Live-cell imaging shows that NiV-G increases axonal transport of NiV-F.

<p>(A–C) Single-frame images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s006" target="_blank">Video S1</a> (thin upper images) and kymographs (bottom images) of the analysis of particles moving along 100 µm of axons in DIV10 rat hippocampal neurons co-transfected on DIV5 with plasmids encoding either NiV-F-GFP (NiV-F) and mCh-Tub (Tub) (panel A), NiV-G-mCh (NiV-G) and GFP (panel B) or NiV-F-GFP and NiV-G-mCh (panel C). Images in (A) and (B) are shown in grayscale. In panel (C), NiV-F-GFP and NiV-G-mCh fluorescence are shown individually in grayscale and as green and red, respectively, in merged images (yellow indicates co-localization). Tracings with negative and positive slopes in kymographs represent anterograde and retrograde movement of particles, respectively; vertical lines represent particles that are stationary during the 60 s recording. (D) Quantification of NiV-F-GFP and NiV-G-mCh axonal transport in neurons expressing these proteins individually or in combination. Data shown represent the number of anterograde (Ant), stationary (Stat) and retrograde (Ret) particles per 100 µm of axon length during the 60 s recording. NiV-F and NiV-G (green and red bars, respectively) show the number of axonal particles containing these two proteins when expressed individually. “NiV-F (+NiV-G)” (light green bars) is the number of NiV-F-GFP particles in neurons co-expressing NiV-G-mCh; “NiV-G (+NiV-F)” (salmon bars) represents the number of NiV-G-mCh particles in neurons co-expressing NiV-F-GFP. Values are the means±SEM of 20–22 independent measurements for each condition and represent the total number of particles (n_p) indicated under the graph. Statistical significance was calculated by one-way ANOVA followed by Dunnett's test. (*) <i>P</i><0.01 when compared to NiV-F-GFP-containing particles in cells expressing only this protein. (E) Single-frame images from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s007" target="_blank">Video S2</a> showing dendritic particles from neurons co-expressing either NiV-F-GFP and mCh-Tub, NiV-G-mCh and GFP, or NiV-F-GFP and NiV-G-mCh (upper panels in grayscale; bottom panel with merged NiV-F-GFP and NiV-G-mCh images in green and red, respectively). The lower color image is a 4× magnification of the boxed area in the above image; color arrows point to particles containing either NiV-F-GFP or NiV-G-mCh (green and red, respectively) or both proteins (yellow). Scale bar: 10 µm. (F) Co-localization of NiV-F-GFP and NiV-G-mCh in axonal and dendritic particles of co-transfected neurons. NiV-F-GFP co-localization in co-transfected neurons (NiV-F (+NiV-G), light green bars) was the percentage of NiV-F-GFP-containing particles that also contained NiV-G-mCh. Similarly, NiV-G-mCh co-localization (NiV-G (+NiV-F), salmon bars) was the percentage of NiV-G-mCh-containing particles that also displayed NiV-F-GFP. Values are the means±SEM of 22 and 33 measurements of axonal and dendritic particles, respectively, and represent the total number of axonal and dendritic particles (n_pA and n_pD) containing NiV-F-GFP and NiV-G-mCherry indicated under the graph.</p

Interaction of the NiV-F cytosolic tail with AP μ subunits.

<p>(A) Scheme of heterotetrameric adaptor protein (AP) complexes depicting the four subunits in each complex along with subunit isoforms. Combinatorial assembly of subunits can originate multiple forms of AP-1, AP-2 and AP-3 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Mattera2" target="_blank">[56]</a>. (B) Y2H analysis showing interaction of the NiV-F cytosolic tail, but not the NiV-G cytosolic tail, with μ1A, μ1B, μ2, μ3A and μ4. Growth of yeast co-transformants on −His plates is indicative of interactions between tail constructs subcloned in a Gal4 binding domain (BD) vector and μ subunits subcloned in a Gal4 activation domain (AD) vector; growth on +His plates is a control for growth/loading of co-transformants. The TGN38 cytosolic tail was used as a positive control for interaction with various μ subunits. Co-transformations of cytosolic tail constructs with SV40 T-Ag and of μ subunits with p53 were used as negative controls. Co-transformation of p53 and SV40 T-Ag constructs provided an additional positive control for interactions. Images are composites of panels from the same experiments and are representative of three independent experiments. (C) Y2H analysis showing that alanine substitution of Y525 or L528 in the YXXØ-based signal or combined substitution of Y542 and Y543 inhibits the interaction of the NiV-F cytosolic tail with μ subunits. Experiments were performed as in panel B.</p

Somatodendritic sorting of NiV-F mediated by its cytosolic tail.

<p>(A) Schematic representation of NiV-F and NiV-G indicating the NIV-F fusion peptide (FP), transmembrane (M) and cytosolic (C) domains, and amino-acid sequences of the cytosolic domains. The F₂-F₁ active form of NiV-F generated by cathepsin L- or B-mediated cleavage of the F₀ inactive precursor is stabilized by a disulfide (S-S) bridge <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Pager1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Diederich2" target="_blank">[30]</a>. Utilized N-linked glycosylation sites <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Moll1" target="_blank">[26]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Biering1" target="_blank">[28]</a> are indicated by branched lines. The YXXØ motif (YSRL, residues 525–528) and a YY pair (residues 542 and 543) in the NiV-F tail are highlighted in red. (B–E) Rat hippocampal neurons were co-transfected on DIV4 with plasmids encoding NiV-F-GFP, NiV-G-HA, Tac or Tac-NiV-F, and mCherry-tubulin (mCh-Tub, marker of both dendrites and axons), fixed on DIV10, and immunostained with rabbit anti-MAP2 and goat anti-ankyrin-G (ANK-G) (to identify dendrites and the AIS, respectively) and with mouse anti-HA or mouse anti-Tac antibodies (to visualize NiV-G-HA or Tac-based constructs). Cells were imaged by confocal microscopy. Grayscale images correspond to NiV-F-GFP fluorescence (B), anti-HA (C) or anti-Tac (D, E) staining. Merged color pictures at the bottom of all panels display mCh-Tub fluorescence (red) and anti-MAP2 (green) staining (axons appear red due to mCh-Tub labeling, while dendrites are yellow due to co-labeling by mCh-Tub and MAP2). Insets show anti-ANK-G labeling (AIS shown in cyan). The AIS and axons are marked by cyan and red arrowheads, respectively. Scale bar: 20 µm. Quantitative analysis of NiV-F and NiV-G polarized sorting was performed through calculation of the dendrite/axon (D/A) polarity index (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-t001" target="_blank">Table 1</a>).</p

Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

<div><p>Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons.</p></div

Binding sites on μ subunits involved in interactions with the NiV-F tail.

<p>(A) Surface representation of the three-dimensional structure of the μ1A C-terminal domain (residues 157–423) (PDB ID 1W63; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Heldwein1" target="_blank">[57]</a>) exhibiting potential binding sites for YXXØ (A-site, red) and YX[FYL][FL][E] (B-site, blue) signals on opposite sides of the molecule. The potential A-site in μ1A was mapped by sequence alignment with μ2 and μ3A, and based on the structure of the complexes between the C-terminal domains of μ2 or μ3A and YXXØ signal-containing peptides <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Owen1" target="_blank">[37]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Mardones1" target="_blank">[38]</a>. Mapping of the potential B-site in μ1A was based on sequence alignment with μ4 and on the structure of the C-terminal domain of this subunit in complex with a YKFFE-containing peptide <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Burgos1" target="_blank">[39]</a>. Model images were built with PyMOL <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-DeLano1" target="_blank">[58]</a>. (B) Effect of alanine substitutions in critical residues of μ subunits A- and B-sites on the interaction with the NiV-F cytosolic tail. Residues in the putative A-sites of μ1A and μ4 were defined by homology with the residues in the corresponding sites of μ2 and μ3A <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Owen1" target="_blank">[37]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Mardones1" target="_blank">[38]</a>. Similarly, residues in the putative B-sites of μ1A, μ2 and μ3A were defined by homology with those in the cognate site of μ4 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Burgos1" target="_blank">[39]</a>. Numbering of μ2 residues corresponds to the variant containing 433 residues (NCBI Reference Sequence NP_001020376). Residues whose substitution resulted in a strong inhibition in NiV-F tail binding are labeled in red (A-site) or dark blue (B-sites); substitutions causing a weaker inhibition are labeled in magenta (A-site) or light blue (B-site). Details of the Y2H analysis are as in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-g003" target="_blank">Figure 3 B–C</a>.</p

Effect of dominant-negative mutants of μ subunits on somatodendritic sorting of NiV-F.

<p>(A–D) Rat hippocampal neurons were co-transfected with plasmids encoding NiV-F-GFP and wild-type (WT) or dominant-negative (DN) mutants of HA-tagged μ subunits (A-site mutants of μ1A, μ2, μ3A and μ4, and B-site mutant of μ4, as labeled on top of images). The effects of μ1A, μ2, μ3A and μ4 constructs are shown in panels A, B, C and D, respectively. Cells were immunostained with mouse anti-HA, rabbit anti-MAP2 and goat anti-ANK-G, and imaged as indicated in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-g001" target="_blank">Figure 1</a>. Grayscale images shown in all panels correspond to NiV-GFP fluorescence. The anti-HA, anti-MAP2 and anti-ANK-G staining is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s002" target="_blank">Figure S2</a>. Two images corresponding to different effects on NiV-F-GFP sorting (representing ∼75 and 25% of the neuronal population) observed following expression of μ2 D174A/W419S are shown in (B). The AIS and axons are marked by cyan and red arrowheads, respectively. Scale bar: 20 µm. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-t001" target="_blank">Table 1</a> for polarity indexes.</p

NiV-F and NiV-G exhibit different rates of biosynthetic trafficking.

<p>HEK293T cells transfected with NiV-F-AU1 (A) or NiV-G-HA (B) were pulse-labeled for 15 min and chased for the indicated times; this was followed by lysis, immunoprecipitation with anti-epitope antibodies and treatment in the presence or absence of endo H. Samples were subjected to SDS-PAGE under reducing conditions and autoradiography. (A) NiV-F₀ displays four utilized sites of N-linked glycosylation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Moll1" target="_blank">[26]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Aguilar1" target="_blank">[27]</a> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-g001" target="_blank">Figure 1A</a>) some of which become endo H-resistant while others remain endo H-sensitive upon transport through the Golgi complex. Golgi processing is evidenced by the appearance of a lower mobility band from 0.5 h of chase onwards in lanes with undigested immunoprecipitates. Treatment of the Golgi-processed form of NiV-F₀ with endo H results in an intermediate mobility shift (apparent from 0.5 h of chase onwards) referred to as “F₀ resistant”, while digestion of the unprocessed form results in a larger shift referred to as “F₀ sensitive” (visible from time 0 of chase onwards) (corresponding lines at right of panel). The NiV-F₁ form, whose production depends on transport of newly synthesized NiV-F₀ to the plasma membrane followed by endocytosis and cleavage in acidic endosomes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Diederich1" target="_blank">[20]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Pager1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Diederich2" target="_blank">[30]</a>, was apparent after 1 h of chase and exhibited partial resistance to endo H (bands indicated by bracket at right of panel). The NiV-F₂ fragment was not detected because the AU1 epitope is located at the C-terminus of the tagged protein (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s004" target="_blank">Figure S4A</a> for NiV-F cleavage scheme). (B) NiV-G displays six utilized sites of N-linked glycosylation <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Biering1" target="_blank">[28]</a> (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-g001" target="_blank">Figure 1A</a>). The position of endo H-resistant and -sensitive forms of NiV-G is shown by brackets. Note the slower production of endo H-resistant NiV-G compared to that of endo H-resistant NiV-F₀ shown in panel A. Data in panels A and B are representative of two independent experiments where NiV-F-AU1- and NiV-G-HA-transfected cells were pulsed, chased and processed simultaneously. The position of molecular mass markers (in kDa) is shown at left. (C) Densitometric analysis of autoradiograms in panels A and B was performed with Image J software (<a href="http://rsbweb.nih.gov/ij" target="_blank">http://rsbweb.nih.gov/ij</a>). Results shown represent the percentage of endo H resistance (100×endo H resistant forms/endo H resistant forms+endo H sensitive forms) at different times of chase (only the F₀ form was considered in the quantitative analysis of NiV-F).</p

Proposed regulation of NiV-F neuronal sorting by NiV-G.

<p>NiV-F (F₀ inactive precursor) and NiV-G (G) exhibit relatively fast and slow biosynthetic trafficking, respectively (full and dashed lines in scheme). In the absence of G, F₀ is first sorted to the somatodendritic plasma membrane and then internalized to early/recycling endosomes located in the soma where it undergoes proteolytic activation (generation of F_2,1). This allows for efficient activation of NiV-F within the somatodendritic domain. The interaction between active F_2,1 and newly synthesized G (exhibiting delayed trafficking out of the TGN) alters the sorting of the fusion glycoprotein and results in the presence of NiV-F•NiV-G complexes (F_2,1G) not only in dendrites but also in the axonal compartment. Although depolarization of NiV-F by NiV-G may also occur when AP-2-dependent endocytosis is affected (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s005" target="_blank">Figure S5</a>), the endocytosis-dependent proteolytic activation of NiV-F <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Diederich1" target="_blank">[20]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Pager1" target="_blank">[29]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Diederich2" target="_blank">[30]</a> and the different rates of biosynthetic trafficking makes endosomes the most plausible site of formation of NiV-F•NiV-G complexes. Localization of NiV-F•NiV-G complexes to axons may facilitate transneuronal spread of NiV. Differential sorting of NiV-F in the presence or absence of NiV-G may coordinate NiV-F processing, axonal transport and anterograde spread between neurons.</p

Interaction with NiV-G abolishes somatodendritic sorting of NiV-F.

<p>(A) Co-immunoprecipitation of NiV-F and NiV-G. HEK293T cells were co-transfected with NiV-F-GFP and NiV-G-HA; approximately 24–27 h post-transfection, cells were lysed and subjected to immunoprecipitation (IP) with rabbit anti-HA. Antigen-antibody complexes were eluted from beads and subjected to SDS-PAGE under reducing conditions and immunoblotting (IB) with either mouse anti-HA or mouse anti-GFP (upper and lower panels at right, respectively). Samples of cell lysates were also subjected to SDS-PAGE and immunoblotting (left panels). Full and dashed lines at right of bottom panels show the mobility of the NiV-F₀ and -F₁ forms (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107.s004" target="_blank">Figure S4</a> for cleavage scheme). Co-IP of the NiV-F₂ fragment with NiV-G could not be evaluated because the GFP moiety is fused to the C-terminus of NiV-F. The position of molecular mass markers (in kDa) is shown at left of blots. (B) Rat hippocampal neurons were co-transfected with NiV-F-GFP and NiV-G-HA, immunostained with mouse anti-HA, rabbit anti-MAP2 and goat anti-ANK-G, and imaged by confocal microscopy. Upper panels show NiV-F-GFP fluorescence (left) and anti-HA staining (right). The left lower panel shows anti-MAP2 (magenta) and anti-ANK-G (cyan) staining; the right lower panel displays merged images of NiV-F-GFP fluorescence (green) and anti-HA immunostaining (red) (yellow indicates co-localization). Cyan and white arrowheads show the position of the AIS and axons, respectively. Scale bar: 20 µm. Co-transfection with NiV-F and NiV-G constructs resulted in the frequent appearance of fused neurons (typically 2–3 cells) consistent with the role of these glycoproteins in cell-to-cell fusion <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Lamp1" target="_blank">[15]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Moll1" target="_blank">[26]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat.1004107-Aguilar1" target="_blank">[27]</a>. Only isolated neurons co-transfected with NiV-F and NiV-G, showing unaltered distribution of MAP2 and ANK-G markers, were selected for quantitative analysis of polarity of the two glycoproteins (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#ppat-1004107-t001" target="_blank">Table 1</a>).</p

Quantification of NiV-F and NiV-G sorting into somatodendritic and axonal domains of rat hippocampal neurons.

<p>Dendrite to axon (D/A) polarity indexes were calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004107#s4" target="_blank">Materials and Methods</a>. Values are expressed as mean <b>±</b> SD (<i>n</i>) (<i>n</i>; number of cells analyzed).</p><p>The notation NiV-F-GFP (+NiV-G-HA) refers to the NiV-F-GFP polarity index in cells co-expressing NiV-G-HA. The same applies to the NiV-G-HA (+NiV-F-GFP) notation.</p><p>Statistical significance for all groups including NiV-F-GFP and NiV-G-HA constructs was calculated by one-way ANOVA followed by Dunnett's test.</p><p>(*)<i>P</i><0.01 when compared to NiV-F-GFP. Significance between group pairs including Tac or NiV-F-GFP Δ104–109 constructs was calculated by Student's <i>t</i>-test.</p>(§)<p><i>P</i><0.01 when compared to Tac;</p>(‡)<p><i>P</i><0.01 when compared to NiV-F-GFP Δ104–109.</p