INTRODUCTION

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The influenza A virus genome consists of eight single-stranded RNA segments of negative polarity (viral RNAs [vRNAs]) (26, 36). In virions, each vRNA segment is assembled into a ribonucleoprotein particle (RNP) by association with the nucleoprotein and the three viral polymerase subunits (PB1, PB2, and PA; herein referred to as P proteins) (30). Other viral proteins mediate morphogenesis of virions by budding at the plasma membrane. Upon entry into a permissive cell by endocytosis and envelope fusion, viral RNPs travel to the nucleus, where transcription and replication of vRNA segments takes place (7, 21). The viral RNPs constitute the active transcription and replication competent unit (25). During infection, incoming vRNAs are initially transcribed into viral mRNAs. Nascent host cell polymerase II pre-mRNA transcripts provide capped oligonucleotides to initiate viral transcription (6). Polyadenylation of viral transcripts to yield mRNA occurs through a polymerase stuttering mechanism involving an oligo(U) signal adjacent to the RNA panhandle structure at the 5' termini of the vRNA genes (40, 45). During replication, the P proteins switch to a primer-independent mode of RNA synthesis, generating full-length copies of cRNA which are used as intermediates for the production of progeny vRNAs.

The P proteins are found largely as heterotrimeric complexes within virions or the nuclei of infected cells (12, 33). Of the three P proteins, PB1 is the best characterized functionally. Biochemical and structural analyses recognize PB1 as responsible for RNA chain elongation. PB1 contains amino acid motifs common to all RNA-dependent RNA polymerases and RNA-dependent DNA polymerases (32). Mutations within these motifs render the complex inactive for transcription and replication in tissue culture cells (5). In cell-free systems, e.g., nuclear extracts of insect cells expressing PB1 alone, the protein can catalyze the synthesis of RNA using synthetic short minigenome model RNAs as templates, in a primer-dependent mode. PB2 activity appears essential for transcription (27). PB2 binds to methylated cap-1 structures at the 5' termini of actively transcribed cellular mRNAs which are subsequently endonucleolytically cleaved by the P complex, producing 10- to 13-mers used for priming of viral mRNA transcription (6). On the other hand, PA seems required for vRNA replication, although its role in this process remains obscure. Temperature-sensitive influenza PA mutants are severely impaired in vRNA replication with normal viral mRNA synthesis (29).

At least one essential role has been tentatively assigned to each P protein in viral replication, yet some of these functions seem to be dependent on the formation of the heterotrimer for optimal viral RNA transcription and replication (24, 25). Although it is conceivable that heterotrimer subunit interactions may allow more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Elucidating the P protein interactions will help understand the integration of P protein catalytic activities in viral RNA replication and mRNA transcription. Using a mammalian two-hybrid system, we have previously identified the N-terminal 48-amino-acid region of PB1, termed domain , as a determinant of interaction with PA (39). The interaction between PA and domain  appeared neither modulated nor altered by other regions of the PB1 polypeptide. This observation is supported by the findings of Toyoda et al. and González et al. (19, 42). Interestingly, a high degree of sequence conservation was found within the domain  of homologous PB1 polymerases from influenza virus types B and C, the salmon orthomyxovirus, and the orthomyxovirus-like insect viruses Thogoto and Dhori (39). Here we report the high-resolution functional map of the PB1 domain  boundaries: only the N-terminal 12 amino acids of PB1 are required for interaction with PA. We used a model influenza virus reporter minigenome (RG) to analyze the role of specific PB1 regions in viral gene expression and genome replication (25, 38). Efficient transcription of viral RG into translatable mRNA requires the PA protein and a functional PA binding  domain on PB1. Influenza viruses bearing these mutant PB1 genes were either nonviable or displayed severely impaired growth. These results indicate that the PB1-PA interaction is essential for virus viability in cell culture.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Enzymes reagents and software. DNA restriction and modification enzymes were purchased from Promega (Madison, Wis.), New England Biolabs (Beverly, Mass.), and Roche Molecular Biochemicals (Indianapolis, Ind.). Reagents for DNA sequencing were obtained from United States Biochemical (Cleveland, Ohio). Fetal bovine serum (FBS), antibiotic solution (containing penicillin and streptomycin), L-glutamine, sodium butyrate, and butiryl coenzyme A (butiryl-CoA) were from Sigma (St. Louis, Mo.). Dulbecco's modified essential medium (DMEM) and Lipofectamine reagent were bought from Gibco/BRL (Gaithersburg, Md.), and [¹⁴C]chloramphenicol and Tran³⁵S-label were bought from ICN (Costa Mesa, Calif.). Oligonucleotides were purchased from Ransom Hill (Ramona, Calif.). Sequence alignments were performed using the Wisconsin Genetics Computer Group package (13) and influenza virus type A, A/WSN/33 (Swissprot accession no. P03430); influenza virus type B, B/Lee/40 (Swissprot accession no. P07832); and influenza virus type C, C/JJ/50 (Swissprot accession no. P19703). Secondary-structure prediction analyses were done with the Jpred multiple approach consensus algorithm of Cuff and colleagues (8, 9). Rabbit monspecific polyclonal antisera against PB1, PB2, and PA proteins were kindly provided by D. Nayak (1).

Cells and vaccinia T7 recombinant virus. CV-1 and Cos-7 African green monkey cells (ATCC CCL-70 and CRL-1651, respectively; American Type Culture Collection, Manassas, Ua.) were maintained in DMEM supplemented with 10% (vol/vol) FBS. MDCK and 293T cells were kindly provided by E. Hoffmann and maintained in DMEM-5% FBS. Cloning and mutagenesis procedures were carried out in Escherichia coli mutS and DH10B strains (2). Recombinant vaccinia virus encoding T7 RNA polymerase (vaccinia-T7 virus) strain vTF7-3 was obtained through the AIDS Research and Reference Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health (catalog no. 356) (18).

Plasmid constructions. Plasmids pSG424, pAASVVP16, and pG5EC for the two-hybrid system in mammalian cells were provided by H. A. Vasavada and G. N. Nallur (Yale University, New Haven, Conn.) (43). pSGPB1t, pSGPB1c, and pVP16PA have been previously described (39). Plasmids pcDNA762/PB2, pcDNA787/PA, pcDNA693/NP, and p2HHCATHdR for the influenza virus minigenome replication (P5) system have been described previously (38). pCMV/SEAP was provided by B. Cullen (3). pGEMT7Luc was acquired from Promega. Plasmids pHW181 through pHW188 for rescue of influenza viruses were kindly provided by E. Hoffman (23). A SmaI/BamHI fragment from pSGPB1t encoding the first 654 amino acids of the PB1 gene from A/WSN/33 was ligated into pSP72 (Promega), previously digested with BglII, blunt ended with Klenow fragment, and subsequently digested with BamHI. The resulting intermediate plasmid was further manipulated to generate p72PB1c by incorporation of a BamHI/HindIII fragment encoding the last 103 amino acids of PB1 from A/PR/8/34 (in pcDNA774/PB1) (38).

Two-hybrid assay. CV-1 cells (5 × 10⁵ cells per well) were plated on 35-mm-diameter dishes (Costar, Cambridge, Mass.) 2 to 4 h before transfection. Cells were transfected with 3 µg of total DNA (1 µg of each two-hybrid plasmid and 0.5 µg of each reporter, pG5EC and pCMV/SEAP) and 20 µg of Lipofectamine reagent (Gibco/BRL, Grand Island, N.Y.) per well in a final volume of 1.2 ml of DMEM. DNA-Lipofectamine complexes were left in contact with cells for 6 h at 37°C in 5% CO₂ according to the directions of the manufacturer (Gibco/BRL). After transfection, cells were supplemented with 1 ml of DMEM containing 10% (vol/vol) FBS and 10 mM sodium butyrate. Eighteen hours after the start of transfection, DNA-Lipofectamine complexes were removed and replaced with fresh 10% FBS-DMEM.

P5 influenza virus RG assay. The P5 influenza virus RG assay was performed as previously described with minor modifications (38). CV-1 cells were inoculated with vaccinia-T7 at an input multiplicity of infection (MOI) of 5 and incubated at 37°C in a moist atmosphere containing 5% CO₂ for 45 min. Lipofectamine (12 µg) in 100 µl of DMEM without serum was mixed with 0.020 µg of pGEMT7Luc and 0.100 µg of each P5 plasmid (pcDNA774/PB1 in the original P5 system was replaced by p72PB1c), and complexes were allowed to form at room temperature (37). Immediately after infection, cells were washed three times with DMEM-serum-free medium, transfected with the DNA-lipid complexes in a final volume of 1.2 ml in DMEM without serum, and incubated for 4 h at 37°C and 5% CO₂. Once transfection was completed, DNA-lipid complexes were removed and cells were incubated for an additional 13 h in the presence of 2 ml of 10% FBS-DMEM. Control replication experiments were performed by substituting the PA encoding plasmid with DNA from pcDNA3neo in the transfection (Invitrogen).

Radiolabeling and coimmunoprecipitation assay. For metabolic radiolabeling of proteins, Cos-7 cells were infected with vaccinia-T7 at an MOI of 10, as explained above, and transfected with a mixture of DNAs, including 1 µg of pcDNA787/PA or pcDNA762/PB2 and 1 µg of the WT or a mutant version of PB1 in p72PB1c (see Fig. 4). At 7 h postinfection (hpi), cells were washed twice with methionine-free medium and incubated in this medium for 1 h. Subsequently, cells were supplemented with Tran³⁵S-label to reach a final concentration of 50 µCi/ml. Labeling was carried out for 2 h at 37°C in a 5% CO₂ moist chamber. After labeling, cells were rinsed twice with 1× phosphate-buffered saline (PBS) and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.5; 2 mM EDTA; 100 mM NaCl; 1% NP-40; 0.5% Na deoxycholate; 0.1% sodium dodecyl sulfate [SDS]; 1% aprotinin). Samples were subdivided into two aliquots and used for coimmunoprecipitation using monospecific polyclonal antibodies against either PB1, PB2, or PA as described (1).

Recovery of influenza viruses with mutant PB1 genes. Transfections with eight plasmids for rescue of influenza virus were performed essentially as described by Hoffman et al. (23). At 72 h posttransfection (hpt), virus in supernatant was removed and used to inoculate confluent MDCK cells (second blind passage). Plaque assays using agar overlay were performed with 10-fold serial dilutions of the second-blind-passage virus. Plaque-purified viruses were propagated on fresh MDCK cells and saved for future use. A 200-µl aliquot of the supernatant of the plaque-purified virus was subjected to RNA isolation using RNEasy (Qiagen, Inc., Valencia, Calif.) and reverse transcription-PCR using avian myeloblastosis virus reverse transcriptase and Taq polymerase (Roche Molecular Biochemicals) and a specific set of primers (available upon request). PCR products were purified using the Qiaquick PCR purification kit (Qiagen) and sequenced as described above. Positive plaques were further propagated at least two more times, and RNA was extracted for PCR and sequencing analysis as explained above. In all cases the predicted mutation was maintained in the virus progeny. To compare virus yield, MDCK cells in 75-cm² flasks were infected with WT or mutant virus at an MOI of 0.1. Two days after infection (6 days in the case of mutants L7D and L10D starting with an MOI of 1 and 3 days in the case of mutant P5L), supernatants were separated from floating cells by low-speed centrifugation, and virus yield was analyzed by plaque assay as explained above. The strategy outlined above was repeated at least one more time with those mutants that produced virus. Virus rescue for PB1 mutants D2V, V3D, L8D, F9D, and A14D was attempted four times, with identical negative results.

CAT, alkaline phosphatase, luciferase, and Western blot analyses. Cells were harvested 48 hpt (two-hybrid mammalian protein-protein interaction) or 18 hpi with vaccinia-T7 virus (influenza virus P5 replication system) in 70 µl of lysis buffer (100 mM KPO₄ pH 7.6; 1 mM dithiothreitol). Chloramphenicol acetyltransferase (CAT) activity was analyzed as described (20) except that butiryl-CoA was used instead of acetyl-CoA and CAT activity was measured by either liquid scintillation counting using a Wallac counter from LKB-Pharmacia (Gaithersburg, Md.) or thin-layer chromatography. Conversion of [¹⁴C]chloramphenicol into the acetylated forms was allowed to progress for 1 h at 37°C and represented approximately 60% of maximal conversion rate for the most active constructs (expressing WT PB1). Thus, only experiments in which CAT activity was in the linear range are reported. Experiments with PB1 mutants that lacked CAT activity per standard assay conditions were subsequently carried out using an extended incubation period (10 h) to increase the sensitivity of the assays.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Refined map of the PA-binding site of PB1. PB1 and PA interact to assemble influenza virus transcription and replication complexes, which also include PB2. A stretch of 48 amino acids at the N terminus of PB1 was shown previously to be sufficient for binding of PA in vivo (39). Our previous experiments also showed that the 709 amino acids outside of this 48-amino-acid N-terminal region of PB1 did not affect binding of PA. The first goal of this work was to refine the mapping of the domain  boundaries using a mammalian two-hybrid interaction assay. To this end, we engineered a set of nested deletion mutants within the PB1 N-terminal 48 amino acids, fused to the GAL4 DNA binding domain (GAL4 DBD), termed GAL4PB1 chimeras (Fig. 1). In some of these deletion mutants, the modification of restriction site-generated DNA ends resulted in the creation of glycine or serine amino acid codons flanking the deletion (Fig. 1). To assess the PA-binding properties of the mutant GAL4PB1 chimeras in two-hybrid assays, they were cotransfected into CV-1 cells with a mixture of the VP16PA transcription activation domain plasmid and the DNA encoding the CAT reporter controlled by the GAL4 promoter. In this system, the interaction of two chimeric proteins activates CAT mRNA transcription (39). The magnitude of two-hybrid reporter activity can be correlated with the strength of the interaction between the two chimeras, as reported by Estojak et al. (15). Efficient PA binding was observed with PB1 mutants carrying various deletions within the region spanning residues 18 to 47 (Fig. 1). The levels of CAT observed for these PB1 mutants were indistinguishable from that obtained with the WT PB1 chimeric protein. In contrast, deletions encompassing all or part of the N-terminal 17 amino acids of PB1 did not mediate PA binding. PB1i18-30 shows interaction despite the alanine-to-glycine substitution at position 17, suggesting that this residue is not essential for binding PA. Taken together, the deletion mapping data suggested that the region responsible for binding PA is located within residues 1 to 17 of PB1.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Amino acid residues essential for the interaction of PB1 with PA in a mammalian two-hybrid system. Polymerase subunits PB1 and PA from influenza virus A/WSN/33 were fused to GAL4 DBD and VP16 AD, respectively, and tested for interaction in a mammalian two-hybrid assay as described (39). Small N-terminal (N) and internal deletions (i) were introduced within the first 48 amino acids of PB1 fused to GAL4. PB1 deletion mutants that interact with PA, such as the WT PB1 chimeric construct GAL4PB1, as revealed by CAT expression, are marked (+); lack of interaction is also shown (). The amino acid positions of the deletion termini are reflected in the nomenclature (e.g., positions 10 to 18 in GAL4PB1i10-18). Except when printed in italics, G (glycine) and S (serine) shown flanking the deletion were absent in wild-type PB1; they arose from codons created by modification of DNA overhangs prior to ligation. The numeric ruler indicates the amino acid positions within PB1 (domain ). Vertical dotted lines indicate the position of -sheet structure boundaries predicted by Jpred analysis of the first 50 amino acids of PB1 (10).

Site-directed mutagenesis of PB1 domain . (i) Effects on PA interaction. In order to identify the individual amino acids within the 16 N-terminal residues of PB1 that are indispensable for binding to PA we expressed mutants in which every residue in this region was replaced (Fig. 2). We engineered mutants carrying aspartic acid in place of the nonpolar residues in the WT sequence. Thus, valine (V) at positions 3 and 12; threonine (T) at position 6; leucine (L) at positions 7, 8, and 10; and phenylalanine (F) at position 9 were individually replaced with aspartic acid (D). Proline (P) residues 5 and 13 were replaced with leucine and aspartate, respectively. The basic residue lysine (K) 11 and the amide residue asparagine (N) 4 were also replaced with aspartate. The only aspartic acid residue in this region, at position 2, was mutated to valine. A double mutant was prepared in which glutamine (Q) 15 and asparagine 16 were supplanted by aspartate and valine.

View larger version (43K): [in this window] [in a new window]   FIG. 2.   Site-directed mutagenesis of the PA-binding domain in GAL4PB1. Single or double amino acid substitutions were introduced within PB1 and GAL4 DBD coding regions. Amino acids are indicated in single-letter code; dots denote unchanged amino acids. Mutant PB1 interaction efficiency with VP16PA, based on CAT transactivation in the two-hybrid assay, is indicated as a percentage relative to the WT GAL4PB1 (set arbitrarily at 100%). A minimum of three independent experiments were carried out for each mutant (the standard deviation for each mutant is shown). Two-hybrid assays were performed with pSGPB1t-derived PB1 mutants, lacking the C-terminal 103 amino acids of PB1. Asterisks indicate mutants that were tested also as GAL4PB1 chimeras encoding the entire ORF of PB1, with similar results (see Materials and Methods).

(ii) Effects on polymerase activity. To assess the role of the PB1-PA interaction in influenza virus genome replication and gene expression in vivo, we utilized a modified plasmid-based influenza virus transcription-replication system. CV-1 cells are infected with vaccinia-T7 virus and transfected with a mixture of five plasmids with transcription units under the control of the bacteriophage T7 promoter. One of the plasmids in the set encodes a model influenza virus reporter gene flanked by two cis-acting ribozymes. Transcription of the precursor reporter gene RNA and subsequent cis cleavage at specific locations result in a mature RNA whose termini are identical to authentic influenza virus A genomic RNA segment 8 and flank the reporter CAT, mimicking an influenza virus vRNA (38). The remaining four plasmids in the set express the influenza virus nucleoprotein and the three-polymerase subunits. In this vaccinia-T7-driven influenza virus RG system, transfection of the five-plasmid set (P5) is necessary and sufficient for CAT expression in cells, as reported previously (38). Omission of a plasmid expressing any of the four proteins causes a >99% reduction in RG CAT accumulation (reference and data not shown). Because total reporter activity from the P5 RG system is the result of transcription and replication, it is important to emphasize that no RG expression is observed in the absence of PA (data not shown), consistent with previous observations (25).

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Expression of PB1 mutants in primate cells. WT and mutant PB1 were expressed in CV-1 cells infected with vaccinia-T7 and transfected with expression plasmids. Cells were harvested at 18 hpi, and lysates were analyzed by Western blotting with a monoclonal antibody to the HA epitope tag (12CA5). WT and representative mutants of PB1 are shown (D2V, V3D, N4D, L7D, L8D, F9D, L10D, and A14D). A lysate from nontransfected cells was used as a negative control (), while a lysate expressing an unrelated HA-tagged protein of 56 kDa served as positive control (+). Proteins were separated on an SDS-10% PAGE gel and transferred to a Hybond-C membrane (Amersham). The luminescent signal (ECL; Amersham) was recorded by exposure to X-ray film, which was subsequently developed, scanned, and digitized using Adobe Photoshop software. Small arrowheads indicate the two PB1 polypeptides that reacted with the anti-HA antibody.

(iii) Effects on virus viability. A correlation between the PA interaction capability of the PB1 mutants and their ability to function in the RG system was observed. These mutants included some with intermediate polymerase function relative to WT (e.g., 30 to 60% activity). The biological significance of intermediate polymerase activities could be best interpreted by evaluating the viability of influenza viruses bearing mutant PB1 genes. New developments in reverse genetics of influenza virus allow the recovery of recombinant or mutant influenza A viruses without the use of a helper virus, extending the application of influenza virus to the engineering of PB1 (16, 23, 35). We engineered PB1 mutants using the method recently described by Hoffman et al. in which eight-plasmid transfections into cells lead to the production of infectious influenza virus particles with high efficiency (10⁶ PFU) (23). Transfection of plasmids with single amino acid substitutions in PB1 along with seven other plasmids encoding the remaining influenza virus genes of the influenza virus A/WSN/33 strain into 293T-MDCK cocultured cells resulted in the production of recombinant viruses for the mutants without lethal mutations. In direct correlation with our polymerase activity data, we were able to rescue mutant viruses that displayed detectable and significant polymerase activity. The exception is mutant F9D, which had only a 3% polymerase activity of the WT (Table 1) and thus might be too low for virus rescue. Supernatants of rescued mutant viruses were subjected to plaque purification and subsequent RNA isolation, reverse transcription-PCR, and sequence analysis of the PB1 gene. All rescued viruses contained the introduced mutation in PB1 and were stable after successive passages in tissue culture (data not shown). An initial characterization of this panel of mutant viruses indicates that their growth characteristics are different from the WT A/WSN/33 virus; all display very small plaque morphology (diameters from 0.5 to 1.0 mm, compared to 2.5 to 3.0 mm in the WT) and low yields of infectious progeny in cell culture. The entire process of virus rescue, from the transfection of eight plasmids to plaque purification and growth phenotype determination was repeated at least one more time for each mutant. The same results were obtained for each mutant in the repetitions, including their growth characteristics, suggesting the absence of spurious substitutions that could arise with these manipulations. However, a detailed molecular characterization of these viruses will be the subject of a future report.

Analysis of PA binding to PB1 mutants by coimmunoprecipitation. The observation that PB1 mutants P5L, L7D, and L10D failed to interact with PA in two-hybrid assays but displayed low polymerase activity and were viable upon plasmid rescue from transfected cells was intriguing (Table 1). Influenza virus mutants L7D and L10D replicated very poorly, requiring 6 days in culture to reach titers on the order of 10³ PFU/ml starting from an MOI of 1. Mutant P5L was only marginally viable, with a titer of <10³ PFU/ml achieved after 3 days in MDCK cells (Table 1). This partial conflict between the two-hybrid interaction data, polymerase acitivity, and virus viability upon rescue was explored further using a biochemical assay. PA was coexpressed in cultured cells with each of the PB1 domain  mutants, and the formation of radiolabeled heteromeric complexes was analyzed by coimmunoprecipitation with monospecific anti-PB1 or anti-PA antibody (1). Interestingly, PA was found to bind to all PB1 mutants, albeit with different efficiencies (Fig. 4). PA binding to these PB1 mutants is specific; PA was not immunoprecipitated in the absence of PB1, ruling out the existence of artifacts caused by the anti-PB1 antibody (Fig 4, lane 8). Although this assay did not allow quantitation of interaction efficiencies it shows that defective binding rather than absence of PA binding is likely responsible for lower polymerase activity. As such, these results explain the discrepancy between the two-hybrid and polymerase data observed for mutants L7D and L10D (Fig. 4A, lanes 3 and 6). We also tested PA binding to D2V and P5L mutants by coimmunoprecipitation. PA binding to the D2V mutant was readily detected, in agreement with our two-hybrid data (Fig. 4A, lane 11). Only a weak binding between PA and P5L was detected (Fig. 4A, lane 14), in general agreement with our two-hybrid data. However, considering that the P5L mutant possesses polymerase activity and a viable virus was rescued, it is conceivable that the interaction between this mutant and PA is unstable and difficult to detect by these methodologies.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Coimmunoprecipitation of PA-PB1 complexes. (A) PA and PB1 proteins were coexpressed in Cos-7 cells using the vaccinia-T7 virus system, radiolabeled, and immunoprecipitated with an anti-PB1 monospecific antibody (see Materials and Methods). Lanes: 1, PA-PB1 WT; 2, PA-T6D; 3, PA-L7D; 4, PA-L8D; 5, PA-F9D; 6, PA-L10D; 7, PB1 WT alone; 8, PA alone; 9, mock-transfected control; 10, PA-PB1/[11]; 11, PA-D2V; 12, PA-V3D; 13, PA-N4D; 14, PA-P5L; 15, PA-K11D; 16, PA-V12D; 17, PA-P13D; 18, PA-A14D; 19, PB1/[11] alone. Proteins were separated on an SDS-7.5% PAGE gel, treated for fluorography, and exposed to X-ray film. Approximately 36 h after exposure, the films were developed, scanned, and digitized using Adobe Photoshop software. *, presence of the second PB1-specific band. (B) Immunoprecipitation as described in panel A except that anti-PA monospecific antibody was used. Proteins were separated on an SDS-6.0% PAGE gel and treated as described in panel A. are, presence of a vaccinia-expressed protein that coprecipitates with the anti-PA antibody and migrates on the polyacrylamide gel like the PB1 protein.

View larger version (24K): [in this window] [in a new window]   FIG. 5.   Coimmunoprecipitation of PB2-PB1 complexes. PB2 and PB1 proteins were coexpressed in Cos-7 cells using the vaccinia-T7 virus system, radiolabeled, and immunoprecipitated with an anti-PB1 monospecific antibody. Lanes: 1, PB2-D2V; 2, PB2-V3D; 3, PB2-P5L; 4, PB2-A14D; 5, PB2-PB1 WT; 6, PB2 alone. *, presence of the second PB1 specific band. After coimmunoprecipitation, proteins were treated as described in Fig. 4A.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

A region of 48 amino acids at the N terminus of PB1 was previously reported to include the interface for recruitment of PA to the polymerase complex (39). Similarly, coimmunoprecipitation of PA protein with deletion mutants of PB1 transiently expressed in mammalian cells pointed to the importance of the N terminus of PB1 to bind PA (this report and reference 42). The alignment of the PB1 genes from influenza virus types A, B, and C, as well as those from the insect and fish orthomyxoviruses, revealed a clustering of amino acid sequence conservation towards the C terminus of this 48-amino-acid region (residues 17 to 48). Because of the putative functional importance of P complex formation, we favored the notion that the PB1 domain that binds PA would be conserved among members of the family. Thus, the interaction was proposed to lie between residues 17 and 48 (39). Contrary to that prediction, the high-resolution mapping of the PB1 domain  entailing functional analyses of two sets of PB1 mutants revealed that the interaction of domain  with PA actually occurs through the N-terminal 12 amino acids. The presence of a -sheet structure spanning residues 6 to 12 is predicted by the Jpred algorithm of Cuff and Burton (data not shown) (8, 9). The loss of interaction resulting from substitutions at proline 5 indicates that the integrity of this structure may be required. Although this N-terminal region is absolutely conserved among all known type A influenza virus PB1 sequences, PB1 genes from different influenza virus types and from more-distant orthomyxoviruses from insects and fish reveal that the N-terminal 12 amino acids are not conserved (data not shown). There are significant amino acid differences between the PB1 domain  of influenza virus type A and those of types B and C virus (e.g., B/Lee/40 and C/JJ/50) within the region that appears to be important for interaction and replication. For example, type B PB1 has F7 in place of the L7 found in influenza virus type A. Another nonconservative substitution can be found at T6, which has been replaced by Y6 in types B and C PB1 orthologs. This substitution for a bulky aromatic amino acid in a region predicted to fold as a -sheet could preclude the interaction with the influenza virus type A PA. Thus, domain  divergence during influenza virus type speciation may have made subsequent intertypic PB1 gene exchange among members of the family impossible, by disrupting the PB1-PA interaction interface. Nevertheless, PB1 proteins from influenza type A, B, and C viruses maintain an overall similarity within this region, including the high content of aliphatic amino acids, the presence of charged amino acids at exactly the same locations, and a predicted secondary -sheet structure (data not shown).

To determine if PA binding is important for the polymerase activity of PB1 complexed with PB2, we measured the reporter expression activity of PB1 mutants with known PA interaction abilities. Polymerase activity in vivo was assessed using an influenza virus plasmid-based transcription-replication system, which revealed that there was a general correlation between PB1-PA interaction ability and polymerase activity. This was especially true for the region extending from N4 to P13. Within this region, regression analysis of relative interaction on polymerase activity yielded values of 0.93 (data not shown). Mutant D2V had a limited impact on PA interaction efficiency yet showed no polymerase activity. We contemplate two major possibilities to explain this result. First, D2V could be binding PA with an abnormal spatial geometry that interferes with the normal function of the P complex. Alternatively, this substitution may have no impact on PA binding but instead may abolish polymerase activity by altering the conformation of a discontinuous PB1 domain important for PA function. The first interpretation would be consistent with the notion that D2 contributes to orient PA in an optimal functional conformation. A similar interpretation can be made about position V3. The A14D mutation that lies outside the region for interaction with PA has no polymerase activity but binds PA with WT efficiency as observed by two-hybrid and coimmunoprecipitation assays. The A14D mutation also appears to possess intact PB2 binding. No role has been assigned to this position for either interaction with other viral components and/or intrinsic polynucleotide extension ability of PB1. The presence of a hydrophobic amino acid at this position appears important since it is conserved within the three influenza virus types (isoleucine in type B and valine in type C). Deleterious effects on the activity of PB1 could result from substitution of a single highly conserved amino acid, as previously reported (5).

We have used reverse genetics to attempt the introduction of representative mutant PB1 genes, bearing mutations within the 12 N-terminal amino acids, into the influenza virus genome. Interestingly, the recovered mutant viruses displayed altered phenotypes and growth characteristics relative to the WT. It would be of interest to establish if any of these PB1 mutants with altered PA interaction and/or polymerase function display novel replication phenotypes and/or modified disease pathogenesis in laboratory animal hosts (35). To the best of our knowledge, the PB1 mutant set described in this report is the first to include a number of mutants that display partial polymerase activity that has been rescued in fully competent viruses. We attribute the failure to recover viable virus from certain PB1 mutant genomes such as F9D to RNA synthesis levels below the threshold required for progeny virion assembly. This conclusion rests on the improvements to the virus rescue system described by Hoffmann et al. (23), which render it comparable to the one described by Neumann et al. in terms of virus yield from a given amount of transfected DNA (35).

Interestingly, our results suggest that the polymerase activity of PB1 is stimulated by PA even in the  domain mutants failing to bind PA efficiently, as evidenced by PB1 L7D and L10D mutants (Fig. 2). However, the RG expression level in these mutants is five- to sixfold below WT levels, suggesting that the PB2-PB1-PA interaction is essential for maximum influenza virus PB1 transcriptase activity. It is possible that PA is mediating this enhancement by way of its ability to induce proteolysis of many cellular proteins as reported by Perales et al. (37). Our experiments also provided additional evidence for a role of PA in transcription and are consistent with previous findings on the direct role of PA in genome transcription and replication by the P complex. Future studies are needed to evaluate the functional properties of PA mutants deficient in their ability to interact with PB1.