Fig 1.
Inclusion bodies (IBs) in NiV-infected cells.
(A) Vero76 cells were infected with wildtype NiV at a multiplicity of infection (MOI) of 0.05. At 24 h p.i. cells were fixed with 4% PFA for 48 h and permeabilized with Triton X-100. For staining of NiV IBs in syncytia, permeabilized cells were incubated with NiV N-specific antiserum. Scale bar, 10 μm. (B-D) For ultrastructural analysis, cells were infected with NiV at a MOI of 2. Infected cells were fixed and processed for transmission electron microscopy at 24 h p.i.. (B) Low magnification overview of a NiV-positive cell containing a perinuclear IB (IBperi) and an IB associated with the plasma membrane (IBPM). (C, D) Enlarged views of IBperi (C) and IBPM (D) marked in the overview in (B) are shown. Boxed areas are shown at higher magnifications. White arrows indicate mostly cross-sectioned viral nucleocapsids within IBperi, or aligned at the plasma membrane in IBPM.
Fig 2.
Colocalization of NiV M protein and inclusion bodies in NiV-infected and transfected cells.
(A) Vero76 cells were infected with wildtype NiV and NiVΔM at a MOI of 0.01 and 0.025, respectively. At 24 h p.i. cells were fixed with 4% PFA for 48 h and permeabilized with Triton X-100. Cells were incubated with a NiV N-specific guinea pig antiserum to visualize IBs (green). NiV M was detected with an M-specific rabbit antiserum (red). Nuclei were counterstained with DAPI (blue). Confocal xy sections of the central regions of syncytia are shown. White dotted lines indicate the lateral borders of syncytia. (B) Cells coexpressing the NiV proteins N, P, G and F in the presence (N/P/G/F + M) or absence of NiV M (N/P/G/F) were fixed and permeabilized with Triton X-100 at 24 h after transfection. Immunostaining was performed as described above. Scale bars, 10 μm. In the right panels, quantifications of IB distribution in syncytia is shown. Using the automated ImageJ analyze particle tool, the total numbers of IBs and the number of IBs located at a maximum distance of 10 μm from the lateral edge of the syncytium were counted in individual sections of 6–10 syncytia from three individual experiments. The average percentage of membrane-proximal IBs in syncytia in the absence and presence of M was calculated. Error bars indicate the standard error of the mean. Statistical significance is indicated by asterisks (unpaired t-test; ***, p < 0.001).
Fig 3.
Formation and localization of IBs in the absence and presence of NiV M.
(A) To induce IB formation plasmid-encoded NiV N and NiV P proteins were coexpressed (N+P). (B) N and P proteins were expressed together with the NiV M protein. 24 h after transfection, cells were fixed, permeabilized with Triton X-100 and incubated with an M-specific peptide serum (red, M) and NiV N-specific antibodies (green, IB). Nuclei were counterstained with DAPI (blue). Scale bars, 10 μm. Right panels: To quantify the intracellular localization of IBs (green) in the perinuclear region and the periphery, an ImageJ based quantification tool (IB-LoM) was used. The average IB distribution (quantified in confocal sections of 5–6 cells from three independent experiments) is shown. N, nucleus; PM, plasma membrane.
Fig 4.
Formation of NiV IBperi and IBPM monitored by live cell imaging.
To follow IB formation in the presence of M over time, Vero76 cells were cotransfected with NiV N and NiV PeGFP together with mCherryNiV M and untagged NiV M (see Materials and Methods). At 14 h p.t., the live cell time-lapse experiments were started. Images were recorded with a Nikon TE2000 microscope. IBs (green) and M proteins (red) were detected via the PeGFP or the mCherryM autofluorescence, respectively. Pictures were taken every 50 sec and processed with Nikon NIS-Elements Microscope Imaging Software. (A) Merged images of the whole cell at different time points are shown. The plasma membrane is indicated by the dotted line. Arrows indicate M-negative IBs in the cytoplasm (green). Arrowheads indicate the site where M-positive IBs are formed at the plasma membrane (yellow, IBPM). Scale bar, 10 μm. (B) Magnification of an IBPM (indicated with an arrowhead in panel a). Scale bar, 1 μm. The full-length video is provided as S1 Movie. For comparison, S2 Movie shows IB formation in the absence of the M protein.
Fig 5.
Colocalization of NiV IBs with mRNA binding proteins.
Vero76 cells were infected with NiV at a MOI of 0.05. 18.5 h p.i. cells were fixed with 4% PFA for 48 h and permeabilized with methanol/acetone. (A) NiV IBs were visualized by using NiV N-specific antibodies (green) and Zenon-labeled anti-M peptide serum (cyan). mRNA (red) was detected with an antibody directed against the PolyA binding protein (PABP). (B) NiV IBs (green) and NiV M (cyan) were costained with an anti-eIF4G antibody (red). Nuclei were counterstained with DAPI (blue). In the zoom panels of the confocal images, enlarged views of IBperi and IBPM in the boxed regions are shown. Scale bars, 10 μm.
Fig 6.
Localization of viral RNA in NiV-infected cells.
(A, B) Colocalization of de novo synthesized Br-UTP labeled RNA with IBs. Vero76 cells were infected with NiV at a MOI of 0.05. 18 h p.i. cells were treated for 1 h with actinomycin D to inhibit cellular transcription or left untreated. Then, cells were transfected with 10 mM Br-UTP. After 20 min, cells were fixed and permeabilized with methanol/acetone. Newly synthesized RNAs were detected using a Br-UTP monoclonal antibody (red). (A) Cellular RNA staining in uninfected control cells (Mock) without (-ActD) and with inhibitor (+ActD) are shown. (B) In actinomycin D-treated NiV-infected cells, IBs were visualized with an NiV N-specific antiserum (green) and Zenon-labeled anti-M peptide serum (cyan). (C) Detection of NiV mRNA by FISH. NiV-infected Vero76 cells were fixed at 18 h p.i. and were probed with Quasar 670-labeled FISH probes targeting the positive-sense NiV RNA from nucleotide 1–6000 (+RNA probes). After hybridization to visualize viral N, P and M mRNA (pseudo-colored in red), the N protein was immunostained to detect IBs (green). Nuclei were counterstained with DAPI (blue). In the zoom panels of the confocal images, enlarged views of IBperi and IBPM are shown. Scale bars, 10 μm.
Fig 7.
Colocalization of NiV IBperi with cellular aggresome markers and unrelated cytosolic proteins.
(A, B) Vero76 cells were transfected with NiV N and NiV PeGFP to form IBs (IBperi). At 24 h p.t. cells were fixed with 4% PFA and permeabilized with methanol/acetone. IBs were detected by PeGFP autofluorescence (IBperi). Cellular aggresome markers (γ-tubulin and vimentin) were detected with specific antibodies (red). (C, D) NiV N and NiV PeGFP were coexpressed with non-related cytosolic proteins (red). Measles virus matrix protein (measles M) was detected with a specific monoclonal antibody (C) and reporter mCherry was detected by autofluorescence (D). Nuclei were counterstained with DAPI (blue). Only merged confocal images are shown. IBs within the boxed areas are shown in higher magnification. Scale Bars, 10 μm.
Fig 8.
Colocalization of NiV inclusion bodies with y-tubulin in virus-infected cells.
Vero76 cells were infected with NiV or NiVΔM at a MOI of 0.05 or 0.01, respectively. At 18.5 h p.i. cells were fixed with 4% PFA for 48 h and permeabilized then with methanol/acetone. Cells were stained with an anti-N serum to visualize IBs (green) and with a Zenon-labeled anti-M peptide serum (cyan) to identify M-positive IBPM. For γ-tubulin detection, cells were incubated with anti-γ-tubulin mouse antibodies (red). (A) A confocal section through a NiV-induced syncytium is shown. Magnifications and individual staining of the boxed areas are presented in the bottom panel. (B) M-negative and y-tubulin positive IBperi. (C) M-positive and y-tubulin negative IBPM. (D) Colocalization of M-positive IBPM and M-negative IBperi with y-tubulin in NiV-induced syncytia (n = 8) was quantified using the ImageJ-based macro IB-Coloc. Error bars indicate the standard error of the mean. Statistical significance is indicated by asterisks (unpaired t-test; ***, p < 0.001). (E) A confocal section through a NiVΔM-induced syncytium is shown. The right panel shows a magnification and individual staining of the IBs in the boxed area.
Fig 9.
Model for IB formation and assembly during NiV infection.
(A) NiV protein synthesis and trafficking in early infection stages. NiV surface glycoprotein F and G are synthesized in the ER and transported to the plasma membrane via the secretory pathway (1). NiV M is synthesized in the cytoplasm and rapidly imported into the nucleus (2a). Only after monoubiquitination at K258, NiV M can exit the nucleus (2b). After nuclear transit, NiV M traffics to the plasma membrane to form a dense matrix underneath the plasma membrane (2c). NiV N, P, and L proteins are synthesized at ribosomes throughout the cytoplasm and concentrate rapidly in perinuclear IBs (3). (B) IB formation and assembly in late infection stages. NiV replication and transcription is assumed to occur in cytoplasmic reticular structures rather than in IBs. Newly synthesized viral genomes are encapsidated by the viral nucleocapsid proteins to form NCs (4). NCs are recruited to IBperi until sufficient M protein is expressed (5). NC are transported to the cell periphery either independently (6a) or via cotransport with M proteins (6b). NCs and M proteins accumulate at the plasma membrane and form IBPM (7). The NiV glycoproteins are clustered at IBPM sites and virus budding is initiated (8). Please refer to the text for a more detailed discussion of the model.