Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome

Abstract

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Figure 1. Live cell imaging of cytoplasmic vRNPs in infected MDCK cells.

(A) For live cell imaging, AF568-conjugated anti-NP mAb61A5 (red, mAb61A5) and AF488-conjugated non-specific mouse immunoglobulin (green, control Ab) were cotransfected to infected MDCK cells. Sequential images were acquired by the dual-color protocol and subsequently by the single-color protocol for kinetic analysis. Images were processed and analyzed by using ImageJ software and MTrackJ plugin (Video S1). A representative frame of the movie was shown (left 3 images). Pseudo-positive signals appeared in yellow in merged image (most left image, arrowheads). An example of signal tracking was shown as trajectories (most right image, mAb61A5 single channel). (B and C) Velocity distribution of vRNP signals. Mean and maximum velocities (V_mean and V_max, respectively) of individual motile events were calculated and shown as histograms (Table S7, total 123 motile events derived from 75 trajectories). (D) Distribution of migration lengths. The migration lengths of individual motile events were shown as a histogram.

Figure 2. Live cell imaging of cytoplasmic vRNPs along microtubules.

(A) Live cell imaging was carried out using MDCK cells expressing AcGFP-α-tubulin. Pseudo-positive signals (yellow), the microtubule networks (green), and vRNPs (red) were indicated as arrows. (B) Cropped and each color-split image of the indicated area (white box in panel A). Sequential images were shown in Video S3. (C and D) Time-split images of the merged images and the mAb61A5 channel images in the cropped area, respectively. Elapsed time from the first acquisition was indicated on each image. A vRNP signal (arrowheads in D) moved (event 1, 33.6 to 36.0 s), paused (36.0 to 38.4 s), and moved again (event 2, 38.4 to 40.8 s). Scale bars  = 5 µm.

Figure 3. Colocalization of punctate vRNP signals with Rab11.

(A–C) Localizations of cytoplasmic vRNPs and transiently expressed human Rab proteins. Influenza A virus was infected to MDCK cells transiently expressing AcGFP-tagged human Rab11A, Rab25, and Rab17 (panels A–C, respectively). At 7 hpi, vRNPs were immunostained with mAb61A5 (center image in each set) and visualized by confocal microscopy with AcGFP-Rab proteins (right images). Enlarged images of indicated areas (white boxed) were also shown (lower images). Scale bars are 10 and 5 µm (upper and lower images, respectively). (D) Localizations of transiently expressed human Rab11A, Rab25, and Rab17. FLAG-Rab25 (upper) and FLAG-Rab17 (lower) (center images) were coexpressed with AcGFP-Rab11A (right images) in MDCK cells. Nuclei were stained with DAPI (blue, left images). Scale bar is 5 µm. (E and F) Colocalization of vRNP with endogenous Rab11. Progeny vRNPs were similarly stained with mAb61A5. Endogenous canine Rab11 (right images) was visualized with rabbit anti-Rab11 polyclonal antibody. (E) XY presentation. Scale bars are 40 and 5 µm (upper and lower images, respectively). (F) XZ presentation. Z-stacks of confocal images were acquired at 0.5 µm z-axis interval. Z-projection of maximum intensities (top image) and reconstitution of a xz plane (lower 3 images) were processed by using ImageJ software. Dotted line indicates the position of the reconstituted xz plane. Scale bar is 10 µm.

Figure 4. Localization of progeny vRNPs to RE in active/GTP-bound Rab11 dependent manner.

(A) Alteration of vRNP localization by transient expression of dominant negative Rab11 mutant. Influenza A virus was infected to MDCK cells transiently expressing the wild type (WT, left images), dominant negative (DN, center images), and constitutively active (CA, right images) forms of FLAG-tagged human Rab11A. At 7 hpi, vRNPs (middle images) and FLAG-Rab proteins (bottom images) were immunostained using mAb61A5 and rabbit anti-FLAG polyclonal antibody (pAb) and observed by confocal microscopy. Scale bar is 10 µm. (B) Production of infectious progeny viruses from infected MDCK cells constitutively expressing human Rab11A and its mutants. Culture supernatants of MDCK cells infected with PR8 strain at moi = 1 to 3 were temporally harvested and titers of infectious viruses were measured and indicated as plaque forming unit (pfu)/ml. Single-round infection experiments were carried out using different lots of viral inoculum in independent experiments.

Figure 5. Coimmunoprecipitation of progeny vRNP segments with active/GTP-bound Rab11A.

(A) Coimmunoprecipitation of viral proteins with FLAG-Rab11A and its mutants. MDCK-Neo (lanes 1 and 5), MDCK-F11A-WT (lanes 2 and 6), -DN (lanes 3 and 7), and -CA (lanes 4 and 8) cells were infected with PR8 strain and harvested at 7 hpi. PNS were subjected to immunoprecipitation assays using anti-FLAG mAb, and 10% input (lanes 1–4) and precipitates (lanes 5–6) were analyzed by Western blotting with mouse anti-HA antiserum and anti-FLAG mAb, rabbit anti-PB2, PB1, PA, NP, and M1 antisera. (B) Coimmunoprecipitation of FLAG-Rab11 CA mutant with viral RNP complexes. Immunoprecipitation assay was carried out using anti-NP mAb61A5. Precipitates were treated with RNase A and eluates were subjected to Western blotting analysis. (C) Coimmunoprecipitation efficiencies of viral RNAs. The amounts of viral RNAs in the immunoprecipitates with anti-FLAG mAb were quantified by polarity-specific reverse transcription followed by segment-specific semiquantitative real-time PCR. Coimmunoprecipitation efficiencies were calculated as percentage of RNA amounts in precipitates relative to those in the input (Figure S3). Segment numbers were indicated at the bottom. Columns indicated the coimmunoprecipitation efficiencies of vRNAs (gray and black columns) and c/mRNAs (hatched and white columns) from MDCK-F11A-DN and -CA. (D) Coimmunoprecipitation of vRNP components in the presence of RNase A. Immunoprecipitation assays using infected MDCK-F11A-CA cells were carried out in the absence (lane 1) or the presence of 1, 10, and 100 ng/µl RNase A (lanes 2–4, respectively). Coprecipitated vRNP components (PB2, PB1, PA, and NP) and direct precipitates (FLAG-Rab11A CA) were detected by Western blotting.

Figure 6. Effects of Rab11-FIP deletion mutants on the localization and trafficking of progeny vRNP segments.

(A) Schematic representation of the functional domains of human Rab11-FIPs (FIPn). Numerals at both ends indicate amino acid residues. The Rab binding domains (RBD) of individual Rab11-FIPs were indicated as gray boxes. Typical Rab11-FIP1 gene products (FIP1A, -B, and -C/RCP) were shown. The RBD fragment tagged with mStrawberry at the amino terminus (mSB-FIPnRBD) and the RBD deletion mutant containing a FLAG epitope tag at the carboxyl terminus (FIPnΔRBD-FLAG) were also illustrated. C2, C2-domain; EF, EF-hand domain. (B) Localization of progeny vRNPs in infected MDCK cells transiently expressing Rab11-FIP deletion mutants. Rab11-FIPs with deletion of RBD (upper two rows) and RBD fragments (lower two rows) were visualized using anti-FLAG mAb and mSB (red), respectively. Progeny vRNPs were also visualized using anti-NP mAb61A5 (green). Confocal merged images (odd rows) and vRNP-channel images (even rows) are shown. All images are shown at the same magnification. Scale bar  = 10 µm. (C) Polarized localization of progeny vRNP. XZ sections of polarized MDCK cells. Nuclei were stained with DAPI (blue) and shown in merged images (left images).

Figure 7. Models for spatial orientation of vRNP segments toward a higher-order assembly.

Putative spatial orientations of progeny vRNP segments in the cytoplasm were illustrated. (A) Diffusive random orientation model, (B) membrane-associated vertical orientation model may occur on RE and/or beneath the APM, and (C) membrane-associated horizontal orientation model may occur on a vesicle and/or beneath the APM precoated with M1. Details were described in the Discussion section.