NiV-M clusters organize into dome-like structures

The intracellular protein NiV-M underneath the plasma membrane is thought to direct the assembly and budding process of NiV VLPs7. It is known that the expression of M alone is sufficient for the production of NiV VLPs4. To study the organization of M, pig kidney fibroblast cells (PK13) expressing NiV-M with green fluorescent protein (GFP) at the N-terminus were fixed, permeablized, and immunostained with Alexa Fluor 647. Three-dimensional (3D) fluorophore localizations were carried out using a home-built SMLM with dual focal planes which allows an imaging depth of several microns while using the fiducial markers on the coverslip for real-time 3D drift correction17. Forty thousand images were acquired at 45 Hz to reconstruct a SMLM image. During image acquisition, sample drift was controlled within 1 nm (root mean square, RMS) in the lateral directions and 3 nm in the axial direction17. The fluorophore localization precisions were ~10 nm (standard deviation, SD) laterally and 30 nm axially17,18. Images taken through the middle of the cell (position 1 in Fig. 1a) show distinct M puncta with a diameter of 20–40 nm inside the cell (Fig. 1b). However, images at the plasma membrane (position 2 in Fig. 1a) reveal clusters with diameters of a few hundred nanometers (Fig. 1c). Our findings provide in situ evidence showing that M proteins further assemble at the plasma membrane, which is consistent with the self-assembly model proposed for the human metapneumovirus M in the presence of 1,2-dioleoyl-sn-glycero-3-phosphocholine in solution19.

Fig. 1 Distribution of NiV-M in PK13 cells. PK13 cells expressing NiV-M were fixed, permeabilized, and labeled with goat anti-GFP and anti-goat Alexa Fluor 647 antibodies. a Schematic illustration of the imaging planes of a cell. b x–y cross section (100 nm thick in z) of a region through the middle (position 1 in a) of a representative cell showing the M clusters. Scale bar: 1 μm. c x–y cross section (100 nm thick in z) of a region at the plasma membrane (position 2 in a) of a representative cell showing larger M clusters. Scale bar: 1 μm. d x–y cross section of a region (position 3 in a) of a cell showing a representative dome-like structure formed by M. Scale bar: 1 μm. e z-stacks of the x–y cross section of the dome-like structure boxed in d. Scale bar: 0.1 μm. f 3D surface reconstructed by using the M localizations in e, with M localization density projected on the 3D surface. A higher brightness indicates a higher localization density. One representative cell image out of three independent experiments (n ≥ 30) is shown Full size image

We observed that some M clusters were organized into dome-like structures at the plasma membrane. An example is shown in the boxed region in Fig. 1d on the dorsal surface of the cell (position 3 in Fig. 1a). These dome-like structures could not be observed at the ventral surface of the cell in contact with the coverslip. The z-stacks of the dome-like structure are shown in Fig. 1e, and the 3D surface reconstructed by using the M localizations is shown in Fig. 1f. The organization of M on these dome-like structures was fragmented, not continuous. This is in agreement with the recent cryo-electron microscopy studies showing fragmented M patches in the virions of Newcastle Disease Virus20.

NiV glycoproteins form clusters on the plasma membrane

To investigate the organization of the viral envelope glycoproteins on the plasma membrane, we used FLAG-tagged F21 and hemagglutinin (HA)-tagged G22 constructs (Supplementary Fig. 1). PK13 cells expressing either F or G were fixed at 24 h post transfection, immunostained for F or G via tags, and imaged at the dorsal surface of the cell without permeabilization (position 3 in Fig. 1a). F formed distinct clusters (Fig. 2a) compared to the more dispersed clusters formed by G (Fig. 2b). Since both F and G were abundant on the plasma membrane, the localizations delineated the entire plasma membrane (Fig. 2a, b). The observed membrane structures resembled those imaged by scanning electron microscopy (Supplementary Fig. 2). Both F (Fig. 2a) and G (Fig. 2b) were detected on the cell body and membrane protrusions, which are shown as tubular structures with a relatively uniform diameter of ~200 nm. To determine whether the localization densities of F or G were different between the cell body and membrane protrusions, we sampled a total of ~20 areas from each type of region and compared their localization densities. To account for the cell-to-cell variation in the expression levels of F and G, the localization densities on the protrusions were normalized against the average density of the cell body. We found that the localization densities of F or G on membrane protrusions were not statistically different from those on the cell body (Fig. 2c). A similar conclusion was obtained for cells fixed at 16 h post transfection (Supplementary Fig. 3c). Therefore, our data show that the distribution of the viral envelope glycoproteins is generally uniform over the plasma membrane.

Fig. 2 Distribution of NiV envelope glycoproteins on the plasma membrane of PK13 cells. PK13 cells expressing NiV-F or/and -G were fixed at 24 h post transfection and immunostained. Without permeabilization, NiV-F was immunostained by using a mouse anti-FLAG primary antibody and -G a rabbit anti-HA primary antibody. For single-color SMLM, Alexa Fluor 647 secondary antibodies were used for detection. For dual-color SMLM, Alexa Fluor 647 secondary antibodies were used for detection of F, and Cy3B secondary antibodies for G. a, b x–y cross section (100 nm thick in z) at the dorsal surface (position 3 in Fig. 1a) of a representative cell expressing F (a) or G (b). The boxed region is enlarged to show the detailed distribution pattern. c Comparison analyses of the localization densities of the F (red) or G (green) at the cell body versus membrane protrusions. Each data point was calculated using an area of 0.2 × 0.2 μm2. All data were normalized to the mean of the cell body. d Hopkins’ index of the F and G localizations from n = 30 cells. Lines represent the mean value and SD. The sample size is indicated in the parentheses. The p values were determined by two-tailed, unpaired t-test with Welch correction. e x–y cross section (100 nm thick in z) of a region at the dorsal surface (position 3 in Fig. 1a) of a representative cell co-expressing F (red) and G (green) with a pixel size of 10 nm. Scale bars: 1 μm. f The distribution of the DoC values between F and G molecules. One representative cell image out of three independent experiments (n ≥ 30) is shown Full size image

F generally exhibited a greater Hopkins’ index than G (Fig. 2d), suggesting more extensive clustering of F than G at the plasma membrane23. This observation is similar to the recent SMLM study on HIV Env and Influenza hemagglutinin, both of which demonstrate clustering behavior at the plasma membrane24,25. Similar distribution patterns of the envelope glycoproteins were obtained on the plasma membrane of PK13 cells at a shorter expression period (16 h). This suggests that the distribution and arrangement of F and G are not significantly dependent on the cell surface expression levels (Supplementary Fig. 3).

To determine the co-localization of F and G, we acquired dual-color SMLM images of F and G at the plasma membrane (Fig. 2e). A numerical co-localization analysis was carried out using the coordinate-based co-localization algorithm previously developed by Malkusch et al26. The degree of co-localization (DoC) is calculated for every single-molecule localization and has a value from −1 for segregation, through 0 for random distributions, to 1 for a high probability of correlated co-localization26,27,28. A random distribution indicates that the co-localization of two molecules is a random event rather than regulated by a specific mechanism. In this algorithm, the DoC value is dependent on the maxima radius (R max ) used for the DoC analysis26,27. A R max of 100 nm was used in this study to reflect the size of a typical VLP. The effect of the selected R max on the DoC values is demonstrated in the Supplementary Fig. 4. The analysis indicates that F and G are mostly localized in segregated clusters as the DoC values show a maximum in the negative range (Fig. 2f). The analysis partially explain that the association of the glycoproteins observed by co-immunoprecipitation assays can be due to random events22,29,30. Furthermore, we found that the coexistence of these two NiV envelope glycoproteins did not affect each other’s clustering behavior (Fig. 2e): F formed distinct clusters and G formed more dispersed clusters. PK13 cells are non-permissive for NiV entry because they lack the NiV receptors ephrinB2/B313. Nonetheless, similar behaviors were also observed on NiV permissive HeLa cells, which expressed the ephrinB2/B3 receptors13 (Supplementary Fig. 5). Therefore, the organization of F and G is independent of the presence of the receptors.

NiV-M does not alter the distribution of glycoproteins

To investigate whether M actively recruits F and G at the plasma membrane, three-color images were collected for cells simultaneously expressing M, F, and G. Both F and G were imaged using SMLM, while diffraction-limited images of M-GFP were used to identify regions with large M clusters. Figure 3a shows the F (red), G (green), and M (blue) clusters at the plasma membrane of the cell body (position 3, Fig. 1a). At a higher z position above the cell body, the membrane protrusions could be seen (Fig. 3b). We observed that the clusters of F and G were situated on dome-like structures (1 and 2 in Fig. 3b, c) in the M-positive regions. The shape of the dome-like structures recapitulated those formed by M localizations when expressed alone in the cell (Fig. 1e, f). Figure 3d shows the 3D reconstructed surface using the localizations of F and G in region 1 of Fig. 3b. These dome-like structures of G and F could not be observed when M was absent (Fig. 2a, b). Therefore, it is plausible that these dome-like structures formed by M are the assembly sites of the VLPs.

Fig. 3 Presence of M does not affect the arrangement of the envelope glycoproteins’ clusters on the membrane. PK13 cells were co-transfected with NiV-M, -F, and -G and fixed at 24 h post transfection. Without permeabilization, F was immunostained using a mouse anti-FLAG primary antibody and an anti-mouse Cy3B secondary antibody, and G an anti-HA primary antibody and an anti-rabbit Alexa Fluor 647 secondary antibody. a, b x–y cross section (100 nm thick in z) of a representative cell shows the superimposition of a wide field image for M (blue) and the corresponding SMLM images of F (red) and G (green) on the cell body (a) and membrane protrusions (b). Scale bar: 1 μm. c x–y cross section (100 nm thick in z) of the M-positive sites (c1, 2) boxed in b. Scale bar: 0.1 μm. d 3D surface reconstruction of the dome-like structure in c1, with F (red) and G (green) localization densities projected on the surface. A higher brightness indicates a higher localization density. e Comparison of the localization densities of F (red) or G (green) on the M-positive and M-negative regions at the dorsal surface of the cell. f Comparison of the Hopkins’ indices of F (red) and G (green) in the M-positive and M-negative regions at the plasma membrane. All data in e and f were normalized to the mean of the M-negative regions of the same cell. Lines represent the mean value and SD. The sample size is indicated in the parentheses. The p values were determined by two-tailed, unpaired t-test with Welch correction. g The distribution of the DoC values between F and G molecules in the M-negative and M-positive regions. h VLPs produced in PK13 cells expressing M, F, and G were adhered to fibronectin-coated coverslips, fixed, and stained for NiV-F and G via tags described above. The z-stacks of the x–y cross section of a VLP show the super-imposition of a wide field image of M (blue) and the corresponding SMLM images of F (red) and G (green). Scale bar: 0.1 μm Full size image

Interestingly, the localization densities of F and G at the M-positive regions were not statistically different from those at the M-negative regions (Fig. 3e). Additionally, the Hopkins’ indices (Fig. 3f) and DoC values (Fig. 3g) of F and G were comparable in the M-positive and M-negative regions. Furthermore, dual-color SMLM images indicate non-correlated distributions among M, F, and G with negative averaged DoC values (Fig. 2f and Supplementary Fig. 6). These observations disagree with the commonly believed model that the viral envelope glycoproteins coalesce to the matrix protein for the assembly of the nascent virions7,8,31,32. Previous studies using western blot analysis suggest that F may facilitate G to incorporate into VLPs7. Nonetheless, we did not observe a significant difference on the DoC values between M and G with or without the presence of F (Supplementary Fig. 6c, d and e). Figure 3h shows the z-stacks of a representative VLP, marked by the GFP signal from M (blue). We found that the spatial organization and distribution of F and G on the VLP membrane were similar to that of the host cell’s plasma membrane (Fig. 2e and 3a–c). This observation indicates that the clusters of the envelope glycoproteins on the plasma membrane have not been considerably rearranged when incorporated into the VLPs. All together, these findings suggest an alternative assembly model for NiV, in which no active recruitment of the envelope glycoproteins to M is involved. Instead, the incorporation of the envelope glycoproteins occurs stochastically upon the envelopment of the M assemblies at the host cell’s plasma membrane.

Incorporation of the glycoproteins into VLPs is stochastic

If the incorporation of the envelope glycoproteins into VLPs occurs stochastically, the model predicts that the amounts of the envelope glycoproteins in VLPs should correlate with their expression levels on the host cell membrane rather than showing a fixed stoichiometry of G/M or F/M. To test this model, we collected images of ~10,000 VLPs at 18 and 45 h post transfection of the viral envelope glycoproteins and analyzed the intensity of M, F, and G on VLPs. Fig. 4a, b shows the intensity distributions of F and G in the VLPs, respectively. Both F and G showed significantly higher intensity at 45 h than those at 18 h post transfection, which was consistent with the cell surface expression levels of F and G on the host cells measured by flow cytometry (Fig. 4e). Moreover, the VLPs collected at 45 h post transfection also showed higher intensity ratios for both G/M and F/M. This observation indicates the stoichiometries of G/M and F/M in the NiV VLPs vary with the expression level on the host cells (Fig. 4c, d). These results confirm that the incorporation levels of the envelope glycoproteins are highly dependent on their expression levels at the host cell membrane and may not be regulated by M.