NSvc2-C hydrophobic fusion-loop motifs are required for cell-cell membrane fusion.

(A) Schematic diagrams showing recombinant NSvc2-N and NSvc2-C. Baculovirus gp64 signal peptide was used to replace the original signal peptide at the N-terminus of NSvc2-N or NSvc2-C. (B) Immunofluorescence labeling of the recombinant NSvc2-N or NSvc2-C in Sf9 cells. The Sf9 cells were infected with baculoviruses expressing the recombinant NSvc2-N or NSvc2-C (MOI = 5). The infected cells were probed with the NSvc2-N or NSvc2-C specific antibody at 48 h post infection followed by a TRITC fluorescence-labeled secondary antibody. Bar, 10 μm. (C–F) Fusogenic activity assays using Sf9 cells expressing the recombinant NSvc2-N, NSvc2-C or both (NSvc2-N + NSvc2-C). The Sf9 cells were infected with individual recombinant baculoviruses (MOI = 5). At 48 h post transfection, the growth medium was replaced with a PBS, pH 5.0, for 2 min and then changed back to the normal growth medium for 4 h. Formation of syncytium was observed under a microscope. (G) The number of cells per syncytium was counted under the inverted microscope. The experiment was repeated three times. **, p < 0.01 by the student t-test. (H) Sequence alignment using the two fusion loop sequences (FL1 and FL2) from the NSvc2-C of five different tenuiviruses [RSV (NC_003754.1), maize stripe virus (MSV; U53224.1), rice hoja blanca virus (RHBV; L54073.1), Iranian wheat stripe virus (IWSV; AY312434.1) and rice grassy stunt virus (RGSV; KF438676.1)]. Red residues are highly conserved and yellow residues are semi-conserved. Residues indicated with asterisks are hydrophobic residues and were selected for site-directed mutagenesis. The resulting mutants were tested for their abilities to induce cell-cell membrane fusion. (I) Two fusion loops (FL1 and FL2, green) were found at the top of the 3D-structure model of NSvc2-C. Three different domains are shown in three different colors, and the boxed region of the 3D-structure is enlarged and shown on the right side. Locations of the hydrophobic residues are shown. (J and K) Analyses of fusogenic activities caused by the WT or mutant NSvc2-C. Sf9 cells were infected with the recombinant baculoviruses expressing the WT or mutant NSvc2-C for membrane fusion assays (J). The number of cells per syncytium was counted and analyzed (K). The experiment was repeated three times. **, p < 0.01 by the student t-test. Fig 6C–6F and 6J are bright field light microscopic images.