Functional organization of cytoplasmic inclusion bodies in cells infected by respiratory syncytial virus

Abstract

Infection of cells by respiratory syncytial virus induces the formation of cytoplasmic inclusion bodies (IBs) where all the components of the viral RNA polymerase complex are concentrated. However, the exact organization and function of these IBs remain unclear. In this study, we use conventional and super-resolution imaging to dissect the internal structure of IBs. We observe that newly synthetized viral mRNA and the viral transcription anti-terminator M2-1 concentrate in IB sub-compartments, which we term "IB-associated granules" (IBAGs). In contrast, viral genomic RNA, the nucleoprotein, the L polymerase and its cofactor P are excluded from IBAGs. Live imaging reveals that IBAGs are highly dynamic structures. Our data show that IBs are the main site of viral RNA synthesis. They further suggest that shortly after synthesis in IBs, viral mRNAs and M2-1 transiently concentrate in IBAGs before reaching the cytosol and suggest a novel post-transcriptional function for M2-1.Respiratory syncytial virus (RSV) induces formation of inclusion bodies (IBs) sheltering viral RNA synthesis. Here, Rincheval et al. identify highly dynamic IB-associated granules (IBAGs) that accumulate newly synthetized viral mRNA and the viral M2-1 protein but exclude viral genomic RNA and RNA polymerase complexes.

Fig. 1

Localization of newly synthesized viral RNA in RSV-infected cells. HEp-2 cells were infected with RSV. Two hours before the indicated times p.i. cells were incubated for 1 h with actinomycin D to inhibit cellular transcription and then 5-ethynyl uridine (5EU) was added for 1 h before cells were fixed. RSV polymerase inhibitor (AZ27) was added 6 h prior actinomycin D incubation. The 5EU incorporated in newly synthesized RNAs was detected using Alexa Fluor 647-azide (red) and cells were stained with an anti-N antibody (gray) and Hoechst 33258 (merge). Representative images from three independent experiments are shown. Images were taken under a Leica SP8 confocal microscope at different times p.i. as indicated. Scale bar 10 µm. The boxed areas enclose IBs that are shown magnified (zoom), scale bar 2 µm. White arrow indicates a 5EU signal spot inside an IB

Fig. 2

Localization of viral RNAs in RSV-infected cells. HEp-2 cells were infected with RSV for 24 h. FISH analyses were performed with specific probes (red) (as described in Methods) to detect polyadenylated RNA (PolyA), NS1 mRNA, N mRNA or viral genomic RNA as indicated on the pictures. A negative control was performed by probing G mRNA of VSV (irrelevant probe). Cells were also stained with an anti-N antibody (gray) and Hoechst 33258 (merge). Representative images from three independent experiments are shown. Images were taken under a confocal microscope. Scale bar 5 µm. The boxed areas enclose IBs that are shown magnified (zoom), scale bar 2 µm. Circles represent the borders of IBs

Fig. 3

Cellular proteins required for translation do not colocalyze with IBs or IBAGs. HEp-2 cells were infected with RSV. At 24 h p.i., cells were immunostained either with an anti-PABP (mRNA interacting protein), an anti-eIF4G (translation initiation factor), an anti-S6 (small ribosomal subunit protein), an anti-L4 (large ribosomal subunit protein) or an anti-eRF1 (translation termination factor) as indicated on the pictures (cellular protein in red). Cells were also stained with an anti-N antibody (gray) to detect IBs and with Hoechst 33258 (merge). Representative images from three to four independent experiments are shown. Images were taken under a confocal microscope. Scale bar 5 µm

Fig. 4

Localization of the N, P, L and M2-1 proteins inside transiently reconstituted IBs. BSRT7/5 cells were transfected with plasmids encoding the N, P, L and M2-1 proteins and the M/Luc subgenomic minireplicon. Tagged protein was expressed instead of the corresponding wild type as indicated on the pictures. For N protein, the plasmids encoding wild type and tagged protein were mixed in equivalent ratio when indicated mGFP-N + N. FISH analyses were performed to detect poly(A) RNA (in red) and were stained with Hoechst 33258 (merge). The expressed tagged proteins are visualized thanks to their spontaneous green fluorescence in the first column. Representative images from three independent experiments are shown. Images were taken under a Leica SP8 confocal microscope. Scale bar 5 µm. The boxed areas enclose IBs that are shown magnified in the fourth column, scale bar 2 µm

Fig. 5

Dual color single-molecule localization microscopy of the viral proteins and mRNA inside IBs. BSRT7/5 cells were transfected with plasmids encoding the N, P, L and M2-1 proteins and the M/Luc subgenomic minireplicon. The mEos tagged protein was expressed instead of the corresponding wild-type protein as indicated on the picture. FISH analyses detecting poly(A)-mRNA were performed in order to visualize IBAGs. PALM-STORM images were realized as indicated in Methods section. The first column from the left shows a PALM image of the viral protein labeled with mEos (green). The second column shows a STORM image of the poly(A)-mRNA (red). The third column shows a superposition of the images from the first and second columns. Representative images from two independent experiments are shown, scale bar 5 µm. The boxed areas enclose IBs that are shown magnified in the fourth column, scale bar 2 µm

Fig. 6

Effect of viral RNA synthesis inhibition on IBAGs formation. BSRT7/5 cells were transfected with plasmids encoding the N, P, L and M2-1 proteins and the M/Luc subgenomic minireplicon. M2-1 mGFP a or PmEos b protein was expressed instead of the corresponding wild-type protein as indicated in the boxes on the left panel. a Plasmids encoding the subgenomic minireplicon (ΔminiG) or the L protein (ΔL) were omitted, or a polymerase inhibitor (+AZ27) was added, or wild-type M2-1mGFP was replaced by the R151D M2-1mGFP (impaired in RNA binding) as indicated by a gray box on the left panel. b Plasmid encoding the M2-1 protein was omitted (ΔM2-1) as indicated by a gray box on the left panel. Poly(A) RNAs (red) were detected by FISH analyses and cells were stained with an anti-N antibody (gray) and Hoechst 33258 (merge). The expressed tagged proteins, visualized by epifluorescence (green), are indicated in the box on the left panel and on the pictures. Representative images were taken under a Leica SP8 confocal microscope, scale bar 5 µm. The boxed areas enclose IBs that are shown magnified in the fourth column, scale bar 2 µm

Fig 7

Concentration of newly synthesized viral RNAs in IBAGs is transient. a Schematic time line of the pulse chase experiments. HEp-2 cells were infected with RSV-M2-1mGFP. At 24 h p.i. cells were incubated for 1 h with actinomycin D to inhibit cellular transcription and 5-ethynyl uridine (5EU) was added for one more hour (pulse). Cells were rinsed and fixed immediately (0 h) or after 3 or 6 h (chase). The 5EU incorporated in viral RNAs was detected using Alexa Fluor 647-azide (red) and cells were stained with an anti-N antibody (gray) and Hoechst 33258 (merge). The M2-1mGFP protein is visualized through its spontaneous green fluorescence. b Representative pictures from four independent experiments are shown. Images of inclusion bodies taken under a Leica SP8 confocal microscope at 0, 3 and 6 h after the 5EU pulse are presented. Scale bar 1 µm. The white lines indicate the track of a line intensity profile across the IBs c

Fig 8

IBAGs are dynamic structures. Time-lapse microscopy of IBAGs in HEp-2 cells infected with RSV-M2-1mGFP. At 24 h p.i., cells were imaged in a chamber heated at 37 °C, with a Leica SP8 confocal microscope. The M2-1mGFP protein was visualized by green fluorescence. Representative images from five independent experiments are shown. a Representative images of IBAG dynamics. White arrows indicate IBs undergoing IBAG growth, followed by IBAG collapse. Scale bar 10 µm. b Time-lapse imaging of an IB showing IBAG disassembly (regions of interest). The dotted line indicates the IB boundary as determined on the first and last image. The central image suggests the release of the IBAG content into the cytoplasm indicated by a white arrow. Scale bar 1 µm

Fig 9

Model of viral mRNA trafficking. (1) Viral mRNA synthesis occurs in IBs in area containing genomic viral RNA and N, L, P and M2-1 (designated as peripheral area). M2-1 binds to nascent mRNA poly(A) tail. (2) These RNA–protein complex together with cellular proteins mediate condensation of IBAGs recruiting the newly synthetized mRNA-M2-1 complexes. (3) IBAGs grow and fuse together. (4) When reaching critical size, IBAGs disassembly releases the mRNAs most probably still bound to M2-1 into the cytoplasm. (5) Newly synthetized M2-1 (and possibly M2-1 released from viral mRNA) bind with P in the cytoplasm and is imported in the peripheral area of IBs