Association of the influenza virus RNA polymerase subunit PB2 with the host chaperonin CCT

Abstract

The RNA polymerase of influenza A virus is a host range determinant and virulence factor. In particular, the PB2 subunit of the RNA polymerase has been implicated as a crucial factor that affects cell tropism as well as virulence in animal models. These findings suggest that host factors associating with the PB2 protein may play an important role during viral replication. In order to identify host factors that associate with the PB2 protein, we purified recombinant PB2 from transiently transfected mammalian cells and identified copurifying host proteins by mass spectrometry. We found that the PB2 protein associates with the cytosolic chaperonin containing TCP-1 (CCT), stress-induced phosphoprotein 1 (STIP1), FK506 binding protein 5 (FKBP5), alpha- and beta-tubulin, Hsp60, and mitochondrial protein p32. Some of these binding partners associate with each other, suggesting that PB2 might interact with these proteins in multimeric complexes. More detailed analysis of the interaction of the PB2 protein with CCT revealed that PB2 associates with CCT as a monomer and that the CCT binding site is located in a central region of the PB2 protein. PB2 proteins from various influenza virus subtypes and origins can associate with CCT. Silencing of CCT resulted in reduced viral replication and reduced PB2 protein and viral RNA accumulation in a ribonucleoprotein reconstitution assay, suggesting an important function for CCT during the influenza virus life cycle. We propose that CCT might be acting as a chaperone for PB2 to aid its folding and possibly its incorporation into the trimeric RNA polymerase complex.

FIG. 1.

Analysis of purified influenza virus PB2 protein. Purified PB2-TAP and NS2/NEP-TAP were analyzed by SDS-8% PAGE and stained with silver. The positions of PB2, Hsp90, and Hsp70 are indicated on the right. Note that NS2/NEP-TAP after TEV cleavage has a molecular mass of approximately 17 kDa, and therefore it is not detectable in the gel system used. The positions of unique bands in the PB2 sample are indicated by asterisks on the right. Molecular mass markers (Bio-Rad) in kDa are indicated on the left.

FIG. 2.

Western blot analyses of the interaction of the influenza virus polymerase proteins with CCT. (A) Purification of TAP-tagged RNA polymerase subunits, dimers, and trimeric complexes from 293T cells transfected with the indicated plasmids. Purified proteins were analyzed by SDS-8% PAGE, followed by staining with silver (upper panel) or by Western blotting using a monoclonal CCTβ-specific antibody (Serotec) (lower panel). The arrowheads indicate the position of copurifying Hsp90. (B) Interaction of CCTβ with PB2 proteins of influenza viruses of various subtypes and origins. TAP-tagged PB2 proteins of the indicated influenza virus strains were purified from transfected 293T cells, and purified proteins were analyzed by SDS-8% PAGE, followed by staining with silver (upper panel) or by Western blotting using a CCTβ-specific antibody (lower panel). Positions of molecular mass markers (Bio-Rad) in kDa are indicated on the left.

FIG. 3.

Mapping of the CCT interaction domain in PB2. (A) Diagrams of wild-type (WT) and deletion mutant PB2-GFP fusion constructs. The known domains of PB2 are indicated (49). (B) Coimmunoprecipitation of wild-type and deletion mutant PB2-GFP fusion proteins with CCT. Wild-type and the indicated deletion mutant PB2-GFP fusion proteins were expressed in transfected 293T cells, and immunoprecipitations were performed with a monoclonal CCTβ-specific antibody (Serotec) (+) or in the absence of an antibody (−). PB2-GFP and CCTβ proteins were detected with a monoclonal GFP-specific antibody (Santa Cruz) and the CCTβ-specific antibody, respectively, in cell lysates, flowthrough (FT), and immunoprecipitates (IP), as indicated. The star indicates the position of residual protein A released from the Sepharose and/or the position of the heavy chain of IgG that cross-reacts with the antibodies used in Western blotting. Positions of molecular mass markers (Bio-Rad) in kDa are indicated on the left.

FIG. 4.

Knockdown of CCT in A549 cells and its effect on virus growth. (A) Western blot analysis of CCTβ in A549 cells treated with no (−) siRNA or treated with GFP- or CCTβ-specific siRNA (siGFP or siCCTβ, respectively). RanBP5 detected with a polyclonal RanBP5-specific antibody (Santa Cruz) was used as a loading control. Positions of molecular mass markers (Bio-Rad) in kDa are indicated on the left. (B) Quantitation of Western blot analyses of the knockdown of CCTβ from panel A. CCTβ intensities in siRNA-treated cells were expressed as a percentage of intensities observed in cells not treated with siRNA, which was set to 100%. CCTβ levels were normalized to the levels of RanBP5. Bars represent standard deviations based on three independent experiments. *, P < 0.05, based on a one-sample Student's t test. (C) Growth curves of influenza A/WSN/33 virus in CCT-silenced A549 cells. Control cells not treated with siRNA (−) or cells treated with GFP- or CCTβ-specific siRNA were infected at an MOI of 0.001. At the indicated points postinfection (24, 36, and 48 h), samples were collected, and virus titers were determined by plaque assay in MDBK cells. The results shown represent an average of two independent experiments, with the range indicated. (D) Viability assay of CCT-silenced A549 cells. Cells treated as in panel A were stained with 1% trypan blue, and viable and dead cells were counted. Cell viability was expressed as the percentage of viable cells in the culture. Results shown are an average of two independent experiments, with the range indicated.

FIG. 5.

The effect of CCT knockdown on PB2 protein and viral RNA levels. (A) Western blot analysis of PB2 and CCTβ in untreated 293T cells [(Control and (−) siRNA] or 293T cells treated with GFP- or CCTβ-specific siRNA (siGFP or siCCTβ, respectively). Cells were also transfected with pcDNA-PB1, pcDNA-PB2, pcDNA-PA, pcDNA-NP, and pPOLI-NA-RT plasmids to express recombinant ribonucleoproteins. No plasmids were transfected in the control cells. PB2 was detected with a polyclonal PB2-specific antibody (3). RanBP5 detected with a polyclonal RanBP5-specific antibody (Santa Cruz) was used as a loading control. Positions of molecular mass markers (Bio-Rad) in kDa are indicated on the left. (B) Quantitation of Western blot analysis of PB2 and CCTβ protein levels from panel A. PB2 and CCTβ intensities in siRNA-treated cells were expressed as a percentage of intensities observed in cells not treated with siRNA (−), which was set to 100%. PB2 and CCTβ levels were normalized to the levels of RanBP5. (C) Primer extension analysis of mRNA, cRNA, and vRNA of the neuraminidase gene in CCTβ-silenced 293T cells. Cells were treated with siRNAs, followed by the transfection of plasmids to reconstitute ribonucleoprotein complexes as in panel A. (D) Quantitation of primer extension analysis of viral mRNA, cRNA, and vRNA levels from panel C. RNA levels in siRNA-treated cells were expressed as a percentage of RNA levels observed in untreated cells [(−) siRNA], which was set to 100%. RNA levels were normalized to the total amount of RNA used in the primer extension assay. Bars represent standard deviations based on four independent experiments. *, P < 0.05, based on one-sample Student's t test.