Differential Roles of vrna and crna in Functional Modulation of the Influenza Virus RNA Polymerase *

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1 JBC Papers in Press. Published on May 23, 2001 as Manuscript M Differential Roles of vrna and crna in Functional Modulation of the Influenza Virus RNA Polymerase * Ayae Honda,1,2,3 Atsushi Endo,4 Kiyohisa Mizumoto5 and Akira Ishihama1 From the 1 Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka , Japan, the 2 Japan Science and Technology Corporation, Kawaguchi, Saitama , Japan, the 4 Daiichi Pharmaceutical Co., Ltd., Exploratory Research Laboratories, Edogawa, Tokyo , Japan, and the 5 Faculty of Phamaceutical Science, Kitasato University, Minato-ku, Tokyo , Japan * This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan, and CREST (Core Research for Evolutional Science) from the Japan Science and Technology Corporation. 1 Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

2 3 To whom correspondence should be addressed. Tel.: ; FAX: ; 2

3 Running title: Differential activation of influenza virus RNA polymerase by vrna and crna 3

4 The RNA-dependent RNA polymerase of influenza virus is composed of three viral P proteins (PB1, PB2 and PA) and involved in both transcription and replication of the RNA genome. For the molecular anatomy of this multi-functional enzyme, we have established a simultaneous expression of three P proteins in cultured insect cells using recombinant baculoviruses. For purification of P protein complexes, the PA protein was expressed as a fusion with a histidine tag added at its N-terminus. By using affinity chromatography, a complex consisting of the three P proteins was isolated from nuclear extracts of virus-infected cells. The affinity-purified 3P complex showed the activities of capped RNA-binding, capped RNA cleavage, viral model RNA-binding, model RNAdirected RNA synthesis, and polyadenylation of newly synthesized RNA. We conclude that a functional form of the viral RNA polymerase with the catalytic specificity of transcriptase is formed in recombinant baculovirus-infected insect cells. Using the viral RNA-free 3P complex, we found that the capped RNA cleavage takes place in the presence of vrna but not of crna, indicating that the vrna functions as a regulatory factor for the specificity control of viral RNA polymerase as well as a template for transcription. The structural elements of RNA directing the expression of RNA polymerase functions were analyzed using variant forms of the model RNA templates. The genome of influenza virus is composed of eight negative-strand viral RNA (vrna) segments, which altogether encodes 10 different viral proteins (1). The genome RNAs are transcribed, in virus-infected cell nuclei (2, 3), into mrna by the virionassociated RNA-dependent RNA polymerase (4). Genetic and biochemical studies indicate that this RNA polymerase is involved in both transcription [vrna-directed mrna synthesis] and replication [vrna-directed synthesis of complementary RNA (crna) and crna-directed vrna synthesis] (5, 6). In transcription, the RNA polymerase catalyzes not only viral RNA synthesis but also the cleavage of capped host cell RNA to generate capped RNA primers for viral mrna synthesis (7-11) and polyadenylation at the 3 -termini of mrna (12-15). The RNA polymerase also carries an apparent proofreading activity for nascent RNA chains (16). 4

5 The RNA polymerase is composed of one molecule each of three viral proteins, PB1, PB2 and PA (17, 18). The PB1 protein plays a central role in RNA polymerase assembly by providing the contact surfaces for both PB2 and PA (19). In the virus particles, this RNA polymerase is bound at a double-stranded region of viral RNA (vrna) formed by base-pairing between its 5 - and 3 -termini (20, 21). Since the RNA polymerase is tightly associated with vrna (18) and since the content of RNA polymerase in virions is very low, i.e., less then 1% of total virion proteins (1, 22), no purification method yielding large amounts of the functional RNA polymerase has been established. Previously Kobayashi et al. (23) demonstrated that the viral RNA polymerase with RNA synthesis activity could be reconstituted in vitro from three P proteins individually purified from recombinant baculovirus-infected insect cells, while Szewczyk et al. (24) succeeded to recover active RNA polymerase after renaturation of the P proteins separated by SDS-PAGE. In both cases, however, it was not enough to yield the efficient reconstitution of functional RNA polymerase in vitro. At present, therefore, the available methods for the analysis of structure-function relationships of this complex enzyme are limited. For detailed analysis of the structure-function relationship of each P protein, large quantities of the RNA-free form of functional RNA polymerase are essential. Here we show that all three P proteins simultaneously expressed in insect cells after coinfection of recombinant baculoviruses are assembled into a complex [the complex consisting of three P proteins is hereafter designated as 3P complex ]. The isolated 3P complex was found to be active in viral model RNA-directed RNA synthesis and capped RNA cleavage. Using the 3P complex, we also found that the capped RNA cleavage takes place only when the 3P complex binds to vrna but not crna. RNA elements for the expression of RNA polymerase functions were then analyzed using a set of variant model RNAs. EXPERIMENTAL PROCEDURES Preparation of Recombinant Baculoviruses --- Autographa california nuclear 5

6 polyhedrosis virus (AcNPV) was used for construction of recombinant viruses. The construction of recombinant baculoviruses for expression of PB1 and PB2 was described previously (23). To construct recombinant virus for expression of PA with a His-tag at N terminus, the cdna for PA was amplified by PCR, and inserted between the NcoI and BglII sites of pachlt-b (Phamigen). The resulting plasmid DNA (pachltpa) was cotransfected with linealized baculovirus DNA into Sf9 insect cells using the liposome method. After 96 h culture at 28 O C, the supernatant was harvested, and used for infection of Sf9 cells. After 96 h culture, the titer of recombinant virus RBVH-PA in the culture medium reached to approximately108 PFU/ml as determined by plaque assay. Purification of P Protein Complexes --- Spodoptera frugiperda Sf9 cells and Trichoplusia ni Tn5 cells were used for recombinant baculovirus infection. Sf9 were grown in the Grace medium with 5% serum while Tn5 were grown in a serum-free medium (Invitrogen). Tn5 or Sf9 cells were coinfected with three species of the recombinant baculovirus each at moi (multiplicity of infection) of 2. After 4 days culture, the 108 cells were harvested, suspended in 5 ml of a disruption buffer which contained 10 mm HEPES (ph 7.6), 0.1% Triton X-100 and 1 mm phenylmethylsulfonyl fluoride (PMSF, Sigma), and disrupted with a Dounce homogenizer. The nuclei were recovered by centrifugation for 5 min at 2,000 rpm, and then homogenized in 3 ml of a nuclear extraction buffer, which contained 10 mm HEPES buffer (ph 7.8), 500 mm NaCl and 20% glycerol. The homogenate was incubated for 30 min on ice with stirring, and centrifuged at 45,000 rpm for 2 h. The supernatant (nuclear extract) was mixed with metal affinity resin (Clontech) and incubated for 30 min at 4 o C with constant rotation. The resin was washed with a washing buffer, which contained 50 mm Na phosphate (ph 7.0), 500 mm NaCl and 5 mm imidazole until proteins could not be detected as measured with a UV spectrometer, and then eluted with an elution buffer which contained 50 mm Na phosphate (ph 7.0), 300 mm NaCl and 100 mm imidazole. Proteins eluted were analyzed by SDS-8% PAGE and gels were stained with Coomassie brilliant blue (CBB). 6

7 Immunoblotting of P Proteins --- P proteins separated by SDS-PAGE were electro-blotted onto polyvinylidene difluoride (PVDF) membranes (Nippon Genetics, Japan) in 10 mm CAPS buffer (ph 11) containing 10% methanol. The blotted filters were incubated with anti-pa, anti-pb1 and anti-pb2 antibodies, and then with peroxidase-conjugated anti-rabbit IgG, which was detected by staining with 3,3 - diaminobenzidine tetrahydrochloride (DAB) (DOJIN). Anti-P protein antibodies were raised in rabbits against each P protein purified from E. coli BL21 expressing recombinant P proteins under the control of T7 promoter (Y. Asano and A. Ishihama, unpublished). In Vitro RNA Synthesis --- In vitro RNA synthesis was carried out for 60 min at 30 O Cin 50 µl of the standard reaction mixture, which contained 50 mm HEPES/KOH (ph 7.6), 100 mm NaCl, 5 mm Mg acetate, 2 mm DTT, 0.3% Triton X-100, 0.25 mm each of ATP, GTP and CTP, 4 µm UTP, 10 µci [α- 32 P]UTP, 2.5 µg BSA, 0.25 mm ApG or 250 ng globin mrna, 1 unit RNasin (Promega), and 1 pmol of v53 [53 nucleotide-long vrna (v-sense or negative-strand RNA)] or c53 [53 nucleotide-long crna (c-sense or positive-strand RNA)] model RNA template [see below for the preparation of model RNAs]. Transcripts were electrophrased on 10% PAGE in the presence of 7 M urea and the gels were exposed to imaging plates to analyze with the BAS2000 image analyzer (Fuji, Tokyo, Japan). vrna and crna Binding Assay --- The purified 3P complex and radioactive RNA (about 10,000 cpm) were incubated for 30 min at 30 O C in 50 µl RNA-binding buffer, which contained 50 mm HEPES/KOH (ph 7.8), 100 mm NaCl, 5 mm Mg acetate, 2 mm DTT, 0.3% Triton X-100 and 10 µg trna. After UV irradiation for 30 min on ice, anti-pb1 was added, and the incubation was continued at 37 O C for 1 h. Antigen-antibody complexes formed were recovered after incubation with protein A- Sepharose (Pharmacia) on ice for 1 h. The protein A-Sepharose complexes were washed once with PBS and then subjected to SDS-8% PAGE. Gels were analyzed as above. 7

8 Capped RNA Binding Assay --- Crosslinking of capped RNA to the P proteins was carried out essentially as described in Honda et al. (25). Capped mrna with 32 P only at 5 cap-1 structure (about 1,000 cpm) and the 3P complex or RNP were incubated in 50 µl reaction mixture containing v-sense model RNA, 50 mm HEPES/KOH (ph 7.6), 100 mm NaCl, 5 mm Mg acetate, 2 mm DTT, 0.3% Triton X-100 and 1 unit of RNasin (Promega) at 30 o C for 30 min, and then exposed to UV for 30 min (26). The RNA-crosslinked proteins were digested with RNase A and RNase T1, and then incubated with anti-pb2 at 37 o C for 1 h. Antigen-antibody complexes formed were precipitated after incubation with protein A-Sepharose (Pharmacia) for 2 h on ice. The precipitates were washed once with PBS, and fractionated by electrophoresis on SDS-8% gels. The gels were analyzed as above. Capped RNA Endonuclease Assay --- The purified 3P complex was incubated in 50 µl of the reaction mixture which contained 50 mm HEPES/KOH (ph 7.8), 100 mm NaCl, 2 mm DTT, 0.3% Triton X-100, 2.5 µg BSA, and radioactive capped RNA with 32 P only at 5 cap structure (about 2,000 cpm) for 30 min at 30 o C. Reaction products were extracted with phenol-chloroform, precipitated with ethanol, and analyzed by 12% PAGE in the presence of 7 M urea (10). The gel was analyzed as above. Detection of Polyadenylated RNA --- Oligotex (dt) 25 (Takara) was added to the reaction mixtures for in vitro RNA synthesis and incubated at 75 o C for 10 min. The heat-treated mixtures were placed on ice for 5 min, and then 5 M NaCl was added to a final concentration of 500 mm. After incubation at 37 o C for 10 min, the samples were centrifuged for 5 min at 2,000 rpm. Precipitates were washed with 10 mm Tris-HCl buffer (ph 7.5) containing 1 mm EDTA, 0.5 M NaCl, and 0.05% SDS, and resuspended in 0.1 ml distilled water. RNA was extracted with phenol-chloroform, ethanol 8

9 precipitated, and then analyzed by PAGE in the presence of 7 M urea. Preparation of Model RNA Templates --- The model templates, v53, and c53, were synthesized by transcribing pv53 and pc53 plasmid DNA, respectively, with T7 RNA polymerase (27). Radioactive v53 and c53 RNAs were prepared by transcribing pv53 and pc53 DNA in the presence of radioactive substrates. Preparation of Capped RNA with 32P only at Cap-1 Structure --- Two types of radioactive capped RNA, globin mrna and capped poly(a), with 32P only at cap-1 structure were synthesized. For radio-labeling, globin mrna was subjected to one cycle of decapping and recapping reactions in the presence of [α-32p]gtp using vaccinia virus capping enzyme (Gibco BRL), while poly(a) was synthesized using T7 RNA polymerase and then capped in the presence of [α- 32 P]GTP using yeast capping enzyme (28). RESULTS Expression of Three P proteins using Recombinant Baculoviruses --- Recombinant baculoviruses for the expression of three different P protein subunits of influenza virus RNA polymerase were constructed using cdnas which were synthesized from vrna segments 1, 2 and 3, each encoding PB2, PB1 and PA protein, respectively. For the purpose of the 3P complex purification, PA was expressed as a fusion with a histidine (His) tag sequence of 40 amino acids in length, added at its N-terminus. As expected from the finding that the N-terminal sequence of PA is not involved in subunitsubunit contact (19), the addition of this His tag did not interfere with the assembly and activity of RNA polymerase (see below). The construction of recombinant baculoviruses for PB1 and PB2 production was described previously (22). To check the expression of the P proteins, the recombinant baculoviruses were 9

10 infected, individually or in various combinations, into Sf9 cells, and the whole cell lysates were analyzed by immunostaining with specific polyclonal antibodies against each P protein. When the recombinant baculovirus RBVH-PA was singly infected, a band crossreactive against anti-pa was detected in both the cytoplasmic and nuclear fractions following SDS-8% PAGE (Fig. 1A, lanes 2 and 3). The migration of H-PA (His-tagged PA) was slightly slower than the authentic PA without His-tag (Fig. 1A, lane 1). We could not detect any immunostained band in mock infected cell extracts (Fig. 1A, lanes 4 and 5). Likewise, the expression of PB1 and PB2 was detected after single infection with the corresponding recombinant virus (data not shown). When all three recombinant viruses, RBVPB1, RBVPB2 and RBVH-PA, were coinfected into Sf9 cells, the expression of not only H-PA but also PB1 and PB2 were detected after immunostaining with anti-pb1 (Fig. 1B) and anti-pb2 (Fig. 1C), respectively. PB1 and PB2 were again recovered in both cytoplasmic and nuclear fractions. Purification of the 3P Complex from Recombinant Virus-Infected Cells --- In order to examine whether the three viral P proteins form complexes in insect cells, the three recombinant baculoviruses were coinfected (at moi 2 for each virus) into Tn5 cells [note that the expression levels of P proteins were higher for Tn5 than Sf9 (Fig.1E )]. After SDS-8% PAGE of the whole cell lysates, the expression of all three P proteins was confirmed by immunostaining using polyclonal antibodies against each P protein (see Fig. 1). Since the recovery was higher for the nuclear fraction than the cytoplasmic fraction, attempts were made to purify P protein complexes from the nuclear fraction. Nuclei were solubilized with a low concentration of a non-ionic detergent, and the nuclear extract was prepared after centrifugation for 1 hr at 45,000 rpm. The supernatant was directly subjected to metal affinity resin purification. Proteins eluated with 100 mm imidazole were fractionated by SDS-8% PAGE, and the gel was stained with CBB. Three stained bands were detected (Fig. 1D, lane 3), which crossreacted with anti-pb1, anti-pa and anti-pb2 antibodies, respectively, in this order of migration. Both PB1 and PB2 migrated on SDS-PAGE with the same mobility as the corresponding authentic proteins associated with viral RNP cores (compare Fig. 1D, lanes 1 and 3). On the other 10

11 hand, the H-PA migrated as fast as the recombinant H-PA recovered from RBVH-PA virus-infected cells (Fig. 1D, lane 2) but more slowly than the RNP-associated untagged authentic PA (Fig. 1D, lane 1). Thus we conclude that at least some of the PB1 and PB2 molecules are associated with the His-tagged PA protein. In order to set up a high-level expression system for these influenza virus P proteins, we next compared their expression levels in two insect cell lines, Tn5 and Sf9, which were cultured under the same conditions at 28 O C. After metal affinity resin purification and SDS-PAGE, all three P proteins were detectable for both preparations even by CBB staining (Fig. 1E). From the staining intensity, the 3P complex is composed of nearly equal amounts of three P proteins. Since the expression level in Tn5 cells was higher than that in Sf9, we used hereafter Tn5 for large scale purification of the 3P complex. Based on the CBB staining intensity, the purification yield of the 3P complex was estimated to be about 5 µg from 10 7 cells. Detection of RNA Synthesis Activity for the 3P Complex --- The influenza virus RNA polymerase solubilized from viral RNP cores shows RNA synthesis activity in the presence of exogenously added model vrna templates which carry terminal conserved sequences of viral RNA segments (29). The RNA synthesis activity of the 3P complex purified from recombinant baculovirus-infected insect cells was tested using two model templates, v-sense (minus-strand) v53 and c-sense (plus-strand) c53, each consisting of 53 nucleotides, and two primers, dinucleotide ApG and globin mrna. When ApG was used as a primer, RNA products were detected for both v53- and c53- directed reactions (Fig. 2A). The major product in v53-directed reaction migrated on PAGE in the presence of 7 M urea as fast as the template v53 (Fig. 2A, lanes 1 and 8), while the major transcript in c53-directed reaction migrated faster than the template c53 (Fig. 2A, lanes 2 and 6). The small-sized RNA may represent a transcript initiated at an internal ApG-binding site within the crna template. The nuclear extract of mockinfected cells was unable to synthesize RNA (Fig. 2A, lanes 3 and 4), indicating that the RNA synthesis as detected with the 3P complex fraction represents the activity of the 11

12 influenza virus RNA polymerase formed in recombinant virus-infected insect cells, but not of cellular enzymes. This prediction was confirmed by the finding that the 3P complex is unable to synthesize RNA when unrelated RNAs without the viral promoter sequences were used as template (data not shown). Thus, we conclude that the 3P complex is able to recognize vrna as does the native viral RNA polymerase. Influenza virus RNA polymerase is interconvertible between transcriptase and replicase, but the capped RNA-primed initiation of RNA synthesis is a unique property of the transcriptase (5, 6). On the other hand, the replicase form of RNA polymerase is considered to initiate RNA synthesis de novo without using primers because the replication product or vrna in virus particles retains 5 -triphosphate (30). In the absence of primer addition, a low level of RNA synthesis was detected for v53-directed reaction (compare Fig. 2A, lanes 7 and 8). With use of c53 template, the 3P complex is virtually inactive in unprimed RNA synthesis (Fig. 2A, lane 5). Thus, we propose that the 3P complex formed in insect cell represent the transcriptase form of viral RNA polymerase. When in vitro RNA synthesis reaction was carried out using globin mrna as a primer, products were detected only for the v53 template (see below). Since the globin mrna preparation used in this experiment was heterogenous in size and 5 terminal structure, it was difficult to estimate the efficiency of transcription initiation between the synthetic primer ApG and the natural primer capped RNA. Transcripts from the globin mrna-primed reaction were heterogenous in size, but the majority migrated slower than a marker RNA of 100 nucleotides in length (see below). Polyadenylation Activity of the 3P Complex --- In capped RNA-primed transcription, the capped oligonucleotides of nucleotides in length, generated after cleavage of capped RNA by the viral RNA polymerase-associated endonuclase (7-11), are used as primers for transcription initiation, and remain associated with 5 termini of transcripts. Thus, the size of transcripts in the v53-directed and globin mrna-primed reaction should be as long as nucleotides. Some of the transcripts formed in v53- directed and globin mrna-primed reaction by the 3P comlex were, however, longer 12

13 than 100 nucleotide-long marker (see below), implying that transcripts contain various sizes of poly(a) sequence. To test whether the slowly migrating RNAs carry poly(a) sequences, in vitro transcripts were mixed with oligo(dt)25 resin, and the resin-bound RNAs were eluted by heat-treatment in the presence of high concentrations of salt. After 6% PAGE in the presence of 7 M urea, radioactive RNAs were detected, of which the majority migrated slower than the marker RNA of about 100 nucleotides in length (Fig. 2B, lanes 5 nd 6). Some of the ApG-primed transcripts also bound to the oligo(dt)25 resin, but the size of these RNAs were shorter than the marker RNA (Fig. 2B, lanes 2 and 3). These results indicate that at least some of the RNA products formed on the vrna model template in the presence of either ApG or mrna primers contain A-rich sequences. Binding Activity to vrna and crna --- Influenza virus RNA polymerase is associated with duplexes formed by both 5 - and 3 -terminal sequences of vrna segments (20, 21), and recognizes specific sequences located at 5 - and 3 -termini of vrna and crna (31, 32). These sequences are conserved among eight RNA segments, and provide the initiation sites for transcription and replication (5, 6). Some intrinsic activities of the RNA polymerase are exposed only after binding to these transcription promoter or replication origin sequences (33-35). Distinct regions of the PB1 subunit are involved in the recognition of vrna and crna (35, 36). To examine the RNA recognition specificity of the 3P complex, we then analyzed its binding activity to vrna and crna. Radio-labeled model templates, v53 and c53, and an unrelated TMV RNA of similar size were incubated with the 3P complex for 30 min at 30 O C in the transcription assay mixture without substrates, then exposed for 30 min to UV light for crosslinking. After treatment with RNases, the 3P complex was immunoprecipitated with a combination of anti-pb1 and protein A, and the immuno-precipitates were analyzed by SDS-8% PAGE. Both vrna (Fig. 3, lanes 2 and 3) and crna (Fig. 3, lanes 5 and 6) model templates were crosslinked to the 3P complex to the same extent, but no crosslinked product was detected for the unrelated TMV RNA (Fig. 3, lanes 8 and 9). 13

14 These results support the notion that the 3P complex formed in insect cell carries the recognition activity of specific sequences on both vrna and crna. Capped RNA-Binding Activity of the 3P Complex --- One unique feature of influenza virus growth is that host cell capped RNA is used as a source of primers for initiation of viral transcription (4). Even more remarkably, the influenza virus RNA polymerase itself is able to cleave the capped RNA at specific positions near the 5 cap structure (5, 6). The finding that globin mrna could serve as a primer for transcription by the 3P complex itself (see above) indicates that this complex also carries the activities of capped RNA binding and cleavage. To confirm this prediction, we next investigated the binding activity of 3P complex to capped RNA. For this purpose, we prepared rabbit globin mrna recapped with [α- 32 P]GTP using vaccinia virus guanylyltransfease and capped poly(a) with 32 P only at cap-1 structure using yeast capping enzyme. The 3P complex or viral RNP were then incubated with capped RNA with 32 P only at cap-1 structure in the presence of v53 template, and immediately exposed to UV irradiation for crosslinking. After subsequent digestion with RNase A and RNase T1, the 3P complex was immunoprecipitated with a combination of anti-pb2 serum and protein A, and fractionated by SDS-8% PAGE. The radioactivity was detected in the PB2 band for both the 3P complex (Fig. 4A, lanes 2 and 3) and RNP (Fig. 4A, lane 4) [note that use of anti- PB1 gave the same results]. The activity of capped RNA binding was not detected in the absence of v53 template (data not shown). The nuclear extract from mock-infected cells produced no radioactive band at the P protein positions (Fig. 4A, lane 1). The results indicate that the 3P complex expresses the capped RNA-binding activity when it binds to vrna. Capped RNA Cleavage Activity of the 3P Complex --- The detection of both mrna-primed transcription (see Fig. 2) and capped RNA-binding activity (see Fig. 4A) indicates the association of capped RNA cleavage activity with the 3P complex. To confirm the prediction, we tested capped RNA cleavage activity for the purified 3P 14

15 complex. In this experiment, capped poly(a) with 32 P only at the cap-1 structure was used as the substrate. In agreement with our previous observation (10), the capped poly(a) was cleaved by the RNP to generate fragments of nucleotides in length (Fig. 4B, lane 1). The same capped poly(a) was cleaved by the purified 3P complex in the presence of v53 template (Fig. 4B, lane 4) but the activity was not detected in the absence of vrna (Fig. 4B, lane 3). To our surprise, however, the enhancement of capped RNA endonuclease was observed only with v53 template, but c53 RNA was virtually inactive in this activation of capped RNA endonuclease (Fig. 4B, lane 6). This observation indicates, for the first time, that the 3P complex discriminates between v- sense and c-sense RNA, even though the 3P complex can bind to both vrna and crna (see Fig. 3). RNA Signals Required for Capped RNA Endonuclease --- As an attempt to identify the structural element on vrna for activation of the RNA polymeraseassociated capped RNA endonuclease, we constructed a set of vrna and crna variants (Fig. 5). Since vrna is active in directing capped RNA endonuclease while crna is virtually inactive (see Fig. 4). We then constructed two chimeric forms of model RNA, v84(3 c) and c84(3 v), in which 3 -terminal sequences of 16 nucleotides in length were replaced by the 3 -terminal sequences of crna and vrna, respectively (Fig. 5). To check the role of 3 -proximal puff on vrna (nucleotides 8 to 10 from 3 terminus of vrna), two types of variant model RNA were constructed. v84(dpuff) does not contain the puff after base substitutions, while the location of puff is shifted to 3 terminus (nucleotides 7 to 8 from 3 terminus) for v84(mpuff) (Fig. 5). The template and effector activities of these model RNAs were examined by measuring the activity of capped RNA cleavage (Fig. 6A), primer ApG-dependent RNA synthesis (Fig. 6B) and capped RNA-binding activity (Fig. 6C). v84 directed the expression of all three activities of the purified 3P complex, and c84 was virtually inactive as template and effector [c84 directs primer-dependent RNA synthesis but products are shorter than the template (see Fig. 2A)]. None of the variants tested directed 15

16 the expression of RNA polymerase functions. Thus we conclude that the influenza virus RNA polymerase functions as the transcriptase only when it is associated with vrna, and the structure formed by 5 - and 3 -terminal sequences of vrna is critical as the RNA signal for expression of the transcriptase function. DISCUSSION Large-scale Purification of Influenza Virus RNA Polymerase --- Several attempts have been made without success for purification of the influenza virus RNA polymerase from virus particles, because the yield was not been satisfactory (18, 37) and/or the contamination with viral RNA was not excluded (38, 39). The difficulty is attributable to tight association of the RNA polymerase with the genomic RNA. For most negative-strand viruses, virion-associated RNA polymerase is dissociated from RNP cores prior to NP during stepwise dissociation of viral protein (40). In contrast, the influenza virus RNA polymerase can not be removed from vrna even after dissociation of NP (18, 37). In parallel with the enzyme purification work, efforts have been made to establish in vitro reconstitution systems of the RNA polymerase from isolated individual P protein, but again the recovery of enzyme activity has so far remained at low levels (23, 24). To overcome the above failures to obtain large amounts of the functional RNA polymerase in RNA-free form, we have developed an expression system of functional form of influenza virus RNA polymerase in methylotrophic yeast Pichia pastoris (41) but the purification of RNA polymerase without contamination of endogenous nucleases was difficult. Here we succeeded to express all three P proteins simultaneously in insect cells after coinfection with three species of the recombinant baculovirus, each coding for PB1, PB2 and His-tagged PA protein (H-PA), respectively. The baculovirus expression system is now being widely used for the production of protein assemblies which are composed of multiple components and which can not be reconstituted in vitro (42-44). Although the expression levels of PB1, PB2 and H-PA were different and varied depending on culture conditions, the purified 3P complex contained nearly equal amounts 16

17 of the three P proteins. This finding supports the concept that the influenza virus RNA polymerase core enzyme is composed of one molecule each of the three P proteins (18). Catalytic Properties of Influenza Virus RNA Polymerase Expressed in Insect Cells --- The purified 3P complex from insect cells was found to possess the following known activities of influenza virus transcriptase: (i) the ability to bind to RNA with the terminal conserved sequences of vrna or crna; (ii) capped RNA-binding activity; (iii) endonucleolytic cleavage of capped RNA; (iv) capped RNA-primed initiation of RNA synthesis on model vrna; and (v) polyadenylation of newly synthesized RNA. The 3P complex is able to bind both vrna and crna (see Fig. 3), and to catalyze ApG-primed RNA synthesis using both vrna and crna templates (see Fig. 2). The activity of ApG (artificial primer)-primed RNA synthesis was lower for c53 template than v53 (see Fig. 2), and the size of major product in c53-directed transcription was shorter than the template c53 (see Fig. 2). Moreover, the capped RNA (natural primer)-primed RNA synthesis was observed only when vrna template was used (see Fig. 2). In good agreement with these findings, the capped RNA cleavage was detected in the presence of vrna but not with crna (see Fig. 4). The activity of model vrnadirected and primer-dependent RNA synthesis by the 3P complex was the highest among those obtained by various methods so far established in this laboratory (18, 23, 38, 41). The de novo initiation of RNA synthesis without primers is the activity characteristic of the replicase (5, 6). RNA synthesis activity by the purified 3P complex was, however, virtualy undetectable in the absence of primer addition. For replication, a host factor(s) appears to be involved (5, 6). In fact, we have identified several species of host protein, which interact with the P proteins (Honda, A., Okamoto, T. and Ishihama, A., in preparation). Accordingly our failure to detect the replicase activity with the 3P complex formed in insect cells may be explained if the putative host factor(s) necessary for conversion of transcriptase to replicase is missing in insect cells or is lost during the 3P complex purification. The purified RNA-free 3P complex could be useful for in vitro search of the putative host factor(s) for replication as well as for the detailed molecular anatomy of the RNA polymease. 17

18 Viral RNA as a Modulator of the RNA Polymerase Functions --- The 3P complex can bind both vrna and crna (see Fig. 3A), but its intrinsic activities of capped RNA cleavage and primer-dependent RNA synthesis are expressed only with vrna (see Fig. 3C, 4B and 5). The activation of RNA polymerase-associated capped RNA endonuclease by binding to vrna template is consistent with the observations by others (35). These observations altogether indicate that: (i) the 3P complex (RNA-free RNA polymerase) discriminates between v-sense and c-sense RNA; and (ii) vrna functions not only as the template for transcription but also as a regulatory factor for activation of the capped RNA endonuclease associated with the influenza virus RNA polymerase. Previously we found that host cell capped RNA has dual functions, i.e., priming of viral transcription and allosteric activation of the RNA polymerase in virion (45). Taken together we propose that both the activity and specificity of the influenza virus RNA polymerase are modulated by interactions with various species of host and viral RNA. Since the capped RNA endonuclease can not be activated by interaction with crna, transcription should not take place even when the RNA polymerase is associated with crna in the late stage of virus infection. Instead the initiation of vrna replication takes place on the crna template without using primers (30). Acknowledgments --- We thank T. Okamoto (Nat. Inst. Genet.) for preparation of model RNA templates, A. Iwata (Nippon Inst. Biol. Sci.) for preparation of anti-p protein antibodies, and R.S. Hayward (Univ. Edinburgh) for critical reading of the manuscript. The work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and the CREST fund from the Science and Technology Corporation. REFERENCES 18

19 1. Palese, P., and Kingsbury, D. W in Genetics of Influenza viruses. Springer-Verlag, Vienna 2. Herz, C., Stavnezer, E., Krug, R., and Gurney, T. (1981) Cell 26, Jackson, D. A., Caton, A.J., McCready, S. J., and Cook, P. R. (1982) Nature 296, Krug, R. M., Alonso-Caplen, F. V., Julkenun, I., and Katze, M. G. (1989) in The influenza viruses, (Krug, R.M., ed.) p Plenum Press, New York 5. Ishihama, A. (1996) Biochimie 78, Honda, A., and Ishihama, A. (1997) Biol. Chem. 378, Bouloy, M., Plotch, S. J., and Krug, R. M. (1978) Proc. Natl. Acad. Sci. USA 75, Beaton, A. R., and Krug, R. M. (1981) Nucleic Acids Res. 9, Plotch, S. J., Bouloy, M., Ulmanen, I., and Krug, M. (1981) Cell 23, Kawakami, K., Mizumoto, K., and Ishihama, A. (1984) Nucleic Acids Res. 11, Shaw, M. W., and Lamb, R. A. (1984) Virus Res. 1, Robertson, J. S., Schubert, M., and Lazzarini, R. A. (1981) J. Virol. 38, Hay, A. J., Skehel, J. J., and McCauley, J. (1982) Virology 116, Luo, G. X., Luytjes, W., Enami, M., and Palese, P. (1991) J. Virol. 65, Pritlove, D. C., Poon, L. L., Fodor, E., Sharps, J., and Brownlee, G. G. (1998) J. Virol. 72, Ishihama, A., Mizumoto, K., Kawakami, K., Kato, A., and Honda, A. (1986) J. Biol. Chem. 261, Horisberger, M. A. (1980) Virology 107, Honda, A., Mukaigawa, J., Yokoiyama, A., Kato, A., Ueda, S., Nagata, K., Krystal, M., Nayak, D., and Ishihama, A. (1990) J. Biochem. 107, Toyoda, T., Adyshev, D. M., Kobayashi, M., Iwata, A., and Ishihama, A. (1996) J. Gen. Virol. 77, Honda, A., Ueda, K., Nagata, K., and Ishihama, A. (1987) J. Biochem. 102, 19

20 Hsu, M.-T., Parvin, J. D., Gupta, S., Krystal, M., and Palese, P. (1987) Proc. Natl. Acad. Sci. USA 84, Kawakami, K., Ishihama, A., and Hamaguchi, M. (1981) J. Biochem. 89, Kobayashi, M.,Tuchiya, K., Nagata, K., and Ishihama, A. (1992) Virus Res. 22, Szewczyk, B., Laver, W. G., and Summers, D. F. (1988) Proc. Natl. Acad. Sci. USA 85, Honda, A., Mizumoto, K., and Ishihama, A. (1999) Genes Cells 4, Mahy, B. W. J., Barrett, T., Nichol, S. T., Penn, C. R., and Wolstenholme, A. J. (1981) in The Replication of Negative Stranded Viruses (Bishop, D. H. L. and Compans, R.W, eds), p Elsevier/North-Holland, New York 27. Parvin, J. D., Palese, P., Honda, A., Ishihama, A., and Krystal, M. (1989) J. Virol. 63, Ito, N., Yamada, H., Kaziro, Y., and Mizumoto, K. (1987) J. Biol. Chem. 262, Scholtissek, C., and Murphy, B. R. (1992) Arch. Virol. 58, Honda, A., Mizumoto, K., and Ishihama, A. (1998) Virus Res. 55, Seong, B. L., and Brownlee, G. G. (1992) J. Gen. Virol. 73, Fodor, E., Seong, B. L., and Brownlee, G. G. (1993) J. Gen. Virol. 74, Hagen, M., Chung, T. D., Butcher, J. A., and Krystal, M. (1994) J. Virol. 68, Cianci, C., Tiley, L., and Krystal, M. (1995) J. Virol. 69, Li, M. L., Ramirez, B. C., and Krug, R. M. (1998) EMBO J. 17, Gonzalez, S., and Ortin, J. (1999) EMBO J. 18, Kato, A., Mizumoto, K., and Ishihama, A. (1985) Virus Res. 3, Seong, B. L., Kobayashi, M., Nagata, K., Brownlee, G. G., and Ishihama, A. (1992) J. Biochem. 111, Galarza, J. M., Peng, Q., and Summers, D. F. (1996) J. Virol. 70,

21 40. Naito, S., and Ishihama, A. (1976) J. Biol. Chem. 251, Hwang, J.-S., Yamada, K., Honda, A., Nakade, K., and Ishihama, A. (2000) J. Virol. 74, Roy, P., Mikailov, M., and Bishop, D. H. (1997) Gene 190, Cai, J., Gibbs, E., Uhlmann, F., Phillips, B., Yao, N., OíDonnell, M., and Hurwitz, J. (1997) J. Biol. Chem. 272, Kimura, M., and Ishihama, A. (2000) Nucleic Acids Res. 28, Kawakami, K., Mizumoto, K., Ishihama, A., Shinozaki-Yamaguchi, K., and Miura, K. (1985) J. Biochem. 97, Zuker, M., Mathews, D.H. and Turner, D.H. (1999) In RNA Biochemistry and Biotechnology (Barciszewski, J. and Clark, B.F.C., eds.), p NATO ASI Series, Kluwer Academic Publ. 21

22 Footnotes Abbreviations used were: CBB, Coomassie brilliant blue; crna, complementary RNA; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RNP, ribonucleoprotein; vrna, viral RNA 22

23 FIGURE LEGENDS FIG. 1. Expression and purification of influenza virus P proteins. [A] The recombinant baculovirus RBVH-PA was infected onto Sf9 cells at moi of 2. After 4 days culture, the cells were harvested and the cell lysate was prepared as described in EXPERIMENTAL PROCEDURES. After centrifugation for 5 min at 2,000 rpm, the supernatant (cytoplasm) and pellet (nuclei) were fractionated by SDS-PAGE, and the gel was subjected to immuno-blotting using anti-pa antibodies. [B and C] Three kinds of recombinant viruses, RBVPB1, RBVPB2 and RBVH-PA, were coinfected onto Sf9 cells at moi of 2 for each virus. After 4 days culture, the cells were harvested and treated as above. Both cytoplasm and nuclear fractions were immunoblotted with anti-pb1 [B] or anti-pb2 [C] antibodies, respectively. RNP was analyzed as a control. [D] Recombinant baculovirus RBVH-PA (lane 2) or all three recombinant baculovirus, RBVPB1, RBVPB2 and RBVH-PA (lane 3), were infected onto Tn5 cells at moi of 2 for each virus, and the cells were cultured for 4 days. The supernatant fractions of whole cell lysates were mixed with metal affinity resin, and the imidazole eluates were fractionated by SDS-8% PAGE. The gel was stained with CBB. Lane 1, RNP isolated from influenza virus A/PR8; lane 2, the imidazole eluate fraction from RBVH-PA-infected cells; lane 3, the 3P complex isolated from cells coinfected with three recombinant baculovirus. [E] All three recomibant viruses were coinfected onto Sf9 (lane 1) or Tn5 (lane 2) cells. After 4 days incubation, the 3P complex was isolated and analyzed as above. FIG. 2. Model template-dependent RNA synthesis in vitro by the 3P complex. [A] RNA synthesis in vitro by the 3P complex purified from the recombinant virus-infected cells (lanes 1-2 and 5-8) or the corresponding fraction from mock-infected cells (lanes 3-4) was carried out using either v53 (lanes 1, 3, 7 and 8) or c53 (lanes 2, 4, 5 and 6) template and either in the presence (lanes 1-4, 6 and 8) or absence (lanes 5, and 7) of ApG primer. The conditions for in vitro RNA synthesis are as described in EXPERIMENTAL PROCEDURES. Products were analyzed by urea-10% PAGE. Samples 1-4 and 5-8 were run on separate gels. [B] Products of in vitro transcription in the presence of 23

24 indicated template (lanes 1-6, v53; and lanes 7-12, c53) and primer (lanes 1-3 and 7-9, ApG; and lanes 4-6 and 10-12, globin mrna) were incubated with oligo(dt) 25 resin. The resin-bound RNA was recovered and analyzed by urea-6% PAGE. Migration position of 100 nucleotide-long marker RNA is shown on left. FIG. 3. Template RNA- and capped RNA-binding activities of the 3P complex. The purified 3P complex was incubated with 32P-labeled v53 (lanes 1-3), c53 (lanes 4-6) or RNA with random sequence (lanes 7-9), and then exposed to UV for crosslinking. After RNase treatment, the RNA-crosslinked proteins were immunoprecipitated with anti-pb1 antibodies and analyzed by SDS-8% PAGE. The gel was exposed to X-ray film. The assay conditions were as described in EXPERIMENTAL PROCEDURES. Crosslinked proteins were analyzed, after RNase T1 and RNase A digestion, by SDS-8% PAGE. The gel was exposed to X-ray film. FIG. 4. Capped RNA endonuclease activity of the 3P complex. [A] RNA-cap binding activity assay: Globin mrna with 32P only at the cap structure was mixed with RNP (lane 4), two different amounts of the purified 3P complex (lanes 2 and 3) or the corresponding fraction from mock-infected cells, and then subjected to crosslinking by exposure to UV under the conditions as described in MATERIALS AND METHODS. After RNase treatment, the samples were analyzed by SDS-PAGE followed by expose to X-ray film. [B] Capped RNA endonuclease assay: Either RNP (lane 1), the 3P complex (lanes 4-6) and the imidazole eluate from mock-infected cells (lanes 2 and 3) were incubated with capped poly(a) with 32 P only at cap-1 structure in the absence (lanes 1 and 4) or presence of 1 pmol each of either v53 (lanes 2 and 5) or c53 (lanes 3 and 6) template. After incubation for 30 min at 30 O C, cleavage products were analyzed as described in EXPERIMENTAL PROCEDURES. The gel was exposed to X-ray film. FIG. 5. Model RNA variants. Model RNA templates, V84 and C84, and variants with the indicated sequences were prepared by transcribing the respective template DNA with 24

25 T7 RNA polymerase (25, 27). The secondary structures of these RNA were predicted using mfold program developed by Zuker et al. (46). 3 -Terminal sequence of 16 nucleotides in length of v84 and c84 were replaced the corresponding sequences of c84 or v84, respectively, to generate v84(3 c) or c84(3 v). The sequence within 3 -proximal puff was mutated so as to prepare v84(dpuff) without puff, while to generate v84(mpuff) with the puff close to the 3 proximal end, nucleotide substitution mutations were introduced at the corresponding region. FIG. 6. Template and effector activities of variant model RNAs. [A] Capped RNA endonuclease assay: Capped poly(a) with 32 P only at 5 terminus was treated with the purified 3P complex in the presence of vriant model RNAs [Lane 1, v84; lane 2, c84(3 v); lane 3, v84(3 c); lane 4, v84(dpuff); lane 5, v84(mpuff); lane 6, c84]. After incubation for 30 min at 30 O C, RNA was analyzed by PAGE in the presence of urea, and the gel was exposed to an imaging plate, which was analyzed with BAS2000 image analyzer. [B] Primer-dependent RNA synthesis assay: ApG-dependent RNA synthesis was carried out with use of the purified 3P complex and the same set of variant model templates as indicated in A. Products were analyzed by PAGE in the presence of urea and the gel was subjected to image analysis with BAS 2000 image analyzer. [C] RNAcap binding assay: Capped poly(a) with 32 P only at 5 terminus was mixed with the purified 3P complex in the presence of various model templates, and the mixtures were exposed to UV for RNA-protein crosslinking. After RNase digestion, the samples were anlyzed by SDS-PAGE. The gel was analyzed with BAS2000 image analyzer. 25

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32 Differential roles of vrna and crna in functional modulation of the influenza virus RNA polymerase Ayae Honda, Atsushi Endo, Kiyohisa Mizumoto and Akira Ishihama J. Biol. Chem. published online May 23, 2001 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts