YB-1 functions as a porter to lead influenza virus ribonucleoprotein complexes to microtubules

Abstract

De novo-synthesized RNAs are under the regulation of multiple posttranscriptional processes by a variety of RNA-binding proteins. The influenza virus genome consists of single-stranded RNAs and exists as viral ribonucleoprotein (vRNP) complexes. After the replication of vRNP in the nucleus, it is exported to the cytoplasm and then reaches the budding site beneath the cell surface in a process mediated by Rab11a-positive recycling endosomes along microtubules. However, the regulatory mechanisms of the postreplicational processes of vRNP are largely unknown. Here we identified, as a novel vRNP-interacting protein, Y-box-binding protein 1 (YB-1), a cellular protein that is involved in regulation of cellular transcription and translation. YB-1 translocated to the nucleus from the cytoplasm and accumulated in PML nuclear bodies in response to influenza virus infection. vRNP assembled into the exporting complexes with YB-1 at PML nuclear bodies. After nuclear export, using YB-1 knockdown cells and in vitro reconstituted systems, YB-1 was shown to be required for the interaction of vRNP exported from the nucleus with microtubules around the microtubule-organizing center (MTOC), where Rab11a-positive recycling endosomes were located. Further, we also found that YB-1 overexpression stimulates the production of progeny virions in an Rab11a-dependent manner. Taking these findings together, we propose that YB-1 is a porter that leads vRNP to microtubules from the nucleus and puts it into the vesicular trafficking system.

Fig 1

Identification of YB-1 as a novel vRNP-interacting proteins. (A) Identification of cellular and viral proteins interacting with vRNP complexes. HeLa cells infected with influenza virus at an MOI of 10 were subjected to immunoprecipitation assays with control IgG (lane 2) or anti-NP (lane 3) antibody-conjugated protein A-Sepharose. The coprecipitated proteins were eluted in 100 mM glycine (pH 2.8), separated through 10% SDS-PAGE, and visualized by silver staining. Molecular mass markers are also shown in lane 1. (B) Intracellular localization of YB-1 in infected cells. Infected MDCK cells were subjected to indirect immunofluorescence assays with anti-YB-1 antibody followed by FISH assays using a probe that hybridizes with segment 1 vRNA at 0, 4, 8, and 12 hpi. For LMB treatment, infected cells were incubated in culture medium containing 20 nM LMB at 7 hpi, and then the intracellular localization of vRNA and YB-1 was visualized by FISH and indirect immunofluorescence assays at 12 hpi. The result for SeV-infected cells at 12 hpi is also shown. Scale bars, 10 μm. (C) Intracellular localization of cellular proteins related to P-bodies and SGs. Mock-infected cells (left panels) or infected MDCK cells at 8 hpi (right panels) were subjected to indirect immunofluorescence assays with anti-RAP55 (upper panels, red), anti-RCK (middle panels, red), or anti-TIAR (lower panels, red) antibodies. Nuclear DNA was stained with TO-PRO-3 iodide (blue). Scale bars, 10 μm.

Fig 2

Colocalization of YB-1 and vRNP export complexes in nuclear speckles. (A) Intracellular localization of YB-1, M1, and NS2. At 8 hpi, infected MDCK cells were subjected to indirect immunofluorescence assays with rabbit anti-YB-1 (red) and either mouse anti-M1 (upper panel, green) or rat anti-NS2 (lower panel, green) antibody. Scale bar, 10 μm. (B) Intracellular localization of vRNA, YB-1, M1, and NS2 in the presence of LMB. At 7 hpi, infected MDCK cells were incubated for 1 h in the presence of 20 nM LMB. Segment 1 vRNA (left panels, green), YB-1 (upper panel, red), M1 (middle panel, red), and NS2 (lower panel, red) were visualized by FISH and indirect immunofluorescence assays. Scale bars, 10 μm. (C) Accumulation of vRNA and YB-1 in PML NBs in the presence of LMB. After treatment of LMB as described for panel B, mock-infected (upper panel) and infected (middle and lower panels) MDCK cells were subjected to FISH assays using the probe that hybridizes with segment 1 vRNA (upper and middle panels, green) and to indirect immunofluorescence assays with rabbit (upper and middle panels, red) and mouse (lower panel, red) anti-PML and rabbit anti-YB-1 antibodies (lower panel, green). Scale bars, 10 μm.

Fig 3

Specific interaction of YB-1 with vRNA but not with either cRNA or viral mRNA. (A) Intracellular localization of YB-1 and viral mRNA/cRNA. At 8 hpi, infected MDCK cells were subjected to FISH assays using a probe that hybridizes with segment 1 cRNA and mRNA (green) and to indirect immunofluorescence assays with anti-YB-1 antibody (red) with or without 20 nM LMB treatment for 1 h. Nuclear DNA was stained with TO-PRO-3 iodide (blue). Scale bars, 10 μm. (B) Coimmunoprecipitation of YB-1 and viral RNA molecules. HeLa cells constitutively expressing FLAG-YB-1 were infected with influenza virus at an MOI of 10. After 8 hpi, cell lysates were prepared and subjected to immunoprecipitation assays in the presence of either control IgG or anti-FLAG antibody as described in Materials and Methods. The immunoprecipitated viral RNAs were eluted with 100 μg/ml FLAG peptide and then quantitatively analyzed by reverse transcription followed by real-time PCR with primers specific for segment 5 vRNA, cRNA, and NP mRNA. To quantitatively evaluate the data, 5% equivalents of mock-infected and infected samples were also observed.

Fig 4

Accumulation of YB-1-vRNP complexes on microtubules with Rab11a-positive recycling endosomes in response to infection. (A) Colocalization of YB-1 and microtubules in cytoplasmic punctate signals. At 12 hpi, mock-infected (upper panels) and infected (lower panels) MDCK cells were subjected to indirect immunofluorescence assays with anti-YB-1 (red) and α-tubulin (green) antibodies. Scale bars, 10 μm. (B and C) Interaction of YB-1 with Rab11a-positive recycling endosomes on microtubules. HeLa cells constitutively expressing FLAG-YB-1 were infected with influenza virus at an MOI of 10. (B) Cell lysates were prepared and subjected to immunoprecipitation assays in the presence of either control IgG (lanes 2 and 5) or anti-FLAG antibody (lanes 3 and 6) at 12 hpi. Coprecipitated proteins were eluted with 100 μg/ml FLAG peptide and detected by Western blotting assays with anti-PB1, anti-Rab11a, anti-α-tubulin, and anti-FLAG antibodies. Ten percent equivalents of mock-infected (lane 1) and infected (lane 4) lysates were also subjected to Western blotting assays. (C) The eluate purified from infected lysate from panel B, lane 6, was reimmunoprecipitated with either control IgG (lane 2) or anti-NP antibody (lane 3), and then the eluate was subjected to Western blotting assays with anti-α-tubulin and anti-Rab11a antibodies. Lane 1, 30% equivalent of proteins immunopurified with anti-FLAG antibody from infected cell lysate.

Fig 5

Effect of YB-1 overexpression on the production of infectious virions. (A) Expression levels of YB-1 and Rab11a proteins. 293T cells were transfected with either pCAGGS empty plasmid (lanes 1 and 3) or pCAGGS-FLAG-YB-1 (lanes 2 and 4) at 24 h after treatment of either nontargeting (control; lanes 1 and 2) or Rab11a (siRab11a; lanes 3 and 4) siRNA. At 24 h after transfection of expression vectors, the cell lysates were prepared and analyzed by SDS-PAGE followed by Western blotting assays with anti-YB-1, anti-Rab11a, and anti-β-actin antibodies. (B) Accumulation levels of viral proteins in cells overexpressing FLAG-YB-1. 293T cells were transfected with either pCAGGS or pCAGGS-FLAG-YB-1. At 24 h posttransfection, cells were infected with influenza virus at an MOI of 10. At 0, 2, 5, and 8 hpi, cell lysates were prepared and analyzed by Western blotting assays with anti-PB1, anti-NP, anti-M1, anti-NS2, and anti-α-tubulin antibodies. (C) Production of infectious virions. Control (open diamonds and filled squares) and Rab11a KD 293T (open triangles and open squares) cells transfected with either pCAGGS (open diamonds and open triangles) or pCAGGS-FLAG-YB-1 (filled and open squares) as described for panel A were infected with influenza virus at an MOI of 0.5. The culture supernatants collected at 3, 6, 12, 16, 20, and 24 hpi were subjected to plaque assays to examine the production of infectious virions in a single-round infection. The average titers and standard deviations determined from three independent experiments are shown. (D) Stimulatory activity of YB-1 on the virus titer in Rab11a KD cells. The slopes of the lines in panel C were determined by the least-squares method, and the ratio of the virus titer from cells overexpressing FLAG-YB-1 to that from cells transfected with pCAGGS is shown.

Fig 6

Accumulation of vRNP on microtubules in YB-1 knockdown cells. (A) Expression level of YB-1 in YB-1 KD cells. HeLa cells transfected with nontargeting (control; lanes 1 to 4) or YB-1 (siYB-1; lanes 5 to 8) siRNA were lysed, and then the lysates (2.5 × 10³, 5 × 10³, 1 × 10⁴, and 2 × 10⁴ cells) were subjected to SDS-PAGE followed by Western blotting assays with anti-YB-1 antibody at 48 h posttransfection. (B and C) Accumulation levels of viral proteins and RNAs in YB-1 KD cells. At 48 posttransfection of siRNA, control and YB-1 KD cells were infected with influenza virus at an MOI of 10. At 0, 2, 5, and 8 hpi, cell lysates were prepared and analyzed by Western blotting assays with anti-PB1, anti-NP, anti-M1, anti-NS2, and anti-α-tubulin antibodies. Total RNAs purified from the cells at 0, 2, 5, and 8 hpi were subjected to reverse transcription followed by quantitative real-time PCR with primers specific for segment 5 vRNA and NP mRNA as described in Materials and Methods. (D and E) Intracellular localization of vRNA in YB-1 KD cells. At 8 hpi with or without LMB treatment for 1 h, infected control and YB-1 KD cells were subjected to FISH assays using a probe that hybridizes with segment 1 vRNA (panel D) (scale bars, 10 μm). Cells were counted, and the localization pattern of vRNA in the absence of LMB was determined (E). The number of cells showing each localization pattern was expressed as the percentage of the total cell number (n = 80) in panel E. The average percentages determined from three independent experiments are shown.

Fig 7

YB-1 functions as a porter bringing the progeny vRNP to microtubules. (A) Direct interaction of YB-1 with viral polymerase complex. vRNP (lanes 4 to 6) or micrococcal nuclease-treated vRNP (mnRNP) (lanes 7 to 9) was incubated in the absence (lanes 2, 5, and 8) or presence (lanes 3, 6, and 9) of purified recombinant His-YB-1 protein at 30°C for 1 h. Complexes were purified using Ni-NTA resin, and then proteins were separated through SDS-PAGE and detected by Coomassie brilliant blue (CBB) staining and Western blotting assays with anti-PB1, anti-PB2, and anti-PA antibodies. Lanes 1, 4, and 7 represent 20% of input amounts. (B) Interaction of vRNP with deletion mutants of YB-1. Each GST-fused deletion mutant of YB-1 was incubated with vRNP at 30°C for 1 h. Complexes were purified using glutathione-Sepharose resins, and then proteins were separated through SDS-PAGE and detected by Western blotting assays using anti-PB1 antibody. Lane 1 represents 20% of input amounts. A schematic diagram of the deletion mutants of YB-1 is at the bottom. (C) Interaction between vRNP and microtubules mediated by YB-1. Reconstituted microtubules were incubated with either vRNP (lanes 2 and 4) or YB-1-vRNP complex (lanes 3 and 5), and then complexes were immunoprecipitated with either a nonspecific IgG (control; lanes 2 and 3) or anti-NP antibody (lanes 4 and 5). The immunoprecipitated proteins were separated through SDS-PAGE and subjected to Western blotting assays with anti-α-tubulin and anti-NP antibodies. Lane 1 represents 10% of input amount. (D) Interaction of vRNP with microtubules in YB-1 KD cells. HeLa cells constitutively expressing FLAG-α-tubulin were infected with influenza virus at an MOI of 10. At 8 hpi, cell lysates were prepared and subjected to immunoprecipitation assays with either control IgG or anti-FLAG antibody. The immunoprecipitated proteins eluted with 100 μg/ml FLAG peptide were separated through SDS-PAGE and then visualized by Western blotting assays using anti-PB1 and anti-α-tubulin antibodies. Five percent equivalents of control (lane 1) and YB-1 KD (lane 4) lysates are also shown.