Comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and RNA polymerase accessory factors

Abstract

The trimeric RNA polymerase complex (3P, for PA-PB1-PB2) of influenza A virus (IAV) is an important viral determinant of pathogenicity and host range restriction. Specific interactions of the polymerase complex with host proteins may be determining factors in both of these characteristics and play important roles in the viral life cycle. To investigate this question, we performed a comprehensive proteomic analysis of human host proteins associated with the polymerase of the well-characterized H5N1 Vietnam/1203/04 isolate. We identified over 400 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), of which over 300 were found to bind to the PA subunit alone. The most intriguing and novel finding was the large number of mitochondrial proteins (∼20%) that associated with the PA subunit. These proteins mediate molecular transport across the mitochondrial membrane or regulate membrane potential and may in concert with the identified mitochondrion-associated apoptosis inducing factor (AIFM1) have roles in the induction of apoptosis upon association with PA. Additionally, we identified host factors that associated with the PA-PB1 (68 proteins) and/or the 3P complex (34 proteins) including proteins that have roles in innate antiviral signaling (e.g., ZAPS or HaxI) or are cellular RNA polymerase accessory factors (e.g., polymerase I transcript release factor [PTRF] or Supt5H). IAV strain-specific host factor binding to the polymerase was not observed in our analysis. Overall, this study has shed light into the complex contributions of the IAV polymerase to host cell pathogenicity and allows for direct investigations into the biological significance of these newly described interactions.

Fig. 1.

Purification of the influenza virus polymerase complex and subunit components from A549 cells. (A) Overview of the influenza virus polymerase purification and expression technique. A549 human lung epithelial cells were infected with adenoviruses (rAd5) expressing the indicated proteins (PA-TAP, PB1, and PB2). Control A549 cell lysates or A549 cell lysates expressing PA, PA-PB1, or the 3P complex from VN/1203 (H5N1), WSN (H1N1), or CA/04 (H1N1) were left untreated (−) or treated (+) with nucleases (DNase and RNase A). The polymerase complex (PA-TAP/PB1/PB2) or the subunit components (PA-TAP, PA-TAP/PB1, and PA-TAP/PB2) were purified by IgG affinity chromatography and elution with tobacco etch virus protease. (B) A549 cells were transduced with adenovirus vectors, encoding the indicated VN/1203 polymerase subunits (top). After 48 h, cell lysates were prepared and subjected to IgG affinity chromatography and elution with tobacco etch virus protease. Nuclease-treated, affinity-purified polymerase subunits were then electrophoretically separated on a 7.5% polyacrylamide gel and detected by silver staining. The polymerase subunits (PB1, PA, and PB2) are indicated, as are the positions of molecular mass markers (left). There were no obvious differences in banding patterns compared to samples not treated with nuclease (data not shown).

Fig. 2.

Cellular PA-binding partners. Venn diagram, representing 651 cellular proteins identified in affinity-purified complexes with PA in the absence of nuclease treatment (−N, Set 1; Set 2) and after nuclease treatment (+N, Set 1). We attribute the identification of more proteins in set 2 than in set 1 to slight differences in sample preparation between experiments. Numbers in the various boxes/sectors denote numbers of unique cellular proteins identified in each category. Highlighted in the center of this figure are the 166 cellular proteins that we defined as shared RNA-independent PA-binding partners (i.e., proteins which bound to PA both in the presence and absence of PA-associated nucleic acid). Highlighted in the top center of this figure are the 140 cellular proteins that we defined as RNA-dependent binding partners (i.e., proteins which bound to PA only when PA was also complexed with nucleic acid). All proteins derived from set 2 were not treated with nucleases.

Fig. 3.

The cellular PA-interaction networks. The cellular interaction networks of PA-specific binding partners are shown. Represented in this analysis is a subset of the 166 cellular proteins which bound to PA in an RNA-independent manner and which were identified in all three protein preparations studied (Fig. 2). The analysis shown here reveals a novel interaction of PA with members of the Ran signaling pathway, the caveolar endocytosis-mediated pathway, and the aminoacyl-tRNA biosynthesis pathway. Orange symbols represent molecules that are involved in molecular transport. Highlighted in green are mitochondrial proteins, and highlighted in yellow are molecules chosen for further analysis (IPO7 and PRKDC) or discussed further in the text (EGFR). Nodes depict proteins in the network, and solid lines represent direct whereas dotted lines represent indirect relationships between the proteins. An arrow pointing from one node to the next signifies an activation event, such as phosphorylation or methylation. For simplicity, self-binding or autoregulation events were not included in this figure.

Fig. 4.

Heat map of 33 mitochondrial proteins that bind to PA. The relative abundance of the 33 mitochondrial proteins that are present in the shared RNA-independent fraction of VN/1203 PA-binding partners is shown here across all samples from set 1 and set 2 in the form of a heat map. AIFM1 was among the most abundant mitochondrial proteins. The molecules are arranged according to their mitochondrial location. Molecules involved in transmembrane transport are indicated. T.T., transmembrane transport; n.d., not defined to specific compartment in mitochondrion.

Fig. 5.

Association of human β-importins (IPO5 and IPO7), AIFM1, and PRKDC with PA and 3P complexes from various strains. (A) The relative abundance (spectral counts) of a subset of PA-binding partners (shared fraction) is shown in form of a heat map, with PRKDC being the most abundant protein. The spectral counts are shown from samples across both experimental sets (set 1 and set 2). (B) A549 cells were transduced with adenovirus vectors encoding the indicated VN/1203 (H5N1) polymerase proteins (3P denotes the PA-PB1-PB2 complex). Non-nuclease-treated PA-containing protein complexes were then purified by affinity chromatography, along with a negative-control eluate (A549), as described in Fig. 1A. Protein content in these complexes was evaluated by Western blotting, using antisera specific for IPO5, IPO7, and AIFM1 as well as anti-PA, anti-PB1, and anti-PB2. (C) A549 cells were transduced with adenovirus vectors encoding the 3P complex from the indicated influenza viruses. Non-nuclease-treated 3P complexes were then purified by affinity chromatography, along with a negative-control eluate (A549), as described in Fig. 1A. The purified polymerase complexes from influenza virus strains WSN, VN/1203, and CA/04 were separated by PAGE and detected by silver staining (top panel). The purified protein samples were also separated by SDS-PAGE and analyzed by immunoblotting with antibodies specific for IPO5, IPO7, AIFM1, and PRKDC.

Fig. 6.

AIFM1 interacts with the viral polymerase in transfected and influenza virus-infected cells. The interaction of AIFM1 to PA or the 3P complex was confirmed by coimmunoprecipitation experiments. (A) HEK293T cells were transfected with an expression construct (0.2 μg) encoding AIFM1-Flag (lanes 2 and 4) or empty vector (lanes 1 and 3) and PA (lanes 1 and 2) or the trimeric polymerase complex (0.6 μg each) (lanes 3 and 4). The viral proteins were derived from the A/WSN/33 strain. Whole-cell extract was prepared at 24 h posttransfection and used for immunoprecipitation (IP) with Flag-specific rabbit polyclonal antibody. The presence of AIFM1-Flag and PA, PB1, and PB2 in the precipitates (upper panel) as well as cell extract (lower panel) was analyzed by Western blotting using Flag (AIFM1-Flag), His (PA-His), and polymerase-specific antibodies (PB1 and PB2). (B) AIFM1 binds to PA in influenza virus (A/WSN/33)-infected A549 cells. Mock-infected (M)and WSN-infected A549 cells were lysed at various time points postinfection (24, 30, and 42 hpi), and total protein lysates (left panel) or PA-coimmunoprecipitated proteins (right panel) were subjected to immunoblot analysis using antibodies specific to AIFM1 and PA. Note that the IgG used to immunoprecipitate PA is also apparent in right panel.

Fig. 7.

Identification of 68 cellular binding partners to PA-PB1 (A) and 34 to the 3P (B) complex. (A) A Venn diagram illustrates cellular proteins identified in affinity-purified complexes with PA (set 1 and set 2) or PA-PB1 (set 1). Most of the proteins that copurified with PA (Set 1 and set 2) also copurified with the PA-PB1 complex from the same virus. Numbers in the various boxes/sectors denote numbers of unique cellular proteins identified in each category. Highlighted in the lower panel of this figure are the 68 cellular proteins that interacted with the H5N1 PA-PB1 heterodimer. The boxes at the bottom of the figure denote proteins that were detected only in complexes not treated with nuclease (−N), only in complexes treated with nuclease (+N), or regardless of nuclease treatment (−/+N). (B) Venn diagram, representing cellular proteins identified in affinity-purified complexes with PA alone or PA-PB1 or the complete heterotrimeric polymerase complex (3P) from the A/Vietnam/1203/2004 (H5N1) virus. Most of the proteins identified in association with the 3P complex from A/Vietnam/1203/2004 were also detected in complex with PA plus PA-PB1. However, 34 proteins associated uniquely with the 3P complex. Numbers in the various boxes/sectors denote numbers of unique cellular proteins identified in each category. Highlighted in the lower panel of this figure are the 34 cellular proteins that interacted uniquely with the H5N1 3P. The boxes denote proteins that were detected only in complexes not treated with nuclease (−N), only in complexes treated with nuclease (+N), or regardless of nuclease treatment (−/+N). An asterisk next to the protein symbol depicts proteins previously reported to associate with the influenza virus polymerase. The proteins were grouped according to their functions. Proteins that are underlined were also detected in the corresponding PA-PB1 or 3P (H5N1) fraction from set 2.

Fig. 8.

Pol II interaction network of PA-PB1- and 3P-associated host factors. (A) Proteins shown were identified in association with the following affinity-purified polymerase subunits/preparations: PA-PB1 fraction, blue; 3P fraction, green; PA fraction, white. Nodes depict proteins in the network, and solid lines represent direct whereas dotted lines represent indirect relationships between the proteins. An arrow pointing from one node to the next signifies an activation event, such as phosphorylation or methylation. (B) The relative abundances (spectral counts) of Pol II and Pol I factors are shown in the form of a heat map. The spectral counts are shown from samples across both experimental sets (set 1 and set 2).

Fig. 9.

Interaction of Supt5H and PTRF with the viral polymerase complex and polymerase subunits in transfected HEK293T cells. (A) To confirm the interaction between Supt5H and PTRF to the 3P complex by coimmunoprecipitation experiments, HEK293T cells were transfected with expression constructs (0.2 μg) encoding PTRF-Flag (lane 2), Supt5H (lane 4), or empty vector (lanes 1 and 2) and the components of the VN/1203 (H5N1) polymerase complex (PA, PB1, and PB2) (0.6 μg each). Whole-cell extract was prepared at 24 h posttransfection and used for immunoprecipitation (IP) with Flag-specific rabbit polyclonal antibody. The presence of Supt5H-Flag and PTRF-Flag, PA, PB1, and PB2 in the precipitates (upper panel) as well as cell extract (lower panel) was analyzed by Western blotting using Flag and polymerase-specific antibodies. Approximately 3% of total cell extract used for IP was loaded. (B) Flag-tagged Supt5H expression vector (lanes 4 to 6) or empty vector (lanes 1 to 3) was transfected into HEK293T cells with plasmid vector encoding PA alone, PA and PB1, or 3P from the VN/1203 strain (H5N1). Whole-cell extract was prepared 24 h posttransfection and used for immunoprecipitation with Flag-specific rabbit polyclonal antibody. The presence of Supt5H-Flag and PA, PB1, and PB2 in the precipitates (upper panel) as well as cell extract (lower panel) was analyzed by Western blotting using Flag and polymerase-specific antibodies. Approximately 3% of total cell extract used for IP was loaded. Numbers at the top of lanes 4 to 6 denote the relative ratios of PA to Supt5H, as determined by densitometric analysis of the immunoprecipitates.