Intracellular transport of the measles virus ribonucleoprotein complex is mediated by Rab11A-positive recycling endosomes and drives virus release from the apical membrane of polarized epithelial cells

Abstract

Many viruses use the host trafficking system at a variety of their replication steps. Measles virus (MV) possesses a nonsegmented negative-strand RNA genome that encodes three components of the ribonucleoprotein (RNP) complex (N, P, and L), two surface glycoproteins, a matrix protein, and two nonstructural proteins. A subset of immune cells and polarized epithelial cells are in vivo targets of MV, and MV is selectively released from the apical membrane of polarized epithelial cells. However, the molecular mechanisms for the apical release of MV remain largely unknown. In the present study, the localization and trafficking mechanisms of the RNP complex of MV were analyzed in detail using recombinant MVs expressing fluorescent protein-tagged L proteins. Live cell imaging analyses demonstrated that the MV RNP complex was transported in a manner dependent on the microtubule network and together with Rab11A-containing recycling endosomes. The RNP complex was accumulated at the apical membrane and the apical recycling compartment. The accumulation and shedding of infectious virions were severely impaired by expression of a dominant negative form of Rab11A. On the other hand, recycling endosome-mediated RNP transport was totally dispensable for virus production in nonpolarized cells. These data provide the first demonstration of the regulated intracellular trafficking events of the MV RNP complex that define the directional viral release from polarized epithelial cells.

Fig 1

Construction of rMVs expressing FLP-tagged L proteins. (A) Genome structures of the rMVs. The six internal boxes indicate the N, P, M, F, H, and L genes of MV. The portions colored in green and red indicate the coding regions of the green FLP (EGFP) and red FLP (mCherry), respectively. (B and C) Growth kinetics of the rMVs in Vero/hSLAM cells. Vero/hSLAM cells were infected with the rMVs at an MOI of 0.01. At various time points, the cells and culture medium were harvested separately, and the PFU in both samples were determined. The data represent the means ± standard deviations of results from triplicate samples. The solid and dashed lines indicate the data for the cell-associated and cell-free titers, respectively. (B) Blue and green symbols indicate the data for IC323 and IC323-EGFPtagL, respectively. (C) Blue and red symbols indicate the data for IC323 and IC323-mCherrytagL, respectively. (D) Syncytium morphology and FLP-tagged L protein expression in rMV-infected Vero/hSLAM cells. Vero/hSLAM cells were infected with the rMVs at an MOI of 0.01. The cells were observed daily under light and fluorescence microscopes. Data at 2 days p.i. are shown.

Fig 2

Intracellular distributions of mCherry-tagged L protein and other viral proteins. (A) Vero/hSLAM cells were infected with IC323 or IC323-mCherrytagL, cultured for 2 days, and then analyzed by immunofluorescence and confocal microscopy. Red fluorescence indicates the mCherry-tagged L protein. The N and P/V proteins were detected with viral protein-specific primary antibodies and secondary antibodies conjugated with Alexa Fluor 488 (green) and Alexa Fluor 647 (blue, pseudocolor), respectively. The upper and lower panels show the results for IC323 and IC323-mCherrytagL, respectively. (B) RGB line profiles along the lines selected in panel A. The left and right panels show the data for IC323 and IC323-mCherrytagL, respectively. (C) Vero/hSLAM cells were infected with IC323-mCherrytagL, cultured for 2 days, and analyzed by fluorescence in situ hybridization assay. Lower panels show the result of MV-infected cells with a probe for genomic RNA. Green and red fluorescence indicates genomic RNA and mCherry-tagged L protein, respectively. Assays were also performed with mock-infected cells (upper panels). Middle panels show the result of MV-infected cells subjected to the fluorescence in situ hybridization assay without a probe for genomic RNA. Nuclei were counterstained with DAPI (blue). The scale bars indicate 10 μm. (D to G) Vero/hSLAM cells were infected with IC323-mCherrytagL, cultured for 2 days, and then analyzed by immunofluorescence and confocal microscopy. Red fluorescence indicates the mCherry-tagged L protein. The C (D), M (E), F (F), and H (G) proteins were detected with viral protein-specific primary antibodies and Alexa Fluor 488-conjugated secondary antibodies (green). Nuclei were counterstained with DAPI (blue). Scale bars indicate 10 μm.

Fig 3

Accumulation of the L protein at the MTOC in an MT-dependent manner. (A) Vero/hSLAM cells were infected with IC323-EGFPtagL, cultured for 24 h, and analyzed by time-lapse florescence microscopy. Five images taken at 4-h intervals are shown. Bars indicate 20 μm. (B and C) Vero/hSLAM cells were infected with IC323-EGFPtagL, cultured for 2 days, and analyzed by immunofluorescence and confocal microscopy. Green fluorescence indicates the EGFPtagL protein. α-Tubulin (B) and γ-tubulin (C) were detected with specific primary antibodies and Alexa Fluor 594-conjugated secondary antibodies (red). Nuclei were counterstained with DAPI (blue). (D) Vero/hSLAM cells were infected with IC323-EGFPtagL and transfected with pTagRFP-Tubulin. At 2 days p.i., the intracellular movement of EGFPtagL was analyzed by time-lapse fluorescence microscopy. A total of 150 still images were taken at 1.1-s intervals. The left panel shows the overall confocal image of the cells at the start of the time-lapse analysis. Green and red fluorescence indicates the EGFPtagL protein and TagRFP-tubulin, respectively. The scale bar indicates 10 μm. The right panels show enlarged time-lapse images and time-stacked images of the area indicated by the box in the left panel. The upper and lower panels show obliquely upward and downward movements of the EGFPtagL-containing dots, respectively, at different time points after starting the time-lapse analysis. Movements of the EGFPtagL fluorescence are indicated with white arrows in the time-stacked images. (E) Vero/hSLAM cells were infected with IC323-mCherrytagL. At 2 days p.i., the cells were treated with 5 μM nocodazole for 15 h (5 μM Noc) or left untreated (control). A total of 50 still images were taken at 1.1-s intervals. The panels show the overall confocal images of the cells at the start of the time-lapse analysis. Red fluorescence indicates the mCherrytagL protein. Scale bars indicate 10 μm.

Fig 4

Colocalization and cotransport of mCherry-tagged L protein with EGFP-tagged Rab11A protein. (A–C) Vero/hSLAM cells constitutively expressing EGFP-Rab11A (A), -Rab5 (B), or -Rab7 (C) (Vero/hSLAM/EGFP-Rab cells) were infected with IC323-mCherrytagL. At 2 days p.i., the cells were fixed and analyzed by confocal microscopy. Green and red fluorescence indicates the EGFP-tagged Rab proteins and mCherrytagL protein, respectively. Nuclei were counterstained with DAPI (blue). The lower panels show the RGB line profiles along the selected lines in the merged images (upper right panels). (D) Vero/hSLAM/EGFP-Rab11A cells were infected with IC323-mCherrytagL. At 2 days p.i., the intracellular movements of EGFP-Rab11A and mCherrytagL were analyzed by time-lapse fluorescence microscopy. The left panel shows the overall confocal image of the cells at the start of the time-lapse analysis. Green and red fluorescence indicates the EGFP-Rab11A and mCherrytagL proteins, respectively. The right panels show enlarged time-lapse images of the area indicated by the box in the left panel. A representative series of images taken at 1.1-s intervals and a time-stacked image are shown. The movement of the mCherrytagL dots with EGFP-Rab11A is indicated by the white arrow in the time-stacked image.

Fig 5

Growth kinetics of the IC323-AddmCherry virus in Rab11ADN-expressing Vero/hSLAM or MDCK cells. (A) Vero/hSLAM cells constitutively expressing EGFP, EGFP-Rab11A, or EGFP-Rab11ADN were infected with IC323, cultured for 1 day, and analyzed by immunofluorescence and confocal microscopy. The N protein was detected by indirect immunofluorescence assay. (B to D) Vero/hSLAM cells constitutively expressing EGFP, EGFP-Rab11A, or EGFP-Rab11ADN were infected with IC323-AddmCherry at an MOI of 0.01. At various time points, the cells and culture medium were harvested separately, and the PFU in both samples were determined. The data for the cell-associated and cell-free titers are plotted in panels B and C, respectively. The data represent the means ± standard deviations of results from triplicate samples. The blue, red, yellow, and green lines indicate the data for Vero/hSLAM, Vero/hSLAM/EGFP, Vero/hSLAM/EGFP-Rab11A, and Vero/hSLAM/EGFP-Rab11ADN, respectively. In panel D, representative CPEs at 48 and 100 h p.i. are shown. Red fluorescence indicates the virus-derived mCherry expression. (E) Vero/hSLAM cells constitutively expressing negative-control shRNA (shNC) or shRNA against Rab11A mRNA (shRab11A) were transfected with pMXsIP-EGFP-Rab11A. At 2 days posttransfection, EGFP-Rab11A expression levels were analyzed by Western blotting. (F) Vero/hSLAM cells constitutively expressing shNC or shRab11A were lysed and endogenous Rab11 expression was analyzed by Western blotting. (G and H) Vero/hSLAM cells constitutively expressing shNC or shRab11A were infected with IC323-AddmCherry at an MOI of 0.01. At various time points, PFU in cells (G) and culture medium (H) were determined. The data represent the means ± standard deviations of results from triplicate samples. The blue, red, and green lines indicate the data for Vero/hSLAM, Vero/hSLAM/shNC, and Vero/hSLAM/shRab11A, respectively. (I to K) MDCK cells constitutively expressing EGFP, EGFP-Rab11A, or EGFP-Rab11ADN were plated on 24-well plates and immediately infected with IC323-AddmCherry at an MOI of 0.2. At various time points, the cells and culture medium were harvested separately, and the PFU in both samples were determined. The data for the cell-associated and cell-free titer are plotted in panels I and J, respectively. The data represent the means ± standard deviations of results from triplicate samples. The blue, red, yellow, and green lines indicate the data for MDCK, MDCK/EGFP, MDCK/EGFP-Rab11A, and MDCK/EGFP-Rab11ADN, respectively. In panel K, representative CPEs at 4 days p.i. are shown. Red fluorescence indicates the virus-derived mCherry expression. (L and M) MDCK cells constitutively expressing EGFP, EGFP-Rab11A, or EGFP-Rab11ADN were seeded on transwell filters with 0.4-μm pores and immediately infected with IC323-AddmCherry at an MOI of 0.2. At various time points, the apical and basolateral media were harvested separately, and the PFU in both samples were determined. The data for the apical and basolateral titers are plotted in panels L and M, respectively. The data represent the means ± standard deviations of results from triplicate samples. The blue, red, yellow, and green lines indicate the data for MDCK, MDCK/EGFP, MDCK/EGFP-Rab11A, and MDCK/EGFP-Rab11ADN, respectively. (N and O) MDCK (N) and Vero/hSLAM (O) cells were infected with IC323-AddmCherry at MOIs of 0.2 and 0.01, respectively. At 4 and 2 days p.i., MDCK cells and Vero cells, respectively, were treated with reagents (5 μM nocodazole [5 μM Noc], 5 μM paclitaxel [5 μM Pac], or dimethyl sulfoxide [DMSO] [as vehicle control]) for 1 day. PFU counts in cells and culture medium were determined. The ratios of cell-free virus titer to cell-associated virus titer are plotted. The data represent the means ± standard deviations of results from triplicate samples. (P and Q) MDCK (P) and Vero/hSLAM (Q) cells, which were transfected with pAcGFP1-Tubulin, were infected with IC323-mCherrytagL at MOIs of 0.2 and 0.1, respectively. At 4 and 2 days p.i., MDCK cells and Vero/hSLAM cells, respectively, were treated with reagents for 1 day. Then, cells were fixed and nuclei were stained with DAPI (blue). Z-stacks of images were acquired by confocal microscopy. Green (Q) and red (P and Q) fluorescence indicates the AcGFP-tagged α-tubulin and mCherrytagL protein, respectively. The Z-projection images of the maximum intensities are shown.

Fig 6

Intracellular localizations of the MV RNP complex and Rab11 in MDCK cells. (A to C) MDCK/EGFP (A), MDCK/EGFP-Rab11A (B), and MDCK/EGFP-Rab11ADN (C) cells were infected with IC323-mCherrytagL at an MOI of 0.2. At 4 days p.i., the cells were fixed and their nuclei were stained with DAPI (blue). Z-stacks of images were acquired by confocal microscopy. Green and red fluorescence in the merged images indicates the EGFP-tagged proteins and mCherrytagL protein, respectively. The left panels show Z-projection images of the maximum intensities, and the right panels show reconstituted images of the XZ plane along the lines in the left panels. (D and E) MDCK/EGFP-Rab11A (D) and MDCK/EGFP-Rab11ADN (E) cells were infected with IC323 at an MOI of 0.2. At 4 days p.i., the cell membrane was stained with CM-Dil (red in the merged images) and the cells were fixed for analyses by immunofluorescence and confocal microscopy. Green fluorescence in the merged images indicates the EGFP-tagged proteins. The N protein was detected with a specific primary antibody and an Alexa Fluor 405-conjugated secondary antibody (blue in the merged images). The left and right panels show Z-projection images and reconstituted images of the XZ plane, respectively, as shown in panels A to C. Arrowheads in panel E indicate the accumulation of the N proteins at the bottom of the cytoplasm. (F) MDCK cells were infected with IC323 at an MOI of 0.2. At 4 days p.i., the cell membrane was stained with CM-Dil (red in the merged images) and the cells were fixed for analyses by immunofluorescence and confocal microscopy. The N protein and endogenous Rab11 were detected with specific primary antibodies and secondary antibodies conjugated with Alexa Fluor 405 (blue in the merged images) and Alexa Fluor 488 (green), respectively. The left and right panels show Z-projection images and reconstituted images of the XZ plane, respectively, as shown in panels A to C.