Morbillivirus and henipavirus attachment protein cytoplasmic domains differently affect protein expression, fusion support and particle assembly

Abstract

The amino-terminal cytoplasmic domains of paramyxovirus attachment glycoproteins include trafficking signals that influence protein processing and cell surface expression. To characterize the role of the cytoplasmic domain in protein expression, fusion support and particle assembly in more detail, we constructed chimeric Nipah virus (NiV) glycoprotein (G) and canine distemper virus (CDV) haemagglutinin (H) proteins carrying the respective heterologous cytoplasmic domain, as well as a series of mutants with progressive deletions in this domain. CDV H retained fusion function and was normally expressed on the cell surface with a heterologous cytoplasmic domain, while the expression and fusion support of NiV G was dramatically decreased when its cytoplasmic domain was replaced with that of CDV H. The cell surface expression and fusion support functions of CDV H were relatively insensitive to cytoplasmic domain deletions, while short deletions in the corresponding region of NiV G dramatically decreased both. In addition, the first 10 residues of the CDV H cytoplasmic domain strongly influence its incorporation into virus-like particles formed by the CDV matrix (M) protein, while the co-expression of NiV M with NiV G had no significant effect on incorporation of G into particles. The cytoplasmic domains of both the CDV H and NiV G proteins thus contribute differently to the virus life cycle.

Fig. 1.

Variability of paramyxovirus attachment glycoprotein cytoplasmic domains. Cytoplasmic domains of selected viruses from officially classified genera in the subfamily Paramyxovirinae. Sequences are shown from the amino-terminus up to the carboxy-terminal transmembrane boundary, indicated on the right. Accession numbers: Nipah virus (NiV) G Malaysia strain, AF212302; Hendra virus (HeV) G, AF017149; canine distemper virus (CDV) H 5804P strain, AY386316; measles virus (MeV) H IC-B strain, NC_001498; rinderpest virus (RPV) H Kabete O strain, NC_006296; peste-des-petits ruminants virus (PPRV) H Tu/00 strain, NC_006383; Sendai virus (SeV) HN Ohita strain, NC_001552; human parainfluenza virus type 1 (HPIV1) HN Washington/1964 strain, AF457102; human parainfluenza virus type 3 (HPIV3) HN, NC_001796; mumps virus (MuV) HN Jeryl Lynn strain, AF201473; parainfluenza virus 5 (PIV5) HN W3A strain, NC_006430; human parainfluenza virus type 2 (HPIV2) HN Toshiba strain, NC_003443; human parainfluenza virus type 4 (HPIV4) Toshiba strain, M34033; and Newcastle disease virus (NDV) HN LaSota strain, AF077761.

Fig. 2.

Expression of chimeric glycoproteins with heterologous cytoplasmic domains. (a) Chimeric glycoproteins constructed for this study. NiV G and CDV H are shown, as are NiV G with the CDV H cytoplasmic domain (NiV G/Hcyt), and CDV H with the NiV G cytoplasmic domain (CDV H/Gcyt). The vertical bar represents the boundary between the cytoplasmic and transmembrane domains. (b) Detection of CDV H, NiV G and the chimeric glycoproteins CDV H/Gcyt and NiV G/Hcyt by Western blot. 293T cells were transfected with control expression plasmids or the respective mutants, and were lysed 48 h after transfection. Proteins were detected with an anti-FLAG M2 antibody directly coupled to HRP. (c, d) Cell surface and total cellular expression of CDV H, NiV G and the chimeric glycoproteins CDV H/Gcyt and NiV G/Hcyt. 293T cells transfected with control expression plasmids and the respective mutants were either permeabilized for total protein (b) or fixed for cell surface (c), then stained with anti-FLAG M2 antibody followed by anti-mouse Alexa Fluor 488 secondary antibody. The mean fluorescence intensities for positively stained cell populations are shown. Bars on graphs represent the mean of a sample group and error bars represent sem. Groups were analysed by one-way ANOVA with Tukey post-test. *** P < 0.001; ns, not significant. Statistical significance is indicated on the graphs compared to CDV H cellular expression in (b) and surface expression in (c).

Fig. 3.

Fusion support activity of CDV H, NiV G and the chimeric glycoproteins CDV H/Gcyt and NiV G/Hcyt. (a) Phase-contrast microscopy of cells transfected with either CDV or NiV F in combination with CDV H, NiV G or chimeric glycoprotein expression plasmid. Pictures were taken at 24 h post-transfection at a magnification of × 100. (b, c) Quantitative fusion assay. Semi-confluent monolayers of VerodogSLAMtag cells were independently transfected in triplicate with firefly luciferase plasmid and either CDV or NiV F in combination with CDV H, NiV G or chimeric glycoprotein expression plasmid as described in Methods. After 6 h, the transfected cells were overlaid with 10⁵ cells per well VerodogSLAMtag cells transfected with T7 RNA polymerase expression plasmid and incubated at 37 °C for 24 h. Cells were lysed and the luciferase signal was read in duplicate for each sample. Graphs show luminescence signals from fusion mediated by CDV F (b) or NiV F (c). Bars on graphs represent the mean of a sample group and error bars represent sem. Groups were analysed by one-way ANOVA with Tukey post-test. *** P < 0.001; ns, not significant. Statistical significance is indicated on the graphs in (b) compared to CDV H, and in (c) compared to NiV G.

Fig. 4.

Expression of cytoplasmic domain deletion mutants. (a, b) Schematic diagrams of (a) CDV H and (b) NiV G cytoplasmic domain deletion mutants constructed for this study. Sequences are shown from the amino-terminus up to the carboxy-terminal transmembrane boundary on the right. Tyrosine residues that act or may potentially act as targeting or signalling motifs are shown in black boxes to highlight which residues have been deleted from constructs containing shorter domains. (c–h) Total cellular and cell surface expression of CDV H, NiV G and the CDV H (c–e) or NiV G (f–h) cytoplasmic deletion mutants. (c, f) Detection of CDV H (c), NiV G (f) and the respective deletion mutants by Western blot. 293T cells were transfected with control expression plasmids or the respective mutants, and were lysed 48 h after transfection. Proteins were detected with an anti-FLAG M2 antibody directly coupled to HRP. Blots are representative of four replicates. FACS analysis was performed with 293T cells transfected with control expression plasmids and the respective mutants, which were either permeabilized for total protein (d, g) or fixed for cell surface (e, h), then stained with anti-FLAG M2 antibody followed by anti-mouse Alexa Fluor 488 secondary antibody. The mean fluorescence intensities for positively stained cell populations are shown. (i, j) Cell–cell fusion of WT and cytoplasmic domain deletion mutant glycoproteins. Semi-confluent monolayers of VerodogSLAMtag cells were independently transfected in triplicate with firefly luciferase plasmid and either CDV in combination with CDV H and the respective deletion mutants (i), or NiV F in combination with NiV G and the respective deletion mutants (j). After 6 h, the transfected cells were overlaid with 10⁵ cells per well VerodogSLAMtag cells transfected with T7 RNA polymerase (T7RNAP) expression plasmid and incubated at 37 °C for 24 h. Cells were lysed and the luciferase signal was read in duplicate for each sample. Graphs show luminescence signals from fusion mediated by CDV F (i) or NiV F (j). Bars on graphs represent the mean of a sample group and error bars represent sem. Groups were analysed by one-way ANOVA with Tukey post-test. * P < 0.05; ** P < 0.01; ns, not significant. Statistical significance is indicated on the graphs compared with CDV H in (d) and (e), compared to NiV G in (g) and (h), compared to CDV F+H in (i), and compared to NiV F+G in (j).

Fig. 5.

Quantification of glycoprotein incorporation into VLPs. Comparison of glycoprotein-only and glycoprotein+matrix VLPs and lysates from CHO-K1 cells transfected with either CDV H and M, and the respective mutants (a) or NiV G and M, and the respective mutants (b). Western blots were performed as described in Methods, and the signals from the lysate and VLP fractions from each replicate were normalized to either CDV H or NiV G expressed alone and are presented as the ratio between the signals in the VLPs and the lysate, and represent three independent replicates. White bars represent glycoprotein incorporation into VLPs in the absence of homologous M protein, and black bars represent glycoprotein incorporation into VLPs when the homologous M protein is expressed. Groups were analysed by one-way ANOVA with Tukey post-test. ** P < 0.01; ns, not significant. Statistical significance is indicated on the graphs compared with CDV M+H in (a), while no statistically significant differences were found in (b).