post transfection, cells were incubated with sulfo-NHS-SS-biotin. Biotinylated cell surface proteins in aliquots of the resulting cell extracts were precipitated with neutravidin (lanes 2, 4, 6, 8, and 10). Lanes 1, 3, 5, 7, and 9 show protein precipitated with equivalent amounts of agarose coupled to protein G as a negative control. PDI in the precipitates was detected with anti-PDI antibody in a Western blot. Top panel, lanes 1 and 2, no PDI cDNA; lanes 3 and 4, 0.25 μg PDI cDNA; lanes 5 and 6, 0.5 μg PDI cDNA; lanes 7 and 8, 0.75 μg PDI cDNA; and lanes 9 and 10, 1 μg PDI cDNA. Bottom panel, the detection of actin in each extract.

Effect of inhibitors of PDI isomerases on cell-cell fusion in cells overexpressing ERdj5 or PDI. The effects of DTNB (A), anti-PDI antibody (B), or anti-ERdj5 antibody(C) were assayed using a ß-galactosidase reporter assay, as described in Materials and Methods. Effector cells cotransfected with HN and F protein cDNAs as well as pTA were mixed with target cells (transfected with pB1-G with or without ERdj5 or PDI) in the presence of DTNB (A), anti-PDI antibody (B), or anti-ERdj5 antibody (C). ß-Galactosidase activity in cell extracts represents content mixing and is shown as a percentage of the activity detected in cells transfected only with HN and F cDNAs (HN+F). The results represent the average of three independent experiments with the error bars representing the range.

Binding of conformation-sensitive antibodies to F protein on cell surfaces in the presence of MPB or neuraminidase (NAase). (A, C, and E) Cells transfected with pSVL vector (top panels) or with pSVL-HN and pSVL-F (bottom panels) were processed for surface immunofluorescence using anti-Fu1a antibody, anti-HR1 antibody, and anti-NDV antibody, respectively (indicated on the left). Column 1 shows cells without any treatment, and columns 2 and 3 show cells treated with MPB and neuraminidase, respectively, as described in Materials and Methods. (B, D, and F) Quantification of the fluorescence intensities of the cells shown in panels A, C, and E, respectively. The results shown are averages of the mean fluorescence intensity of 10 different cells and are normalized for background fluorescence by subtracting the mean fluorescence of cells transfected with empty vector. Error bars represent the range of fluorescence intensity of 10 different cells within one experiment, and the experiment shown is representative of three similar experiments.

Model for the role of free thiols in NDV F protein. (A) F protein is present in a prefusion, metastable conformation with intact disulfide bonds. Disulfide bonds are cleaved by PDI-like isomerase, resulting in free thiols in F protein (B) before the major conformational changes in F protein required for fusion (C). MPB binds to the free thiols in F protein, and the binding of MPB prevents further conformational changes and the fusion of membranes. Anti-Fu1a antibody binds to F protein in the prefusion state, while anti-HR1 antibody binds preferentially to the activated or postfusion form of F protein. The enhanced expression of PDI or ERdj5 favors the conformation shown in panel C at steady state. S-S, disulfide bond; SH, free or reduced cysteine.