The Cytoplasmic Tail of Ovine Herpesvirus 2 Glycoprotein B Affects Cell Surface Expression and Is Required for Membrane Fusion

Abstract

Ovine herpesvirus 2 (OvHV-2) causes the fatal veterinary disease malignant catarrhal fever (MCF). Fusion is an essential step in the host cell entry of enveloped viruses and is an important target for vaccine development. OvHV-2 cannot be propagated in vitro, so a robust virus-free cell-cell membrane fusion assay is necessary to elucidate its entry mechanism. OvHV-2 cell-cell fusion requires three conserved herpesviral envelope glycoproteins: gB, gH, and gL. OvHV-2 fusion activity is detectable but low. We hypothesize that enhancing the cell surface expression of gB, which is the core herpesviral fusogen, will increase cell-cell fusion. We generated C-terminal truncation mutants of gB and determined their cell surface expression, subcellular distribution, and fusion activity. Two mutants, including one that lacked the entire cytoplasmic tail domain, failed to function in the cell-cell fusion assay, despite wild-type levels of surface expression. This suggests that the OvHV-2 gB cytoplasmic tail is critical for fusion. A gB mutant truncated at amino acid 847 showed increased surface expression and fusion relative to the wild type. This suggests that the robust fusion activity of gB847 is the result of increased surface expression. gB847 may be used in place of wild-type gB in an improved, more robust OvHV-2 fusion assay.

Keywords: fusion; glycoprotein B; ovine herpesvirus 2; surface expression.

Figure 1

Schematics of OvHV-2 gB and truncation mutants. (A) Amino acid sequence of the C-terminus of OvHV-2 glycoprotein B. The predicted transmembrane domain (bold) spans amino acids 741–760. Two potential tyrosine-based endocytosis signaling motifs are shaded in grey. (B) Schematic representation of gB truncation mutants. Mutants are denoted by the amino acid length of the construct. TM, transmembrane domain (violet), cytoplasmic tail (pink).

Figure 2

Structural models of OvHV-2 gB in (A) pre-fusion and (B) post-fusion conformations. Homology models were derived using SWISS-MODEL based on available published structures (see Materials and Methods). Domain I (cyan) is predicted to span residues 114–326; domain II (green) is predicted to span residues 103–113 and 327–418; domain III (yellow) is predicted to span residues 83–102 and 454–518; domain IV (orange) is predicted to span residues 518–625; domain V (red) is predicted to span residues 626–684; the membrane proximal region (dark green) is predicted to span residues 713–740; the transmembrane domain (violet) is predicted to span residues 741–760; and the cytoplasmic domain (pink) is predicted to span residues 761–863. TM, transmembrane. CTD, cytoplasmic domain.

Figure 3

Expression and detection of OvHV-2 gB truncation mutants. Transfected CHO-K1 cell lysates were analyzed using (A) conventional (denaturing and reducing conditions) or (B) “native” SDS-PAGE, followed by western blot with anti-V5 or anti-OvHV-2 gB monoclonal antibodies F1.2A or F2.15E. Tubulin was used as a loading control in the middle and right panels of (A). Equal volumes of cell lysate were loaded for each lane. Molecular weight markers in kilodaltons are indicated to the left. EV, empty vector.

Figure 4

Microscopic detection of OvHV-2 gB truncation mutants in cells. Transfected CHO-K1 cells expressing (A) gBWT, (B) gB847, (C) gB775, or (D) gB760, or (E) those transfected with pJP007 empty vector (EV), were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton-X 100. OvHV-2 gB monoclonal antibody F1.2A was added and then detected with Alexa Fluor 647-conjugated goat anti-mouse antibody. Nuclei were counterstained with DAPI (overlay, bottom row). Magnification, 40×; scale bar, 20 µm.

Figure 5

Total and surface expressions of OvHV-2 gBWT and the truncation mutants gB847, gB775, and gB760. Transfected CHO-K1 cells were fixed in 3% formaldehyde and either (A) permeabilized with 0.003% Triton-X 100 or (B) left untreated. Anti-gB antibodies F1.2A and F2.15E were added, followed by a fluorescent secondary antibody. The median fluorescence intensity (MFI) of gBWT was set to 100%. (C) Correlation of total and surface expressions of gBWT and each truncated mutant. Pearson correlation coefficient; r = 0.82, r² = 0.68. Results are the mean of three independent experiments. *, p < 0.05; **, p < 0.01; One-way ANOVA with Dunnett’s test was used for multiple comparisons.

Figure 6

(A) Effect of OvHV-2 gB cytoplasmic tail length on fusion. Transfected CHO-K1 effector cells expressing gBWT, gB847, gB775, or gB760 with gH, gL, and T7 polymerase were co-cultured for 18 h with A113 target cells transfected with the luciferase plasmid. Luciferase-induced luminescence was detected as an indicator of fusion. Luminescence of gBWT fusion was set to 100%. Results are the means of three independent experiments. *, p < 0.05; **, p < 0.01; One-way ANOVA with Šídák’s multiple-comparison test. (B) Relative surface expression and fusion activity of OvHV-2 gB mutants. (C) Correlation of surface expression and fusion of gBWT and each truncated mutant. Pearson correlation coefficient: r = 0.97, r² = 0.94.