Journal: The Journal of Biological Chemistry

Article Title: Functional Interaction between Paramyxovirus Fusion and Attachment Proteins * S⃞

doi: 10.1074/jbc.M801018200

Figure Lengend Snippet: CDV F-ODP but not CDV F-Lederle is triggered by MV H. A, microphotographs of Vero-dogSLAM cells co-transfected with equal amounts of plasmid DNA encoding MV or CDV glycoproteins as specified. The cells were photographed at a magnification of 200× after incubation at 37 °C for seven to 11 h. Mock infected cells expressed only the H protein. B, quantification of cell-to-cell fusion activity using a firefly luciferase reporter-based fusion assay. The values reflect the average luciferase activities of at least three independent experiments ± S.D. per glycoprotein combination and are expressed as the percentages of activity measured for MV F and the respective H. C, CDV F glycoprotein variants show different strengths of interaction with MV H. Co-immunoprecipitation of CDVF-ODP and Lederle with MV H. The lysates of co-transfected cells were subjected to immunoprecipitation using specific antibodies directed against an epitope in the MV H ectodomain. Co-precipitated F (upper panel) was detected in comparison with F present in the lysates prior to precipitation (lower panel) by immunoblotting using a specific antiserum directed against an epitope in the cytosolic tail of CDV F.

Article Snippet: Introducing this region from F-Lederle into F-ODP resulted in an ODP chimera that entirely lost its ability to form functional fusion complexes with MV H, whereas, importantly, productive homotypic interaction with CDV H was fully maintained ( , constructs XII and XIII ).

Techniques: Transfection, Plasmid Preparation, Incubation, Infection, Activity Assay, Luciferase, Single Vesicle Fusion Assay, Immunoprecipitation, Comparison, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Functional Interaction between Paramyxovirus Fusion and Attachment Proteins * S⃞

doi: 10.1074/jbc.M801018200

Figure Lengend Snippet: Identification of a minimal domain responsible for productive interaction of CDV F variants with MV H. A, quantification of cell-to-cell fusion activity of reciprocal chimeras between CDV F-ODP and Lederle upon co-transfection with MV H (black bars) or CDV H (gray bars) using the luciferase reporter-based fusion assay as outlined in Fig. 1B. The values were normalized for fusion activity observed upon co-transfection of cells with unmodified F-ODP and MV H or CDV H, respectively. The mean values ± S.D. of three independent experiments are shown, and the extent of differential triggering (based on the percentage of homotypic activity/percentage of heterotypic activity) is specified (see text for details). F constructs are schematized below the graph. The black boxes represent regions derived from F-Lederle, and the gray boxes represent F-ODP. The location of characteristic F protein domains, the site of the disulfide bridge linking the F1 and F2 subunits (black line), and the position of natural (BlpI, PvuII, EcoRV, and SmaI) and engineered (BglII and KpnI, in gray) restriction sites used for chimera generation are shown on the left. PS, N-terminal precursor sequence of CDV F; FP, fusion peptide; HR-A, N-terminal heptad repeat; HR-B, C-terminal heptad repeat; TM, transmembrane domain; CT, cytosolic tail. B, focused chimeras based on engineered restriction sites identify a domain in the N-terminal part of F-ODP (between restriction sites KpnI and EcoRV, construct XIII) to determine productive interaction of F-ODP with MV H. Quantification of fusion activity, calculation of differential triggering and color coding as described in A.

Article Snippet: Introducing this region from F-Lederle into F-ODP resulted in an ODP chimera that entirely lost its ability to form functional fusion complexes with MV H, whereas, importantly, productive homotypic interaction with CDV H was fully maintained ( , constructs XII and XIII ).

Techniques: Activity Assay, Cotransfection, Luciferase, Single Vesicle Fusion Assay, Construct, Derivative Assay, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Functional Interaction between Paramyxovirus Fusion and Attachment Proteins * S⃞

doi: 10.1074/jbc.M801018200

Figure Lengend Snippet: Four point mutations disrupt productive interaction of CDV F-ODP with MV H. A, amino acid sequence alignment of the identified fragment between the KpnI and EcoRV sites of F-Lederle and ODP. The black letters indicate residues that differ between the strains. The fusion peptide (red) and the relative position of the PvuII site are shown. A vertical line marks the furin cleavage site. B, quantitative comparison of fusion activity of individual F-ODP point mutants. The values are expressed as percentages of fusion activity observed upon co-transfection with unmodified F-ODP and either MV or CDV H. The averages of four experiments ± S.D. and the extent of differential triggering are shown. C, visualization of the identified residues in a structural model of the prefusion CDV F-ODP trimer. In the left panel, all four residues (164, 219, 233, and 317) are highlighted in red. Residues 164, 219, and 233 are predicted to be surface-exposed; residue 317 is predicted to be completely buried in the trimer (visible only in the ribbon models; center panel, side view; right panel, top view). For clarity, residues 164, 219, and 233 are not highlighted in the ribbon models. D, co-immunoprecipitation of selected F-ODP mutant constructs with MV H reveals a limited reduction in co-precipitation efficiency as compared with unmodified F-ODP. The experimental conditions were the same as described for Fig. 1C. The values below the blot represent averages of densitometric quantification of co-precipitated F1 material from two independent experiments. They are corrected for the amount of F1 material present in cell lysates prior to precipitation, which serves as internal standard (lower panel), and are expressed as percentage of co-precipitated CDV F1-ODP.

Article Snippet: Introducing this region from F-Lederle into F-ODP resulted in an ODP chimera that entirely lost its ability to form functional fusion complexes with MV H, whereas, importantly, productive homotypic interaction with CDV H was fully maintained ( , constructs XII and XIII ).

Techniques: Sequencing, Comparison, Activity Assay, Cotransfection, Residue, Immunoprecipitation, Mutagenesis, Construct