Role of ran binding protein 5 in nuclear import and assembly of the influenza virus RNA polymerase complex

Abstract

The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin beta3, importin beta3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.

FIG. 1.

Identification of RanBP5 as an interactor of influenza virus RNA polymerase subunit PB1. (A and B) Purification of TAP-tagged RNA polymerase subunits, dimers, and trimeric complex from 293T cells transfected with the indicated expression plasmids. Purified proteins were analyzed by silver staining of an SDS-8% PAGE gel (upper panel) or by Western blotting using a polyclonal anti-karyopherin β3 antibody (Santa Cruz) (lower panels). Positions of PB1tap, PB2tap, PAtap, PB1, PA, and RanBP5 are indicated. The identity of the polymerase subunits was established in previous experiments by Western blot analysis using PB1-, PB2-, and PA-specific antibodies (12). Sizes of protein standards (Bio-Rad) in kilodaltons are shown on the left. The open circles indicate the positions of Hsp90 identified by both matrix-assisted laser desorption ionization-time of flight and LC/MS/MS (12). (C) Sequence of RanBP5 (accession number NM_002271) with peptides identified by LC/MS/MS shown in bold. The sequence coverage was 20%.

FIG. 2.

(A) The interaction between RanBP5 and PB1 is disrupted in the presence of RanGTP. PB1tap or PB1/PAtap dimer were bound to IgG-Sepharose and incubated with Ran(Q69L)GTP or buffer. The immobilized material was released by cleavage with TEV protease and analyzed by silver staining of SDS-8% PAGE gels (upper panel) or Western blotting with a polyclonal anti-karyopherin β3 antibody (Santa Cruz) (lower panel). Positions of PB1, PB1tap, PAtap, and RanBP5 are indicated. Sizes of protein standards (Bio-Rad) in kilodaltons are shown on the left. (B) RanGTP-treated PB1-PA dimer assembles with PB2 into a transcriptionally active polymerase complex. PB1tap, PB1/PAtap dimer, or PAtap, bound to IgG-Sepharose and treated with Ran(Q69L)GTP or buffer, was incubated with crude cell lysates from 293T cells containing PB2. Bound material was released by cleavage with TEV protease and analyzed by silver staining of SDS-8% PAGE gels (upper panel) or by Western blotting with a rabbit polyclonal anti-PB2 antibody (13) (middle panel). Transcriptional activity of the reconstituted RNA polymerase complex was assayed by in vitro ApG-primed transcription (lower panel). Positions of RanBP5, PB1, PAtap, PB2, and transcription products (TP) are indicated on the right. The position of PB1tap and the size of protein standards in kilodaltons are shown on the left. The open circles indicate the positions of Hsp90 identified by both matrix-assisted laser desorption ionization-time of flight and LC/MS/MS (12).

FIG. 3.

Knock-down of RanBP5 in 293T cells. (A) Quantitation of RanBP5 mRNA by quantitative reverse transcription-PCR. Cells were transfected with RanBP5- or CAT- and GFP-specific siRNA, and 48 h posttransfection they were infected with influenza A/WSN/33 virus. Total RNA was harvested 3.0, 4.5, or 6.0 h postinfection, and RNA concentration was determined by measuring the optical density at 260 nm. RanBP5 mRNA was determined by quantitative PCR. The relative values shown are an average of three measurements of three independent RNA samples. Values for the RNA sample harvested at 6 h postinfection from cells treated with control siRNA were set to 1. Standard deviations are shown. (B) Western blot analysis of knock-down levels of RanBP5. Karyopherin α3, detected with a goat polyclonal anti-KPNA3 antibody (Abcam), was used as a loading control. Positions of RanBP5 and karyopherin α3 are indicated on the right. Sizes of protein standards (Bio-Rad) in kilodaltons are shown on the left.

FIG. 4.

Effect of RanBP5 knock-down on viral RNA levels in infected cells. 293T cells transfected with RanBP5- or CAT- and GFP-specific siRNA were infected with influenza A/WSN/33, and total RNA was isolated at the time points indicated. (A) Primer extension assay analyzing NA-specific viral RNAs. The positions of transcription products derived from NA mRNA, cRNA, vRNA, and 5S rRNA, used as an internal control, are indicated on the right. Positions of size markers (³²P-labeled 1-kb ladder; Invitrogen) in nucleotides are indicated on the left. (B and C) Statistical analysis of NA-specific (B) and NS-specific (C) viral RNAs in infected cells transfected with RanBP5 siRNA. An average of three measurements of three independent RNA samples is shown with standard deviations. Values were standardized to the 5S rRNA signal, and for each time point, values were expressed as a percentage of the control values that were set to 100% (samples treated with CAT- and GFP-specific siRNA). One sample Student's t test was performed to assess whether the values were significantly different from 100%. Values significantly different from 100% (P < 0.05) are indicated by an asterisk (*).

FIG. 5.

Effect of RanBP5 knock-down on RNA polymerase localization. CAT- or RanBP5-specific siRNA-treated cells, expressing PB1-GFP and PA (PB1-GFP/PA), PB1-GFP, or PB2-GFP, were analyzed by fluorescence microscopy. Cells were scored for the localization pattern of the GFP-tagged polymerase subunit: N, predominantly nuclear; N/C, distributed throughout the cell; C, predominantly cytoplasmic. The number of cells showing each localization pattern was expressed as a percentage of the total cell number. The average of three experiments with standard deviations is shown. Examples of the localization patterns of GFP-tagged polymerase subunits (GFP) are shown under the corresponding charts. The bottom panels show the same fields of cells as the upper panels, stained for DNA, to indicate the location of nuclei. Note that cells expressing PB2-GFP, which exhibited a strong nuclear signal and a fainter mitochondrial signal, as observed previously (6, 18), were scored as cells with predominantly nuclear localization.

FIG. 6.

Model for the transport and assembly of the influenza virus RNA polymerase complex (see text for details).