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. Author manuscript; available in PMC: 2013 Apr 25.
Published in final edited form as: Virology. 2012 Feb 8;426(1):51–59. doi: 10.1016/j.virol.2012.01.015

Molecular interactions and trafficking of influenza A virus polymerase proteins analyzed by specific monoclonal antibodies

Leslie MacDonald 1, Shilpa Aggarwal 1, Kendra A Bussey 1, Emily A Desmet 1, Baek Kim 1, Toru Takimoto 1,*
PMCID: PMC3285403  NIHMSID: NIHMS352072  PMID: 22325937

Abstract

The influenza polymerase complex composed of PA, PB1 and PB2, plays a key role in viral replication and pathogenicity. Newly synthesized components must be translocated to the nucleus, where replication and transcription of viral genomes take place. Previous studies suggest that while PB2 is translocated to the nucleus independently, PA and PB1 subunits could not localize to the nucleus unless in a PA-PB1 complex. To further determine the molecular interactions between the components, we created a panel of 16 hybridoma cell lines, which produce monoclonal antibodies (mAbs) against each polymerase component. We showed that, although PB1 interacts with both PA and PB2 individually, nuclear localization of PB1 is enhanced only when co-expressed with PA. Interestingly, one of the anti-PA mAbs reacted much more strongly with PA when co-expressed with PB1. These results suggest that PA-PB1 interactions induce a conformational change in PA, which could be required for its nuclear translocation.

Keywords: Influenza virus, polymerase, monoclonal antibody, protein interactions

Introduction

Influenza A virus has been a major threat to public health for centuries. The emergence of new human infections occurs through reassortment of human and avian viral genes or by direct mutation of avian or swine viruses. It is becoming increasingly clear that specific mutations in avian virus polymerase genes can expand the viral host range, although molecular mechanisms of host adaptation by polymerase mutation are not known (Bussey et al., 2010; Bussey et al., 2011; Naffakh et al., 2008; Yamada et al., 2010). The influenza A virus polymerase, which is responsible for genome replication and transcription, is composed of three components: PA, PB1 and PB2. PB1 contains the catalytic site for RNA synthesis, while PB2 binds the cap structure of cellular pre-mRNA, which is cleaved by PA and used as a primer for viral mRNA synthesis (Boivin et al., 2010). Unlike other RNA viruses, influenza genome replication and transcription takes place in the nucleus of infected host cells. Therefore, nuclear translocation of the components and assembly of the polymerase complex in the nucleus are essential for viral growth. Each polymerase component has a putative nuclear localization signal (NLS) (Boulo et al., 2007). When expressed alone, PB2 is efficiently translocated to the nucleus, while PA and PB1 are distributed both in the cytoplasm and in the nucleus. However, it has been shown that co-expression of PA and PB1 significantly enhances nuclear accumulation of these proteins (Fodor and Smith, 2004). It is proposed that PA and PB1 form dimers in the cytoplasm, which are imported into the nucleus where they associate with PB2 to form the trimeric polymerase complex (Deng et al., 2006; Deng et al., 2005; Fodor and Smith, 2004; Huet et al., 2010). A recent study using a bimolecular fluorescence complementation assay, however, suggested a direct interaction between PA and PB2 occurs in the cytoplasm, and that this dimer is subsequently transported into the nucleus (Hemerka et al., 2009).

Most studies on polymerase protein interactions have been performed using proteins tagged with either eGFP or TAP, which could influence the molecular interactions of the proteins (Deng et al., 2006; Deng et al., 2005; Fodor and Smith, 2004). To further characterize the native polymerase proteins, we created a panel of 16 mAbs against polymerase components. We then used the mAb panel to analyze the molecular interactions between the components and also to determine the localization of the proteins in the host cell. Our results are consistent with the previous model suggesting that the PA-PB1 interaction is required for nuclear localization of the proteins. We also found an anti-PA mAb that preferentially binds to PA only when it is in complex with PB1, suggesting a conformational difference between monomeric PA and PA in a heterodimer with PB1, which could be required for nuclear translocation of the complex.

Results

Production and characterization of mAbs

Hybridoma cell lines were produced using published protocols (Fuller, Takahashi, and Hurrell, 2001) by immunizing Balb/c mice with the purified polymerase complex of A/chicken/Nanchang/3-120/01 (H3N2)(Nan) expressed by recombinant baculoviruses (Aggarwal et al., 2010). After a series of fusions between splenocytes isolated from immunized mice and Sp2/0 mouse myeloma cells, we obtained a total of 16 hybridoma cell lines which secrete mAbs specific for the polymerase proteins. Among them, 6 hybridomas produce anti-PA, 4 anti-PB1 and 6 produce anti-PB2 mAbs (Table 1). All mAbs were of the IgG1 subtype, except F1-2F6 which was IgG2b. Polymerase components recognized by the mAbs were identified by immunofluorescence assay (IF), using 293T cells transfected with cDNAs encoding the Nan polymerase genes. Western blot analysis showed variations in reactivity of the mAbs with denatured proteins. Anti-PA mAbs F1-2C3, F1-2F6, F4-296, and F5-32, but not F1-2A5 or F7-236 reacted with PA protein in Western blot analysis. All of the anti-PB1 mAbs reacted with the viral antigen; however, none of the anti-PB2 mAbs were positive by Western blot (Table 1).

Table 1.

Characterization of the monoclonal antibodies against polymerase proteins

Monoclonal antibodies Isotype Bind to Reactivity by
Reactivity with**
Western IF IP WSN Aichi Cal Nan
F1-2A5 IgG1 PA − + + + + + +
F1-2C3 IgG1 PA + + + + + + +
F1-2F6* IgG2b PA + + + + + − +
F4-296 IgG1 PA + + + + + + +
F5-32 IgG1 PA + + − + + + +
F7-236 IgG1 PA − + + + + + +
F5-10 IgG1 PB1 + + − + + + +
F5-19 IgG1 PB1 + + + + + + +
F5-46 IgG1 PB1 + + + − − − +
F7-87 IgG1 PB1 + + + + + + +
F5-59 IgG1 PB2 − + + + + + +
F5-116 IgG1 PB2 − + + + + + +
F5-122 IgG1 PB2 − + + + + + +
F5-195 IgG1 PB2 − + + + + + +
F6-36 IgG1 PB2 − + + + + + +
F7-168 IgG1 PB2 − + + + + + +
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*

In addition to PA, mAb F1-2F6 binds to multiple cellular proteins in IF and IP, but not in Western.

**

Reactivity was determined by Western blotting for mAbs that react with denatured proteins. All other mAbs were tested by radioimmunoprecipitation.

We also tested the specificity of the mAbs with polymerase proteins from three strains, A/WSN/33 (WSN), A/Aichi/2/68 (Aichi), and A/California/04/09 (Cal), in addition to Nan. Sequencing analysis revealed that the polymerase proteins of these strains are well conserved. Nan PA protein is 96, 95, and 97% identical to WSN, Aichi and Cal, respectively. Nan PB1 shares 96, 97, and 95% identity, and Nan PB2 is 93, 93, and 95% identical to WSN, Aichi and Cal, respectively. However, our mAb panel exhibited specificity against the polymerase proteins from these strains (Table 1). The anti-PA mAb F5-32 reacted equally well with all of the strains tested, while F1-2F6 did not react with Cal PA. The anti-PB1 mAb F5-46 recognized Nan, but not the other PB1 proteins tested. All the anti-PB2 mAbs reacted with PB2 from all 4 strains tested (Table 1).

Interactions between polymerase components

Using these mAbs, we next determined the molecular interactions between untagged polymerase components. Previous structural analyses of the purified domains of polymerase proteins have identified interacting sites between the components. The C-terminal domain of PA interacts with the N-terminal region of PB1, and the C-terminal region of PB1 interacts with the N-terminal domain in PB2 (He et al., 2008; Obayashi et al., 2008; Sugiyama et al., 2009). In addition, a recent report suggests a direct interaction between PA and PB2, although this has not been confirmed by another group (Hemerka et al., 2009).

Most of the previous studies on the interactions between polymerase components have been conducted using proteins tagged with relatively large TAP or GFP tags, which may affect heterotrimer complex formation. To analyze the molecular interactions between the polymerase components, we co-expressed multiple components of the polymerase complex in various combinations and performed immunoprecipitation reactions with specific mAbs. When PB1 was co-expressed with PA, both PA and PB1 were co-immunoprecipitated in the presence of the anti-PA mAb F4-296 (Fig. 1A, top panel, lane 4). Similarly, both PB1 and PA co-immunoprecipitated in the presence of the anti-PB1 mAb F5-19 (Fig. 1A, middle panel, lane 4). In addition, when PB1 and PB2 were co-expressed, the anti-PB1 mAb F5-19 or the anti-PB2 mAb F6-36 co-immunoprecipitated PB2 or PB1, respectively (Fig. 1A, middle and bottom panels, lane 6). These results are consistent with the available structural information indicating PA-PB1 and PB1-PB2 interactions.

Figure 1.

Figure 1

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Radioimmunprecipitation of polymerase proteins expressed in various combinations. A) [35S]Met/Cys-labeled proteins in total lysates were used for immunoprecipitation with indicated mAbs. B) Co-immunoprecipitation of polymerase proteins. Total lysates of radiolabeled cells expressing the indicated polymerase components were applied for immunoprecipitation with anti-PB2 mAb (F6-36). Half of each sample was analyzed by PhosphorImager, and the rest was applied for Western blot analysis using anti-PA mAb (F1-2F6). The additional band (*) could be Hsp90 associated with PB2 (Deng et al., 2005).

PA and PB2 co-migrate in SDS-PAGE analysis (Fig. 1A, lane 5), making it necessary to determine direct PA-PB2 interactions using co-immunoprecipitation followed by Western blot analysis. Either an anti-PA or anti-PB2 mAb in complex with Protein G-Dynabeads was reacted with cell lysates expressing PA alone, PB2 alone, PA and PB2 together, or PA, PB1 and PB2 together. The presence of PA or PB2 was then determined by Western blotting. The anti-PB2 mAb F6-36 immunoprecipitated PA only when all three components, PA, PB1, and PB2, were expressed together (Fig. 1B, lane 4). When only PA and PB2 were expressed together, the anti-PB2 mAb did not co-immunoprecipitate PA (lane 3). The same results were obtained when anti-PA mAb was used for immunoprecipitation (data not shown). Our experiment failed to detect the PA-PB2 interaction recently reported using the bimolecular fluorescence complementation assay (Hemerka et al., 2009), which is consistent with reports suggesting no direct interaction between PA and PB2 in transfected cells (Ohtsu et al., 2002; Toyoda et al., 1996).

Translocation of polymerase components

We next determined the distribution of the polymerase components expressed alone or as a complex in HeLa cells by IF using our mAbs. As expected from recent studies, PA and PB1, when expressed individually, localized both in the nucleus and the cytoplasm of HeLa cells (Fig. 2). In sharp contrast, the majority of PB2, when expressed alone, localized in nuclei. However, when expressed as a PA-PB1-PB2 complex, both PA and PB1 were detected mainly in the nucleus, suggesting that the interactions between the components affect the distribution of the proteins in cells (Fig. 2). Previous studies showed that PA-PB1 complex formation is required for the nuclear translocation of both proteins (Fodor and Smith, 2004). PB1 also interacts with PB2 (Fig. 1), which translocates to nuclei by itself, but the effect of a PB1-PB2 interaction on nuclear translocation of PB1 has not been clearly identified, except in studies using tagged proteins (Huet et al., 2010). To determine the effects of co-expressed PA and PB2 on nuclear translocation of PB1, we expressed PB1 fused with eGFP at its C-terminus (PB1eGFP) together with PA or PB2 and visualized the localization of the proteins by fluorescent microscopy (Fig. 3). First, we confirmed that the fusion of an eGFP tag to PB1 did not influence the interaction with PA and PB2 by co-immunoprecipitation assay. As shown in Fig. 3A, PB1eGFP, when expressed with PA or PB2, was immunoprecipitated with PA or PB2 mAbs, indicating that neither PB1-PA nor PB1-PB2 interactions were interrupted by the eGFP tag at the C-terminus of PB1. We next expressed PB1eGFP together with PA to determine the localization of PB1eGFP in cells co-expressing PA using IF analysis. In cells expressing PB1eGFP alone, PB1eGFP localized mainly in the cytoplasm and, to a lesser extent, in the nucleus (Fig. 3B arrow). However, in cells co-expressing both proteins, the majority of PB1eGFP was detected in the nuclei (arrowheads in Fig. 3B). In sharp contrast, co-expression of PB2 did not significantly affect the localization of PB1eGFP (arrowheads in Fig. 3C), as observed with those transfected with PB1eGFP alone (data not shown). These results indicate that although PB1 can interact with both PA and PB2, PB1 must interact with PA to allow nuclear translocation of PB1.

Figure 2.

Figure 2

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IF analysis of the localization of polymerase components expressed alone or altogether in cells. HeLa cells were transfected with cDNAs that express WSN PA, PB1 or PB2, alone (right columns) or all together (left columns), and localization of the proteins was determined by IF using the specific mAbs indicated in the figure.

Figure 3.

Figure 3

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Nuclear translocation of PB1eGFP by PA, but not PB2. A) Cells transfected with expression vectors that contain PB1eGFP, PA, or PB2 were labeled with [35S]Met/Cys and lysates were used for immunoprecipitation using the indicated mAbs. B) and C) HeLa cells transfected with PB1eGFP together with PA (B) or PB2 (C) were processed for IF analysis using anti-PA or anti-PB2 mAbs. Arrowheads indicate cells expressing both PB1eGFP with PA or PB2, and an arrow in (B) indicates a cell expressing PB1eGFP alone. The additional faint bands present (*) could be cleaved PB1eGFP fragments.

Enhanced reactivity of an anti-PA mAb to the PA-PB1 complex

The molecular mechanism that allows efficient nuclear translocation of the PA-PB1 complex, but not PA or PB1 alone, is not clearly understood. Previous studies, however, have shown that RanBP5 binds to the PA-PB1 complex, and that this interaction is required for efficient nuclear translocation of the PA-PB1 complex (Deng et al., 2006). Conversely, a PB1 mutation that abolishes the interaction with RanBP5 reduced nuclear localization of the complex. However, the mutation did not completely prevent nuclear translocation, suggesting that other host cell factors that interact with the complex are involved in nuclear import of the proteins (Hutchinson et al., 2011). It is possible that the PA-PB1 interaction induces a conformational change in the complexed proteins, which allows an interaction with cellular proteins leading to efficient nuclear import. Therefore, experiments were conducted to investigate whether any of the anti-PA mAbs show higher affinity to the PA-PB1 complex than to monomeric proteins. First, we compared the reactivity of the anti-PA mAbs to PA or the PA-PB1 complex by enzyme-linked immunosorbent assays (ELISA) using purified proteins expressed by recombinant baculoviruses. PA was purified using a recombinant baculovirus which expresses TAP-tagged Nan PA (Aggarwal et al., 2010). The PA-PB1 complex was purified from insect cells co-infected with recombinant baculoviruses expressing TAP-tagged PB1 and tag-free PA by TAP purification techniques, which allowed preparation of PA-PB1 complex free of PA monomer. ELISA plates were coated with purified proteins containing the same amount of PA and reactivity of the anti-PA mAbs was tested. The mAb F5-32 reacted equally well with both PA alone, as well as with the PA-PB1 complex (Fig. 4). F1-2F6 also reacted well with both antigens, although it reacted with PA alone slightly better than with the complex. The mAbs F1-2A5 and F4-296 reacted more strongly with the PA-PB1 complex than with PA alone. F7-236 reacted with the PA-PB1 complex, but not with PA expressed alone. However, this loss of reactivity could be due to the location of the TAP tag on the C-terminal of PA, as described below. F1-2C3, in contrast, reacted more strongly with PA alone than with the PA-PB1 complex. Anti-PB1 mAb recognized the PA-PB1 complex, but not PA alone, while anti-PB2 mAb F6-36 reacted with neither antigen, as expected.

Figure 4.

Figure 4

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Reactivity of mAbs against purified PA or PA-PB1 complex determined by ELISA. Purified PA or PA-PB1 complex containing an equivalent amount of PA was coated on ELISA plates and reacted with the indicated mAbs at various dilutions. Solid line: PA monomer, dashed line: PA-PB1 complex.

Because some of the mAbs reacted more strongly to the PA-PB1 complex than to the PA monomer, we further determined the reactivity of the mAbs by radioimmunoprecipitation (RIP) using untagged polymerase proteins expressed in 293T cells. We expressed PA alone, or PA and PB1 together in 293T cells, and the same amounts of radiolabeled lysates were used for immunoprecipitation. As found with the ELISA results, the mAb F1-2A5 immunoprecipitated greater amounts of PA in complex with PB1 than PA expressed alone (Fig. 5). The mAb F1-2C3 immunoprecipitated PA expressed alone, but not the PA-PB1 complex, although the reactivity was very weak. In contrast with the ELISA results, F4-296 immunoprecipitated both PA and the PA-PB1 complex. The mAb F1-2F6, which reacted well to both PA and PA-PB1 in ELISA, immunoprecipitated not only PA, but also many other cellular proteins nonspecifically (data not shown). F5-32 failed to immunoprecipitate PA either expressed alone or in complex with PB1. F7-236 immunoprecipitated both the PA-PB1 complex and the PA monomer, although reactivity with the PA monomer was weaker than with the PA-PB1 complex. In ELISA using C-terminal-tagged proteins, F7-236 did not react with tagged PA (Fig. 4), suggesting that F7-236 reacts with an epitope that is masked by the peptide tag at the C-terminus.

Figure 5.

Figure 5

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Immunoprecipitation of PA or the PA-PB1 complex by the indicated anti-PA mAbs. 293T cells expressing the indicated polymerase components were radiolabeled and the same amounts of total lysates were applied for immunoprecipitation using the five different anti-PA mAbs.

The reactivity of the PA mAbs was also tested by IF assay (Fig. 6). In agreement with the ELISA and RIP results, F1-2A5 reacted with PA in complex with PB1 much more strongly than with PA alone. F4-296 and F7-236 reacted with PA alone, as well as with the PA-PB1 complex, although fluorescent signals were stronger in cells expressing both PA and PB1. F1-2C3 reacted strongly with PA alone and weakly with the PA-PB1 complex, while F5-32 reacted equally well with PA alone and with the PA-PB1 complex (Fig. 6A). In cells infected with WSN virus, F4-296, F5-32 and F7-236 detected PA in both the nucleus and cytoplasm at 9 h post infection (Fig. 6B). F1-2A5 and F1-2C3, however, did not react well with PA in virus-infected cells, suggesting the interaction with PB2 or NP may block the PA epitope recognized by these mAbs.

Figure 6.

Figure 6

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IF analysis of anti-PA mAb reactivity in transfected or infected cells. A) 293T cells were transfected with pCAGGS-WSNPA alone or together with pCAGGS-WSNPB1. PA in the cells was stained with various anti-PA mAbs and anti-mouse Texas Red followed by counterstaining with DAPI. The same exposure time was used for each mAb for detection of PA or PA-PB1. B) HeLa cells were infected with WSN for 9 h and reacted with various anti-PA mAbs, as described above.

We also confirmed the reactivity of the mAbs using 2009 pandemic H1N1 (Cal) PA. 293T cells expressing Flag-tagged Cal PA alone or together with untagged Cal PB1 were processed for IF. As expected, F1-2A5 reacted with PA only when co-expressed with PB1 (Fig. 7). In addition, PA proteins recognized by F1-2A5 were detected only in nuclei. Similarly, signals of F4-296 and F7-236 were detected mainly in the nuclei of cells expressing both PA and PB1. Interestingly, signal from F1-2C3 was found mainly in the cytoplasm of cells expressing both PA and PB1, suggesting that PA not in complex with PB1 remains in the cytoplasm.

Figure 7.

Figure 7

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IF analysis of mAb reactivity and cellular localization of antigens. 293T cells were transfected with pCAGGS expressing the indicated genes and reacted with anti-PA mAbs or anti-Flag Ab. PA, PA1–257 and PA258–716 were tagged with Flag. Cells were counterstained with DAPI. N: nuclear localization, C: cytoplasmic localization, N+C: both nuclear and cytoplasmic localization, -: no reactivity.

To further examine the location of the epitopes recognized by the mAbs, Cal PA fragments containing either residues 1–257 or 258–716 were expressed individually and reactivity with the mAbs were determined by IF. F1-2C3, F4-296, and F7-236 reacted with an epitope in residues 258–716, and F5-32 reacted with the N-terminal domain, containing residues 1–257. F1-2A5 did not react with either domain. Our data derived from the three different methods indicate the presence of an epitope on PA, recognized by F1-2A5, which becomes available only when PA and PB1 form a complex. These results suggest a conformational change in PA induced by binding to PB1 or the presence of an epitope composed of both PA and PB1.

Discussion

Recent studies have highlighted the importance of polymerase proteins in host adaptation of influenza A viruses. Specific mutations in PB2 and PA allow the avian influenza virus polymerase to be strongly active in mammalian cells, where otherwise an avian polymerase would be inactive (Bussey et al., 2010; Bussey et al., 2011; Gabriel et al., 2005; Hatta et al., 2001; Li et al., 2005; Yamada et al., 2010; Yao et al., 2001). The mechanism of how specific mutations in polymerase proteins confer functionality in mammalian hosts is not known. However, previous work suggests that a mutation at PB2 residue 627 affects the interaction between the polymerase complex and NP, which is essential for viral genome replication and transcription (Labadie et al., 2007; Mehle and Doudna, 2008; Rameix-Welti et al., 2009). Other studies suggest that specific PB2 mutations enhance binding to importin-α and affect nuclear translocation (Boivin and Hart, 2011; Gabriel, Herwig, and Klenk, 2008). In addition, the contribution of mammalian-specific interacting proteins is suggested indicating that either an inhibitory factor blocks the avian polymerase or a co-factor enhances the human polymerase (Mehle and Doudna, 2008; Moncorge, Mura, and Barclay, 2010). So far, however, any conclusive mechanism that explains host adaptation by polymerase mutations has not been proposed. The availability of reagents, such as the mAbs produced for this study, will contribute to the understanding of the role of the influenza polymerase in host adaptation and pathogenicity.

To facilitate further analysis of the functions and molecular interactions of polymerase proteins, we created 16 hybridoma cell lines which secrete mAbs against polymerase components. This panel of mAbs allows direct analysis of the molecular interactions between native viral components without the addition of a tag. Recent analyses of polymerase proteins have revealed the structure of a direct interaction site between the C-terminal region of PA with the N-terminal peptide of PB1 (He et al., 2008; Obayashi et al., 2008). Also, the C-terminal region of PB1 and the N-terminal region of the PB2 peptide were shown to form a complex by X-ray analysis (Sugiyama et al., 2009). Consistent with structural data, PB1 was co-immunoprecipitated with PA or PB2 (Fig. 1A), which agrees with previous reports (Deng et al., 2005; Gonzalez, Zurcher, and Ortin, 1996; Perez and Donis, 2001; Toyoda et al., 1996). A recent study suggests a direct interaction between PA and PB2 without PB1 using a bimolecular fluorescence complementation assay (Hemerka et al., 2009). However, a majority of reports suggest no direct interaction between PA and PB2. Our studies also did not detect the formation of a PA-PB2 complex in the absence of PB1 (Fig. 1B), suggesting that PA-PB2 direct binding, if it does exist, would be a low affinity interaction.

Unlike in the majority of RNA viruses, influenza genome replication and transcription take place in the nucleus, requiring nuclear translocation of the polymerase components for virus replication (Boulo et al., 2007). Early studies suggest that individually expressed PA, PB1, and PB2 can enter the nuclei (Akkina et al., 1987; Jones, Reay, and Philpott, 1986; Mukaigawa and Nayak, 1991; Nath and Nayak, 1990; Nieto et al., 1994; Nieto et al., 1992), and nuclear localization signals (NLS) in each polymerase component have been identified (Mukaigawa and Nayak, 1991; Nath and Nayak, 1990; Nieto et al., 1994). Unlike earlier studies, but in agreement with the study by Fodor and Smith (2004), individually expressed PA and PB1 were found to be distributed mainly in the cytoplasm but also to some extent in the nucleus, while PB2 accumulated only in the nucleus (Fig. 2). Co-expression of the three components was shown to result in the accumulation of all three components in the nucleus, supporting the hypothesis that interaction between the components enhances nuclear translocation of PA and PB1. Using GFP-tagged PB1, we confirmed the previous finding that co-expression of PA, but not PB2, significantly enhances nuclear localization of PB1 (Fodor and Smith, 2004). It is still unclear why interaction with PB2, which can be detected in transfected cells (Fig. 1), does not allow the nuclear translocation of PB1 (Fig. 3). Since co-expression of PB1eGFP did not affect the nuclear accumulation of PB2, it is possible that the PB1-PB2 interaction we detected by co-immunoprecipitation could only occur in the nucleus between PB2 and the fraction of PB1 located in the nucleus.

The binding of PB1 to PA dramatically changed the localization of both PB1 and PA proteins (Fig. 3), which was not the case with the interaction of PB1 with PB2, supporting the hypothesis that PA-PB1 complex formation precedes nuclear translocation (Deng et al., 2006; Fodor and Smith, 2004). A recent study showed that interaction of the PA-PB1 dimer with the nuclear import factor RanBP5 plays a major role in the nuclear translocation of the complex (Deng et al., 2006; Hutchinson et al., 2011). Mutation at the PB1 NLS abolished the interaction with RanBP5 and reduced, but did not prevent, nuclear localization of the complex. Their data suggest that interaction with RanBP5 plays an important role in the nuclear translocation of the PA-PB1 complex. However, this also indicates that other factors are involved in the nuclear translocation of the PA-PB1 complex, since a PB1 mutation that abolished the interaction with RanBP5 did not completely prevent nuclear translocation of the PA-PB1 complex. It is possible that the NLS of PA is involved in the nuclear translocation of the complex. Because PA was not able to translocate to nuclei efficiently when expressed alone (Fig. 2), the proposed PA NLS, which exists within the N-terminal 247 residues of PA (Nieto et al., 1994), could be exposed by a structural change upon binding to PB1. In fact, mAbs F1-2A5 reacted with the PA-PB1 complex much stronger than with PA alone, which may reflect the structural difference in PA (Figs. 4–7). As far as we are aware of, this is the first mAb that was found to recognize the PA-PB1 complex better than the PA monomer. It is also possible, however, that F1-2A5 recognizes an epitope composed of both PA and PB1. The PA molecule is composed of two domains separated by a long linker peptide that can be cleaved by limited tryptic digestion (Guu et al., 2008; Hara et al., 2006). PA in complex with PB1 was reported to be highly resistant to tryptic digestion as compared to monomeric PA, also suggesting a possible conformational change in PA upon PB1 binding (Guu et al., 2008). Together with the previous studies, our data suggest that PA binding to PB1 induces a structural change, which may enhance an interaction with cellular proteins resulting in nuclear translocation of the PA-PB1 complex. Further study on the role of the NLS in PA is expected to unveil the mechanism of nuclear translocation of the PA-PB1 complex.

Materials and Methods

Preparation of polymerase complex

Polymerase complex was prepared as described previously (Aggarwal et al., 2010). Briefly, PA, PB1, and PB2 genes of A/chicken/Nanchang/3-120/01 (H3N2) were cloned into pVL1392 (Invitrogen) and recombinant baculoviruses were produced by the protocols provided by the vendor (BD Biosciences Pharmingen). The PA gene was tagged at the C-terminus with the tandem affinity purification (TAP) tag, which consisted of a thrombin cleavage site followed by a 6X His tag, a tobacco etch virus (TEV) cleavage site and finally an IgG-binding domain. Trimeric polymerase complex was prepared from Tni insect cells infected with the three recombinant viruses by the TAP purification technique.

Immunization of mice and preparation of hybridoma cell lines

Balb/c mice were injected subcutaneously with 20 μg of the purified polymerase complex mixed with Freund’s Incomplete Adjuvant. Two booster immunizations (20 μg of protein) were given intraperitoneally at day 10 and 18. Splenocytes were isolated on day 21. Hybridoma cells were generated by standard procedures using Sp2/0 myeloma cells (Fuller, Takahashi, and Hurrell, 2001). The hybridoma culture supernatants were screened by ELISA using purified polymerase proteins as antigen. The isotyping of obtained mouse mAbs was performed using IsoStrip™ Mouse Antibody Isotyping Kit (Santa Cruz Biotechnology). Cell lines secreting the mAb of interest were cloned using limiting dilutions. The mAbs were produced from the hybridoma cells as ascites fluid, culture supernatant, or concentrated culture supernatant using the CELLine bioreactor system (Sigma). All of the hybridoma cells were deposited to BEI resources.

cDNAs

WSN PA, PB1, and PB2 genes in pCAGGS were generously provided by Y. Kawaoka (University of Wisconsin, Madison). WSN PB1 fused with the eGFP gene was created as follows. First, a KpnI site was created at the end of the PB1 coding region using the QuickChange Mutagenesis Kit (Stratagene). The eGFP gene was amplified by PCR from pEGFP-N1 (Clontech) using primers containing KpnI sites flanking the gene, and was inserted into the PB1 gene in pCAGGS. Cal PA and PB1 genes were synthesized by RT-PCR from RNA extracted from cells infected with A/California/04/2009 (H1N1). The PB1 gene was directly cloned into pCAGGS. PA gene was first subcloned into pCMV-Tag4a (Stratagene) to obtain a Flag-tagged gene before insertion into the pCAGGS vector. Flag-tagged CalPA1–257 was constructed from pCAGGS-CalPA by PCR using a forward primer containing a SacI site and reverse primer containing the Flag tag sequence and a SphI site. Similarly, CalPA258–716 was constructed using appropriate primers that amplify the PA gene encoding residues 258–716 with SacI and SphI sites in forward and reverse primers, respectively.

Immunological assays

To identify the polymerase component recognized by each mAb, 293T cells were transfected with pCAGGS vectors containing Nan PA, PB1, or PB2 by Lipofectamine 2000 (Invitrogen). Twenty-four h after transfection, cells were fixed and permeabilized with methanol/acetone (1:1), and reacted with the culture supernatants of the hybridomas, followed by detection with anti-mouse IgG-Texas Red (TR). For Western blot analysis, 40 μg of purified virus (Nan) grown in eggs were used as antigen. After separation by SDS-PAGE, viral proteins were transferred to a PVDF membrane, and reacted with each mAb.

Immunoprecipitation

To compare the reactivity of mAbs with PA alone or with the PA-PB1 complex, 293T cells were transfected with either pCAGGS-WSNPA and pCAGGS, or pCAGGS-WSNPA and pCAGGS-WSNPB1 by Lipofectamine 2000. After 16 h incubation, cells were labeled with [35S]Met/Cys (Perkin Elmer) for 6 h, and lysed with a Nuclear Extraction Triton buffer (20mM Hepes pH7.9, 1.5mM MgCls, 500mM NaCl, 0.2mM EDTA, 20% Glycerol, 1% Triton X-100). Labeled proteins in lysates were immunoprecipitated using specific mAbs and Dynabeads Protein G (Invitrogen).

Enzyme-linked immunosorbent assay (ELISA)

PAtap and the PA-PB1tap complex were purified from Tni insect cells infected with recombinant baculoviruses, as described above. Purified proteins were analyzed by SDS-PAGE, stained with SimplyBlue SafeStain (Invitrogen), and aliquots containing the same amount of PA protein were coated to 96-well plates. The plates were incubated with dilutions of each mAb, followed by anti-mouse IgG-horseradish peroxidase (1:5,000 dilution)(PIERCE) and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(Sigma). The optical density of the samples at 405 nm was measured using SpectraMax Plus (Molecular Devices). The original mAbs were diluted as follows: F1-2A5 (ascites, 1:100), F1-2C3 (ascites, 1:1,000), F1-2F6 (ascites, 1:3,000), F4-296 (concentrated supernatant, 1:300), F5-32 (concentrated supernatant, 1:100), F7-236 (culture supernatant, 1:30), F7-87 (culture supernatant, 1:10), and F6-36 (culture supernatant, 1:30).

Immunofluorescence analysis

Reactivity of the mAbs and localization of the antigen in cells transfected with PA or PA-PB1 or infected with WSN were analyzed by IF. 293T or HeLa cells were transfected with the polymerase genes in pCAGGS using Lipofectamine 2000 (Invitrogen) or infected with WSN at a MOI of 0.3. After 24 h transfection or 9 h infection, cells were fixed with 3.5% formaldehyde in PBS and permeabilized with Methanol/Acetone (1:1) at −20°C. These cells were incubated with each mAb or anti-Flag rabbit serum (Sigma) followed by anti-mouse or anti-rabbit IgG-Texas Red (Invitrogen) and counterstained with DAPI. Dilutions of the mAbs used for the reaction were F1-2A5 (ascites 1:1,000), F1-2C3 (ascites 1:1,000), F4-296 (concentrated supernatant, 1:1,000), F5-32 (concentrated supernatant, 1:1,000), F6-36 (concentrated supernatant, 1:100), F7-87 (culture supernatant, 1:10), F7-168 (culture supernatant, 1:30), and F7-236 (culture supernatant, 1:30). All the images were taken using an Olympus inverted microscope.

Highlights.

  • New mAbs against influenza polymerase proteins were produced.

  • PA-PB1 and PB1-PB2, but not PA-PB2 interactions were confirmed by co-immunoprecipitation.

  • PA and PB1 were localized in nuclei only when they were co-expressed.

  • Structural change of PA when in complex with PB1 was suggested based on the reactivity with some anti-PA mAb.

Footnotes

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