Skip to main content
PMC home page
The Journal of General Virology logoLink to The Journal of General Virology
. 2016 May;97(Pt 5):1066–1076. doi: 10.1099/jgv.0.000415

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

Bevan Sawatsky 1,2,3,4, Dennis A Bente 2,3, Markus Czub 5, Veronika von Messling 1,4,✉
PMCID: PMC7482510  PMID: 26813519

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.

Introduction

The paramyxovirus attachment protein plays a critical role in viral receptor interaction and the activation or triggering of its homologous fusion (F) protein. The attachment proteins of different paramyxoviruses have adapted to a wide spectrum of target receptors, including terminal sialic acids on cell surface glycoproteins for mumps and parainfluenza viruses (Villar & Barroso, 2006), as well as proteinaceous receptors such as signalling lymphocyte activation molecule (SLAM; Tatsuo et al., 2000) and nectin-4 (Mühlebach et al., 2011; Noyce et al., 2011) for morbilliviruses, and ephrin-B2 (Bonaparte et al., 2005; Negrete et al., 2005) and ephrin-B3 (Negrete et al., 2006) for henipaviruses.

Detailed structures have been solved for several paramyxovirus attachment proteins, including the haemagglutinin-neuraminidase (HN) proteins of Newcastle disease virus (Crennell et al., 2000), parainfluenza virus type 5 (Yuan et al., 2005) and human parainfluenza virus type 3 (Lawrence et al., 2004), the haemagglutinin (H) protein of measles virus (MeV; Colf et al., 2007; Hashiguchi et al., 2007) and the glycoproteins (G) of Nipah virus (NiV; Xu et al., 2008) and Hendra virus (HeV; Bowden et al., 2010). These structures reveal a common architecture where the β-sheets of the globular head are arranged symmetrically around a central axis as a six-bladed propeller. The attachment protein-mediated initiation of the F protein rearrangement and subsequent membrane fusion seem to be highly conserved, despite widely diverse receptors (Chang & Dutch, 2012). Interaction with the respective cellular receptors induces conformational changes in the attachment protein globular head (Bose et al., 2012; Liu et al., 2013; Navaratnarajah et al., 2011), which are in turn transferred to the stalk region (Bose et al., 2011; Liu et al., 2013, 2015; Navaratnarajah et al., 2012; Yuan et al., 2011). This F-triggering function of the attachment protein can be dissociated from its other functions, such as intracellular transport, association with F and receptor binding, which indicates that it resides in a distinct region. Indeed, the attachment protein stalk domain is still able to trigger the F protein even when the globular head is removed (Bose et al., 2012, 2014; Brindley et al., 2013; Liu et al., 2013).

Paramyxovirus glycoprotein functions are also regulated by intracellular processes, where the amino-terminal cytoplasmic domain serves as a signal peptide for the translocation of the nascent protein chain into the endoplasmic reticulum (ER) (Spriggs & Collins, 1990), thereby modulating protein maturation and surface transport. While the morbillivirus F and H proteins associate early during biosynthesis in the ER and are transported to the cell surface as a complex (Plemper et al., 2001), the henipavirus F and G proteins are transported independently (Whitman et al., 2009), indicating that differences in interactions between glycoproteins may influence the assembly and cell–cell fusion process. Paramyxovirus HN/H/G and F protein cytoplasmic domains also contain tyrosine motifs, which result in transport of proteins to the basolateral surface of polarized cells (Runkler et al., 2009; Weise et al., 2010). These signals, however, are overridden by the homologous matrix (M) protein, resulting in direction of the glycoproteins to the appropriate cellular compartment for virus assembly (Dietzel et al., 2011; Naim et al., 2000).

Despite the critical role of the cytoplasmic domain, several paramyxovirus attachment proteins tolerate substantial deletions in this domain without their receptor-binding and fusion support properties being adversely affected (Moll et al., 2002; Porotto et al., 2006). To investigate the contribution of the cytoplasmic domain to the different functional aspects of the attachment protein, we characterized cell surface expression and particle assembly support in a series of NiV G and canine distemper virus (CDV) H glycoprotein mutants with heterologous cytoplasmic domain substitutions or progressive deletions in this region.

Results

The NiV G cytoplasmic domain is required for protein expression

The cytoplasmic domains from diverse paramyxoviruses vary widely in length and sequence (Fig. 1), with no obvious common structural elements. To evaluate the extent of functional complementarity, we exchanged the cytoplasmic domains between the CDV H and NiV G glycoproteins (Fig. 2a). The parental FLAG-tagged CDV H and NiV G proteins were readily detected by Western blot, as was the chimera CDV H/Gcyt, whereas the NiV G/Hcyt chimera was very poorly expressed (Fig. 2b). To quantitatively compare the overall and cell surface expression levels of the chimeric glycoproteins, transfected cells were permeabilized or left untreated, stained with the anti-FLAG antibody and analysed by flow cytometry. The expression of the mutant CDV H protein bearing the NiV G cytoplasmic domain (CDV H/Gcyt) was comparable to that of the parental CDV H protein in both cases (Fig. 2c, d). However, the chimera NiV G/Hcyt was again very poorly expressed and was not detected on the cell surface (Fig. 2c, d), which confirms the Western blot results (Fig. 2b), indicating that the NiV G cytoplasmic domain plays a much more important role in protein expression or stability than the corresponding region of CDV H.

Fig. 1.

Fig. 1.

Open in a new tab

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.

Fig. 2.

Open in a new tab

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).

NiV G cannot tolerate exchange of its cytoplasmic domain

Since the fusion-triggering function of paramyxovirus attachment glycoproteins resides in the extracellular domain, fusion support is dependent on correct expression of functional protein on the cell surface (Bose et al., 2012; Liu et al., 2013; Navaratnarajah et al., 2011). We therefore assessed the fusion support function of the parental and cytoplasmic domain exchange mutants in a quantitative fusion assay with the homologous or heterologous F proteins. While CDV H/Gcyt induced levels of fusion similar to those of the parental CDV H protein (Fig. 3a, b), the mutant NiV G/Hcyt protein induced virtually no fusion when it was co-expressed with the homologous NiV F protein (Fig. 3a, c), which is consistent with its low level of surface expression (Fig. 2d). The CDV glycoproteins induced much higher levels of fusion in VerodogSLAMtag cells than the NiV glycoproteins when the absolute levels of luminescence produced in the assay were compared (Fig. 3b, c). VerodogSLAMtag cells stably express high levels of the CDV receptor canine SLAM, but only endogenous levels of the NiV receptor ephrin-B2. Furthermore, the NiV glycoproteins are known to be less fusogenic in most cell lines than the glycoproteins from other paramyxoviruses (Bossart et al., 2002). None of the heterologous glycoprotein combinations CDV F+NiV G and NiV F+CDV H induced fusion, which confirms the specificity of our assay. The cytoplasmic domain of the NiV G protein is thus essential for protein expression, which directly affects fusion support activity, whereas the fusion support function of CDV H is relatively insensitive to substitutions in this region providing protein expression is maintained. This illustrates that the cytoplasmic domain has no direct role in this function of paramyxoviral attachment proteins.

Fig. 3.

Fig. 3.

Open in a new tab

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.

The cytoplasmic domain is not required for CDV H surface expression and fusion support function

To investigate these different tolerances for structural modifications of CDV H and NiV G cytoplasmic domains in more detail, we next generated a series of FLAG-tagged mutants with 10-residue deletions of the respective proteins (Fig. 4a, b). The total amount of protein detected by Western blot and FACS staining for the CDV H cytoplasmic domain deletion mutants was comparable to the parental CDV H (Fig. 4c, d), and yielded similar levels of cell surface expression (Fig. 4e). Overall NiV G expression was generally lower than that of CDV H, and deletion of the first 10 residues of NiV G was sufficient to result in a dramatic reduction of protein levels (Fig. 4f, g). However, NiV GcytΔ10 surface expression was equivalent to that of parental NiV G, suggesting a regulatory function of this region in cell surface transport or protein turnover (Fig. 4h). All further deletions abolished protein expression, demonstrating an essential role for the cytoplasmic domain of NiV G, but not CDV H, in biosynthesis.

Fig. 4.

Fig. 4.

Open in a new tab

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).

We then assessed the CDV H and NiV G cytoplasmic deletion mutants for their ability to promote fusion. All CDV H deletion mutants showed a modest reduction in fusion promotion that was not statistically significant, with the exception of CDV HcytΔ32 (Fig. 4i). Deletion of the first 10 residues of NiV G resulted in a similarly modest reduction in fusion (Fig. 4j), but further deletions in the NiV G cytoplasmic domain significantly reduced fusion to background levels (Fig. 4j). Since the level of fusion promotion corresponds to the amount of protein detected on the cell surface, this indicates that the level of fusion activity is regulated through modulation of the amount of protein on the cell surface.

Efficient CDV particle formation involves interactions of the H protein cytoplasmic domain with the M protein

Interaction between the M protein and the cytoplasmic domain is involved in the incorporation of attachment proteins into viral particles (Cathomen et al., 1998a; Tahara et al., 2007), and many paramyxovirus glycoproteins carry motifs in their cytoplasmic domains that mediate these interactions (Schmitt et al., 1999, 2002; Takimoto et al., 1998; Waning et al., 2002). However, for some paramyxoviruses, expression of the attachment protein alone is sufficient to result in the release of virus-like proteins (VLPs) into the culture supernatant (Diederich et al., 2008; Patch et al., 2007), indicating an intrinsic particle formation capacity of the protein. To investigate the role of the cytoplasmic tails, we analysed the VLP formation upon expression of either the CDV H or NiV G glycoproteins, or the respective deletion mutants, in the absence or presence of the homologous M proteins. We then measured relative incorporation of the attachment proteins into VLPs containing the M protein, and compared these levels to the amount of attachment protein that buds into the culture supernatant in the absence of M. Indeed, co-expression of the CDV M and H proteins resulted in a four- to fivefold enhancement of H incorporation compared with H protein expression alone (Fig. 5a), but this enhancement was completely lost when the first 10 residues of CDV H were deleted (Fig. 5a). There was a slight, albeit non-significant, enhancement of NiV G incorporation into particles when it was co-expressed with NiV M (Fig. 5b). However, there was no difference in NiV G deletion mutant particle incorporation regardless of the presence of NiV M (Fig. 5b). The CDV H protein thus contains a motif in its extreme amino-terminus that enhances its incorporation into particles containing the M protein, while the incorporation of NiV G into VLPs seems to be less dependent on the M protein.

Fig. 5.

Fig. 5.

Open in a new tab

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).

Discussion

In addition to their role in signal transduction, the cytoplasmic domains of surface glycoproteins frequently influence protein processing and transport (Moll et al., 2002; Parks & Lamb, 1990; Spriggs & Collins, 1990; Wilson et al., 1990). Generally, a minimal domain of 10–15 amino acids is required for type II glycoproteins to ensure correct translocation of the nascent protein chain into the ER and subsequent post-translational modifications (Parks & Lamb, 1990; Spriggs & Collins, 1990; Wilson et al., 1990). Here we show that the CDV H cytoplasmic domain is dispensable for protein expression and fusion promotion activity, but has an important role for its incorporation into VLPs. In contrast, the same domain of NiV G is critically important for protein expression and consequently fusion promotion, but has no obvious role in incorporation of the protein into VLPs. The assembly of morbillivirus thus appears to be a highly coordinated process in which the glycoproteins are directed into particles by the M protein, whereas G–M protein interactions do not seem to be important for henipavirus assembly.

Disruption of NiV G cytoplasmic signalling motifs decreases protein expression

The MeV and Sendai virus attachment protein cytoplasmic domains can be reduced to 14 amino acids without loss of function (Moll et al., 2002; Waning et al., 2002), but further deletions result in a considerable decrease of cell surface expression and lack of incorporation into virus particles (Cathomen et al., 1998b). Likewise, using an alternative in-frame start codon, the HeV G protein cytoplasmic domain can be reduced to only 13 residues while still yielding a functional protein that induces fusion more efficiently than the WT protein (Palomares et al., 2013; Porotto et al., 2006). We found that the NiV G protein clearly cannot tolerate deletions of this length, which is largely due to greatly decreased expression of the truncated proteins. Some early studies of henipavirus fusion demonstrated that the homologous HeV glycoproteins fuse more efficiently than the NiV glycoproteins (Bossart et al., 2002), but it has not been clear why this is the case. Since CDV and NiV attachment protein mutants with cytoplasmic domains of 15 residues or fewer lack tyrosine motifs important for post-translational sorting to the basolateral surface of polarized cells (Moll et al., 2001; Runkler et al., 2009; Weise et al., 2010), the resulting loss of appropriate intracellular transport likely disrupts protein homeostasis and decreases protein expression levels.

Independence of CDV H protein expression and fusion support function from the cytoplasmic domain

While the amount of cell–cell fusion observed when paramyxovirus glycoproteins are co-expressed generally corresponds to the relative amounts present on the cell surface, the amount of attachment protein is usually believed to be the more important determinant since it triggers the F protein after reaching the appropriate threshold of binding to the cellular receptor (Hasegawa et al., 2007; Whitman & Dutch, 2007). This held true for both the CDV H and NiV G cytoplasmic domain deletion mutants, where the levels of surface expression corresponded to the overall expression levels of each protein. NiV G was more sensitive to alterations in this domain than CDV H; the expression of CDV H was slightly reduced when deletions were made from the cytoplasmic domain, regardless of the length, while exchange or even deletions of modest length dramatically reduced NiV G expression. CDV H may thus represent an outlier among paramyxoviruses in this respect, since the MeV H protein is affected by cytoplasmic domain deletions in a manner similar to that which we observed for NiV G (Moll et al., 2002).

Incorporation of NiV G into VLPs is non-specific

Consistent with previous reports (Ciancanelli & Basler, 2006; Li et al., 2009; Patch et al., 2007), expression of the CDV and NiV M proteins alone was sufficient for VLP formation. The incorporation of many paramyxovirus glycoproteins into nascent viral particles is a coordinated process and requires interaction with the homologous viral M protein. While both attachment proteins were enriched to varying degrees in VLPs containing either CDV or NiV M, deletion of 10 residues at the amino-terminus of CDV H abolished this enrichment, and the incorporation pattern for CDV H mutants with further deletions was similar to those for VLPs that did not contain the CDV M protein. The morbillivirus H protein therefore has a functional motif at its amino-terminus that directs preferential incorporation into viral particles. While morbillivirus F and H proteins associate very early during biosynthesis (Plemper et al., 2001), F also forms VLPs when co-expressed with M (Cathomen et al., 1998a; Pohl et al., 2007), and recombinant MeVs with partially deleted cytoplasmic tails were viable (Cathomen et al., 1998b), it remains unclear which glycoprotein interacts more strongly with the homologous M protein to drive morbillivirus particle formation. In contrast, henipavirus glycoproteins are transported independently and only associate transiently on the cell surface (Lamp et al., 2013; Whitman & Dutch, 2007; Whitman et al., 2009). Recent studies on VLP formation by the NiV M protein have demonstrated that henipavirus glycoprotein incorporation is a haphazard process, where at least half of the VLPs produced have no surface glycoproteins (Landowski et al., 2014). The M-protein-mediated incorporation of NiV G is also much less efficient than that of the F protein (Landowski et al., 2014), further supporting our findings. In addition, infectious NiV particles are released into the culture supernatant even when the entire M ORF is deleted (Dietzel et al., 2016), demonstrating that the role of the henipavirus M protein in virus assembly is less prominent than for other paramyxoviruses. This implies a greater contribution of the henipavirus glycoproteins to the budding process, suggesting a fundamentally different process of henipavirus assembly compared with other members of this virus family.

Methods

Cells and transfections

Vero E6 (ATCC CRL-1586), CHO-K1 (ATCC CCL-61) and 293T (ATCC CRL-3216) cells and Vero cells stably expressing the canine SLAM protein (VerodogSLAMtag) (von Messling et al., 2003) were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 5 % heat-inactivated FBS (Gibco). For the biochemical analysis of recombinant proteins, 6- or 12-well plates of VerodogSLAMtag or Vero E6 cells were transfected at approximately 90 % confluence as described previously (Sawatsky & von Messling, 2010; Sawatsky et al., 2012). Briefly, each well was transfected with 2 μg of the parental CDV H or NiV G protein expression plasmid, or the respective mutants, using Turbofect (Thermo Fisher). For fusion assays, CDV or NiV F protein expression plasmid containing a C-terminal haemagglutinin tag (HA, YPYDVPDYA) was included in the transfection and fusion activity was evaluated after 24 h. Cells were lysed in 200 μl RIPA buffer [50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 % (v/v) NP-40 and 0.5 % (w/v) sodium deoxycholate] containing protease inhibitor cocktail (Complete; Roche) 48 h after transfection, and the clarified lysate was stored at − 20 °C. For evaluation of protein expression by FACS staining, 293T cells were seeded into six-well dishes and transfected with the respective CDV H or NiV G expression plasmid using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions.

Production of chimeric CDV and NiV glycoproteins

CDV H genes or gene fragments were amplified from the expression plasmid pCG-H5804PZeo (von Messling et al., 2005), while NiV G genes or gene fragments were amplified from the plasmid pczCFG5-NiV G (Sawatsky et al., 2007) and cloned into the BamHI and SphI sites of the pCG1-IRESzeomut expression plasmid (von Messling et al., 2003). FLAG epitope tags (DYKDDDDK) were introduced into the 3′ primer immediately upstream of the stop codon of each gene to facilitate detection (Plemper et al., 2001). FLAG tags were also introduced into the carboxy-termini of all subsequent CDV H and NiV G mutants made for this study. The cytoplasmic domain exchange genes for the mutants CDV H/Gcyt and NiV G/Hcyt were produced by overlapping PCR and assembled before cloning into the BamHI and SphI sites of pCG1-IRESzeomut. CDV H and NiV G cytoplasmic domain truncation mutants were produced by PCR amplification using primers in which blocks of 10 residues in the N-terminal region immediately downstream of the start codon were progressively deleted. The CDV M (5804P strain) gene was amplified from the genomic plasmid p5804PeGFPH (von Messling et al., 2004), while the NiV M (Malaysia strain) gene was amplified from purified viral RNA. The 3′ primers for the NiV M gene contained the coding sequence for the c-myc epitope tag (EQKLISEEDL) immediately upstream of the stop codon (Salditt et al., 2010), while the corresponding primer for CDV M contained no tag. The CDV F gene was amplified from the expression plasmid pCG-F5804PZeo (von Messling et al., 2005), while the NiV F genes or gene fragments were amplified from the plasmid pczCFG5-NiV F (Sawatsky et al., 2007). The 3′ primers for the CDV and NiV F expression plasmids both contained the coding sequence for the haemagglutinin epitope tag (HA, YPYDVPDYA).

FACS staining

Six-well dishes of 293T cells were transfected with the respective NiV G and CDV H mutant expression plasmids as detailed above. Cells were treated with 0.05 % trypsin–EDTA 48 h after transfection, resuspended in DMEM containing 10 % FBS and then transferred to centrifuge tubes and washed with PBS. For total protein staining, cell pellets were treated with Cytofix/Cytoperm (GE Healthcare) for 30 min at 4 °C, and were then washed with PBS. Mouse anti-FLAG M2 primary (Sigma) and goat anti-mouse Alexa Fluor 488 secondary antibodies (Thermo Fisher) were used. For cell surface staining, cell pellets were first stained with the mouse anti-FLAG M2 antibody, and then fixed with 4 % paraformaldehyde before incubation with the secondary antibody.

Quantitative fusion assays

Confluent monolayers of VerodogSLAMtag cells in a six-well dish were transfected with 5 μg per well pCAGGS-T7RNAP, which expresses the bacteriophage T7 DNA-dependent RNA polymerase (T7RNAP) under the control of the chicken β-actin promoter. The transfected cells were trypsinized 18 h later and pooled for use as target cells in the assay. In parallel, VerodogSLAMtag cells were seeded into 24-well dishes at 40–50 % confluence. These cells were transfected with 1 μg per well of plasmid that expresses the firefly luciferase under the control of the T7RNAP promoter (pTM1-ffLuc) along with 0.5 μg per well of expression plasmid for either the CDV or NiV FHA proteins, and 0.5 μg per well of expression plasmid for either CDV H, NiV G or the respective cytoplasmic domain exchange and truncation mutants. After incubation for 6 h at 37 °C, 105 T7RNAP-expressing target cells per well were added to the 24-well dishes and were incubated at 37 °C for 24 h. The cells were lysed with 100 μl per well Cell Culture Lysis Reagent (Promega) at room temperature, transferred to fresh Eppendorf tubes, and briefly centrifuged at 20 000 g to pellet cell debris. The clarified supernatant was used for the reporter assay. Each sample (20 μl) was read in duplicate in black clear-bottom 96-well plates using the Luciferase Assay System (Promega) according to the manufacturer's recommendations. Luminescence was read on an Infinite M200Pro automated plate reader (Tecan).

VLPs

VLPs were produced based on a protocol previously used to purify NiV VLPs (Ciancanelli & Basler, 2006). Confluent monolayers of CHO-K1 cells in six-well plates were transfected with 3 μg CDV M or NiV Mmyc protein expression plasmid and 3 μg of the respective FLAG-tagged CDV or NiV glycoprotein expression plasmid. All VLP experiments were incubated at 32 °C. Control wells were transfected with either M or H/G expression plasmid and an equal amount of empty pCG1-IRESzeomut vector. For glycoprotein-only VLPs, CHO-K1 cells in six-well plates were transfected with 3 μg of the respective FLAG-tagged glycoprotein expression plasmids. The transfection medium was removed after 16 h and replaced with fresh DMEM supplemented with 5 % FBS. At 48 h post-transfection, cells and medium were resuspended and centrifuged at 250 g for 20 min to pellet cellular material. CDV M+H VLPs were incubated for 72 h. The pellet was washed once with PBS and then lysed in 250 μl RIPA buffer. The clarified culture supernatant was overlaid onto 20 % sucrose cushions in TNE buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA) and centrifuged for 2 h at 4 °C in a Beckman SW-41Ti rotor at 30 500 r.p.m. (approximately 160  000 g). The supernatant was aspirated and the VLPs were resuspended in 50 μl 1 ×  SDS-gel loading buffer (GLB) containing 10 % β-mercaptoethanol per tube. An equal volume of 2 ×  SDS-GLB containing 20 % β-mercaptoethanol was mixed with the cellular lysate before separation in parallel on polyacrylamide gels. The NiV Mmyc and CDV M proteins were detected using monoclonal anti-myc clone 9E10 (Millipore) or anti-measles M protein antibodies (MAB8910; Millipore), respectively, and a monoclonal goat anti-mouse secondary antibody conjugated to HRP (Southern Biotech). The FLAG-tagged glycoproteins were detected with an anti-FLAG antibody directly conjugated to HRP. β-Actin was detected in cell lysates using an anti-actin antibody directly conjugated to HRP (mAbcam 8226; Abcam).

Acknowledgements

This paper is dedicated to the late Ron Sawatsky, father of Bevan Sawatsky. We thank Chantal Thibault for technical support, Alexander McAuley for assistance with the luminometer, Yvonne Krebs for FACS staining and all members of the laboratory for constructive discussions and comments on the manuscript. This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (MOP-66989), the Natural Sciences and Engineering Research Council of Canada (NSERC) (1255375-10), the Canadian Foundation for Innovation (CFI) (9488) and the German Federal Ministry of Health to V. v. M., and UTMB start-up funds for D. A. B. The funding agencies had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

References

  1. Bonaparte M. I., Dimitrov A. S., Bossart K. N., Crameri G., Mungall B. A., Bishop K. A., Choudhry V., Dimitrov D. S., Wang L.-F., other authors Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc Natl Acad Sci U S A. 2005;102:10652–10657. doi: 10.1073/pnas.0504887102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bose S., Welch B. D., Kors C. A., Yuan P., Jardetzky T. S., Lamb R. A. Structure and mutagenesis of the parainfluenza virus 5 hemagglutinin-neuraminidase stalk domain reveals a four-helix bundle and the role of the stalk in fusion promotion. J Virol. 2011;85:12855–12866. doi: 10.1128/JVI.06350-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bose S., Zokarkar A., Welch B. D., Leser G. P., Jardetzky T. S., Lamb R. A. Fusion activation by a headless parainfluenza virus 5 hemagglutinin-neuraminidase stalk suggests a modular mechanism for triggering. Proc Natl Acad Sci U S A. 2012;109:E2625–E2634. doi: 10.1073/pnas.1213813109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bose S., Song A. S., Jardetzky T. S., Lamb R. A. Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells. J Virol. 2014;88:3925–3941. doi: 10.1128/JVI.03741-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bossart K. N., Wang L.-F., Flora M. N., Chua K. B., Lam S. K., Eaton B. T., Broder C. C. Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins. J Virol. 2002;76:11186–11198. doi: 10.1128/JVI.76.22.11186-11198.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowden T. A., Crispin M., Harvey D. J., Jones E. Y., Stuart D. I. Dimeric architecture of the Hendra virus attachment glycoprotein: evidence for a conserved mode of assembly. J Virol. 2010;84:6208–6217. doi: 10.1128/JVI.00317-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brindley M. A., Suter R., Schestak I., Kiss G., Wright E. R., Plemper R. K. A stabilized headless measles virus attachment protein stalk efficiently triggers membrane fusion. J Virol. 2013;87:11693–11703. doi: 10.1128/JVI.01945-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cathomen T., Mrkic B., Spehner D., Drillien R., Naef R., Pavlovic J., Aguzzi A., Billeter M. A., Cattaneo R. A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain. EMBO J. 1998a;17:3899–3908. doi: 10.1093/emboj/17.14.3899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cathomen T., Naim H. Y., Cattaneo R. Measles viruses with altered envelope protein cytoplasmic tails gain cell fusion competence. J Virol. 1998b;72:1224–1234. doi: 10.1128/jvi.72.2.1224-1234.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chang A., Dutch R. E. Paramyxovirus fusion and entry: multiple paths to a common end. Viruses. 2012;4:613–636. doi: 10.3390/v4040613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ciancanelli M. J., Basler C. F. Mutation of YMYL in the Nipah virus matrix protein abrogates budding and alters subcellular localization. J Virol. 2006;80:12070–12078. doi: 10.1128/JVI.01743-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Colf L. A., Juo Z. S., Garcia K. C. Structure of the measles virus hemagglutinin. Nat Struct Mol Biol. 2007;14:1227–1228. doi: 10.1038/nsmb1342. [DOI] [PubMed] [Google Scholar]
  13. Crennell S., Takimoto T., Portner A., Taylor G. Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nat Struct Biol. 2000;7:1068–1074. doi: 10.1038/81002. [DOI] [PubMed] [Google Scholar]
  14. Diederich S., Thiel L., Maisner A. Role of endocytosis and cathepsin-mediated activation in Nipah virus entry. Virology. 2008;375:391–400. doi: 10.1016/j.virol.2008.02.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dietzel E., Anderson D. E., Castan A., von Messling V., Maisner A. Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence. J Virol. 2011;85:7162–7168. doi: 10.1128/JVI.00051-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dietzel E., Kolesnikova L., Sawatsky B., Heiner A., Weis M., Kobinger G. P., Becker S., von Messling V., Maisner A. Nipah virus matrix protein influences fusogenicity and is essential for particle infectivity and stability. J Virol. 2016;90:2514–2522. doi: 10.1128/JVI.02920-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hasegawa K., Hu C., Nakamura T., Marks J. D., Russell S. J., Peng K.-W. Affinity thresholds for membrane fusion triggering by viral glycoproteins. J Virol. 2007;81:13149–13157. doi: 10.1128/JVI.01415-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hashiguchi T., Kajikawa M., Maita N., Takeda M., Kuroki K., Sasaki K., Kohda D., Yanagi Y., Maenaka K. Crystal structure of measles virus hemagglutinin provides insight into effective vaccines. Proc Natl Acad Sci U S A. 2007;104:19535–19540. doi: 10.1073/pnas.0707830104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lamp B., Dietzel E., Kolesnikova L., Sauerhering L., Erbar S., Weingartl H., Maisner A. Nipah virus entry and egress from polarized epithelial cells. J Virol. 2013;87:3143–3154. doi: 10.1128/JVI.02696-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Landowski M., Dabundo J., Liu Q., Nicola A. V., Aguilar H. C. Nipah virion entry kinetics, composition, and conformational changes determined by enzymatic virus-like particles and new flow virometry tools. J Virol. 2014;88:14197–14206. doi: 10.1128/JVI.01632-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lawrence M. C., Borg N. A., Streltsov V. A., Pilling P. A., Epa V. C., Varghese J. N., McKimm-Breschkin J. L., Colman P. M. Structure of the haemagglutinin-neuraminidase from human parainfluenza virus type III. J Mol Biol. 2004;335:1343–1357. doi: 10.1016/j.jmb.2003.11.032. [DOI] [PubMed] [Google Scholar]
  22. Li M., Schmitt P. T., Li Z., McCrory T. S., He B., Schmitt A. P. Mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles. J Virol. 2009;83:7261–7272. doi: 10.1128/JVI.00421-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu Q., Stone J. A., Bradel-Tretheway B., Dabundo J., Benavides Montano J. A., Santos-Montanez J., Biering S. B., Nicola A. V., Iorio R. M., other authors Unraveling a three-step spatiotemporal mechanism of triggering of receptor-induced Nipah virus fusion and cell entry. PLoS Pathog. 2013;9:e1003770. doi: 10.1371/journal.ppat.1003770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu Q., Bradel-Tretheway B., Monreal A. I., Saludes J. P., Lu X., Nicola A. V., Aguilar H. C. Nipah virus attachment glycoprotein stalk C-terminal region links receptor binding to fusion triggering. J Virol. 2015;89:1838–1850. doi: 10.1128/JVI.02277-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moll M., Klenk H.-D., Herrler G., Maisner A. A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells. J Biol Chem. 2001;276:17887–17894. doi: 10.1074/jbc.M010183200. [DOI] [PubMed] [Google Scholar]
  26. Moll M., Klenk H.-D., Maisner A. Importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses. J Virol. 2002;76:7174–7186. doi: 10.1128/JVI.76.14.7174-7186.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mühlebach M. D., Mateo M., Sinn P. L., Prüfer S., Uhlig K. M., Leonard V. H. J., Navaratnarajah C. K., Frenzke M., Wong X. X., other authors Adherens junction protein nectin-4 is the epithelial receptor for measles virus. Nature. 2011;480:530–533. doi: 10.1038/nature10639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Naim H. Y., Ehler E., Billeter M. A. Measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells. EMBO J. 2000;19:3576–3585. doi: 10.1093/emboj/19.14.3576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Navaratnarajah C. K., Oezguen N., Rupp L., Kay L., Leonard V. H. J., Braun W., Cattaneo R. The heads of the measles virus attachment protein move to transmit the fusion-triggering signal. Nat Struct Mol Biol. 2011;18:128–134. doi: 10.1038/nsmb.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Navaratnarajah C. K., Negi S., Braun W., Cattaneo R. Membrane fusion triggering: three modules with different structure and function in the upper half of the measles virus attachment protein stalk. J Biol Chem. 2012;287:38543–38551. doi: 10.1074/jbc.M112.410563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Negrete O. A., Levroney E. L., Aguilar H. C., Bertolotti-Ciarlet A., Nazarian R., Tajyar S., Lee B. EphrinB2 is the entry receptor for Nipah virus, an emergent deadly paramyxovirus. Nature. 2005;436:401–405. doi: 10.1038/nature03838. [DOI] [PubMed] [Google Scholar]
  32. Negrete O. A., Wolf M. C., Aguilar H. C., Enterlein S., Wang W., Mühlberger E., Su S. V., Bertolotti-Ciarlet A., Flick R., Lee B. Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus. PLoS Pathog. 2006;2:e7. doi: 10.1371/journal.ppat.0020007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Noyce R. S., Bondre D. G., Ha M. N., Lin L.-T., Sisson G., Tsao M.-S., Richardson C. D. Tumor cell marker PVRL4 (nectin 4) is an epithelial cell receptor for measles virus. PLoS Pathog. 2011;7:e1002240. doi: 10.1371/journal.ppat.1002240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Palomares K., Vigant F., Van Handel B., Pernet O., Chikere K., Hong P., Sherman S. P., Patterson M., An D. S., other authors Nipah virus envelope-pseudotyped lentiviruses efficiently target ephrinB2-positive stem cell populations in vitro and bypass the liver sink when administered in vivo . J Virol. 2013;87:2094–2108. doi: 10.1128/JVI.02032-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parks G. D., Lamb R. A. Defective assembly and intracellular transport of mutant paramyxovirus hemagglutinin-neuraminidase proteins containing altered cytoplasmic domains. J Virol. 1990;64:3605–3616. doi: 10.1128/jvi.64.8.3605-3616.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Patch J. R., Crameri G., Wang L.-F., Eaton B. T., Broder C. C. Quantitative analysis of Nipah virus proteins released as virus-like particles reveals central role for the matrix protein. Virol J. 2007;4:1. doi: 10.1186/1743-422X-4-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Plemper R. K., Hammond A. L., Cattaneo R. Measles virus envelope glycoproteins hetero-oligomerize in the endoplasmic reticulum. J Biol Chem. 2001;276:44239–44246. doi: 10.1074/jbc.M105967200. [DOI] [PubMed] [Google Scholar]
  38. Pohl C., Duprex W. P., Krohne G., Rima B. K., Schneider-Schaulies S. Measles virus M and F proteins associate with detergent-resistant membrane fractions and promote formation of virus-like particles. J Gen Virol. 2007;88:1243–1250. doi: 10.1099/vir.0.82578-0. [DOI] [PubMed] [Google Scholar]
  39. Porotto M., Doctor L., Carta P., Fornabaio M., Greengard O., Kellogg G. E., Moscona A. Inhibition of Hendra virus fusion. J Virol. 2006;80:9837–9849. doi: 10.1128/JVI.00736-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Runkler N., Dietzel E., Carsillo M., Niewiesk S., Maisner A. Sorting signals in the measles virus wild-type glycoproteins differently influence virus spread in polarized epithelia and lymphocytes. J Gen Virol. 2009;90:2474–2482. doi: 10.1099/vir.0.012575-0. [DOI] [PubMed] [Google Scholar]
  41. Salditt A., Koethe S., Pohl C., Harms H., Kolesnikova L., Becker S., Schneider-Schaulies S. Measles virus M protein-driven particle production does not involve the endosomal sorting complex required for transport (ESCRT) system. J Gen Virol. 2010;91:1464–1472. doi: 10.1099/vir.0.018523-0. [DOI] [PubMed] [Google Scholar]
  42. Sawatsky B., von Messling V. Canine distemper viruses expressing a hemagglutinin without N-glycans lose virulence but retain immunosuppression. J Virol. 2010;84:2753–2761. doi: 10.1128/JVI.01813-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sawatsky B., Grolla A., Kuzenko N., Weingartl H., Czub M. Inhibition of Henipavirus infection by Nipah virus attachment glycoprotein occurs without cell-surface downregulation of ephrin-B2 or ephrin-B3. J Gen Virol. 2007;88:582–591. doi: 10.1099/vir.0.82427-0. [DOI] [PubMed] [Google Scholar]
  44. Sawatsky B., Wong X.-X., Hinkelmann S., Cattaneo R., von Messling V. Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression. J Virol. 2012;86:3658–3666. doi: 10.1128/JVI.06414-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schmitt A. P., He B., Lamb R. A. Involvement of the cytoplasmic domain of the hemagglutinin-neuraminidase protein in assembly of the paramyxovirus simian virus 5. J Virol. 1999;73:8703–8712. doi: 10.1128/jvi.73.10.8703-8712.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schmitt A. P., Leser G. P., Waning D. L., Lamb R. A. Requirements for budding of paramyxovirus simian virus 5 virus-like particles. J Virol. 2002;76:3952–3964. doi: 10.1128/JVI.76.8.3952-3964.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Spriggs M. K., Collins P. L. Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein. J Cell Biol. 1990;111:31–44. doi: 10.1083/jcb.111.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tahara M., Takeda M., Yanagi Y. Altered interaction of the matrix protein with the cytoplasmic tail of hemagglutinin modulates measles virus growth by affecting virus assembly and cell-cell fusion. J Virol. 2007;81:6827–6836. doi: 10.1128/JVI.00248-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Takimoto T., Bousse T., Coronel E. C., Scroggs R. A., Portner A. Cytoplasmic domain of Sendai virus HN protein contains a specific sequence required for its incorporation into virions. J Virol. 1998;72:9747–9754. doi: 10.1128/jvi.72.12.9747-9754.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tatsuo H., Ono N., Tanaka K., Yanagi Y. SLAM (CDw150) is a cellular receptor for measles virus. Nature. 2000;406:893–897. doi: 10.1038/35022579. [DOI] [PubMed] [Google Scholar]
  51. Villar E., Barroso I. M. Role of sialic acid-containing molecules in paramyxovirus entry into the host cell: a minireview. Glycoconj J. 2006;23:5–17. doi: 10.1007/s10719-006-5433-0. [DOI] [PubMed] [Google Scholar]
  52. von Messling V., Springfeld C., Devaux P., Cattaneo R. A ferret model of canine distemper virus virulence and immunosuppression. J Virol. 2003;77:12579–12591. doi: 10.1128/JVI.77.23.12579-12591.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. von Messling V., Milosevic D., Cattaneo R. Tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system. Proc Natl Acad Sci U S A. 2004;101:14216–14221. doi: 10.1073/pnas.0403597101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. von Messling V., Oezguen N., Zheng Q., Vongpunsawad S., Braun W., Cattaneo R. Nearby clusters of hemagglutinin residues sustain SLAM-dependent canine distemper virus entry in peripheral blood mononuclear cells. J Virol. 2005;79:5857–5862. doi: 10.1128/JVI.79.9.5857-5862.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Waning D. L., Schmitt A. P., Leser G. P., Lamb R. A. Roles for the cytoplasmic tails of the fusion and hemagglutinin-neuraminidase proteins in budding of the paramyxovirus simian virus 5. J Virol. 2002;76:9284–9297. doi: 10.1128/JVI.76.18.9284-9297.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weise C., Erbar S., Lamp B., Vogt C., Diederich S., Maisner A. Tyrosine residues in the cytoplasmic domains affect sorting and fusion activity of the Nipah virus glycoproteins in polarized epithelial cells. J Virol. 2010;84:7634–7641. doi: 10.1128/JVI.02576-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Whitman S. D., Dutch R. E. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: role for Hendra G protein trafficking and degradation. Virology. 2007;363:419–429. doi: 10.1016/j.virol.2007.01.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Whitman S. D., Smith E. C., Dutch R. E. Differential rates of protein folding and cellular trafficking for the Hendra virus F and G proteins: implications for F-G complex formation. J Virol. 2009;83:8998–9001. doi: 10.1128/JVI.00414-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wilson C., Gilmore R., Morrison T. Aberrant membrane insertion of a cytoplasmic tail deletion mutant of the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. Mol Cell Biol. 1990;10:449–457. doi: 10.1128/MCB.10.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Xu K., Rajashankar K. R., Chan Y. P., Himanen J. P., Broder C. C., Nikolov D. B. Host cell recognition by the henipaviruses: crystal structures of the Nipah G attachment glycoprotein and its complex with ephrin-B3. Proc Natl Acad Sci U S A. 2008;105:9953–9958. doi: 10.1073/pnas.0804797105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yuan P., Thompson T. B., Wurzburg B. A., Paterson R. G., Lamb R. A., Jardetzky T. S. Structural studies of the parainfluenza virus 5 hemagglutinin-neuraminidase tetramer in complex with its receptor, sialyllactose. Structure. 2005;13:803–815. doi: 10.1016/j.str.2005.02.019. [DOI] [PubMed] [Google Scholar]
  62. Yuan P., Swanson K. A., Leser G. P., Paterson R. G., Lamb R. A., Jardetzky T. S. Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk. Proc Natl Acad Sci U S A. 2011;108:14920–14925. doi: 10.1073/pnas.1111691108. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Virology are provided here courtesy of Microbiology Society

RESOURCES