Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its crna and Viral RNA Promoters during Viral RNA Replication

Transcription

1 JOURNAL OF VIROLOGY, Mar. 2006, p Vol. 80, No X/06/$ doi: /jvi Copyright 2006, American Society for Microbiology. All Rights Reserved. Different De Novo Initiation Strategies Are Used by Influenza Virus RNA Polymerase on Its crna and Viral RNA Promoters during Viral RNA Replication Tao Deng, Frank T. Vreede, and George G. Brownlee* Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom Received 27 October 2005/Accepted 5 December 2005 Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of crna and viral RNA (vrna) differed. To do this, two in vitro replication assays, which generated transcripts that had 5 triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppapg synthesis internally on the model crna promoter, whereas it initiated pppapg synthesis terminally on the model vrna promoter. The internally synthesized pppapg could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model crna and vrna promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a crna chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its crna and vrna promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells. Most RNA viruses encode their own RNA-dependent RNA polymerases in order to catalyze viral genome transcription and replication (3). The structures solved to date show that all viral RNA-dependent RNA polymerases form a similar basic right-hand-like structure with fingers, palm, and thumb subdomains (45). However, different viral RNA polymerases also have unique structural features which are important for efficient and accurate initiation of RNA-dependent RNA synthesis (25, 45). An amazing variety of initiation mechanisms for viral RNA-dependent RNA polymerization have been demonstrated. Any given RNA virus may use either one or more of these initiation mechanisms (25, 45). In general, initiation mechanisms can be classified into two different categories, primer-dependent and primer-independent (de novo) initiation (25, 45). For primer-dependent initiation, the most often used primers are either (i) an oligonucleotide covalently linked to a protein, as used by picornaviruses (e.g., poliovirus) (38) or (ii) a capped primer cleaved from the 5 end of the host mrna by a cap-snatching mechanism, which is used for viral genome transcription by some segmented negative-stranded RNA viruses such as influenza virus (4) and Dugbe nairovirus (24). In general, viral RNAs without cap-snatched primers or terminal proteins are initiated by a de novo initiation mechanism, which is common for many positive, negative, double-stranded, and ambisense RNA viruses (25). This mechanism is, clearly, a critical step to * Corresponding author. Mailing address: Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom. Phone: Fax: george.brownlee@path.ox.ac.uk. T. Deng and F. T. Vreede contributed equally to this work. ensure efficient production of new viral RNA and to maintain the integrity of the viral genome (25, 45). In most cases, de novo initiation starts at residue 1 of the template and is immediately followed by elongation. However, in some cases, e.g., Tacaribe arenavirus, it has been observed that short RNA oligonucleotides may be produced by successive rounds of abortive de novo transcription directed by either terminal nucleotides or internal nucleotides of the template. These oligonucleotides are subsequently used as primers for further elongation, as in the so-called prime-and-realign mechanism (7, 17 19, 26). A similar mechanism has also been proposed to explain 3 end repair in turnip crinkle virus (37). Influenza A virus RNA polymerase is a heterotrimeric complex composed of three subunits, PB1, PB2, and PA. No crystal structure is available for this complex, although low-resolution electron microscopic images have been described (1). The PB1 subunit is the key component of the complex, containing conserved motifs of RNA-dependent RNA polymerases (13, 30). The PA and PB2 subunits are also required for both viral RNA transcription and replication (13, 30). The polymerase catalyzes both primer-dependent RNA transcription (vrna to mrna synthesis) and primer-independent replication (vrna to crna and crna to vrna synthesis) in the nucleus of infected cells (13, 30, 46). The mechanism of transcription (vrna to mrna synthesis) has been well studied since both in vivo and in vitro assays are available (10, 14, 41). In contrast, the mechanism of replication remains unclear. The reason for this is that it is difficult to distinguish replication directed by the vrna versus the crna promoters in vivo because of the interdependence of the two promoters in the replication cycle. Existing in vitro primer-independent replication assays (22, 32) are inefficient and may, therefore, not be authentic. 2337

2 2338 DENG ET AL. J. VIROL. The current model for the secondary structure of both the vrna and crna promoters is the so-called corkscrew model (2, 8, 12). This model proposes a partially duplex region formed by the conserved noncoding sequences at both the 5 and 3 ends of each gene segment and two hairpin-loop structures, each with a stem of 2 bp and a tetraloop formed by residues close to the 3 and 5 termini. The secondary structure of the crna promoter proposed by Crow et al. (8) was based on the results of both a mutagenic analysis of the crna promoter in an in vivo minireplicon system, and in vitro promoter binding studies. However, there remained unexplained differences between the in vivo and in vitro results. In particular, point mutations at positions 4, 5, and 7 of the 3 strand of the crna promoter inhibited vrna, crna, and mrna synthesis in vivo, whereas in vitro approaches showed that RNA polymerase can bind all three of these point mutants in a UV cross-linking assay (8) and transcribe them in an ApG-primed transcription assay (40). According to the corkscrew structure of the crna promoter, the nucleotides at positions 4, 5, and 7 of the 3 strand of the promoter are located in a hairpin loop region, and point mutations at these positions would not be expected to disrupt the secondary structure and functions of the promoter in vivo. To resolve differences between the in vivo and in vitro data, an authentic in vitro primer-independent replication assay is needed. Here, we developed two in vitro primer-independent RNA synthesis assays to study primer-independent RNA replication on model promoters. By mutational analyses of the two promoters, we found that influenza virus RNA polymerase uses internal initiation and realignment on its crna promoter but terminal initiation and elongation on its vrna promoter during replication. These results shed new light on previous in vivo data on the crna promoter (8). MATERIALS AND METHODS Plasmids. The pcdna-pb1, pcdna-pb2, pcdna-pa, and pcdna-np protein expression plasmids for the three polymerase subunits and NP of influenza A/WSN/33 virus have been described (14). The ppoli-ccat-rt (8), pcdna- PB2tap (9), and active-site mutant (ASM) pcdna-pb1-d445a/d446a (46) plasmids have been previously described. The pcdna-pb1, pcdna-pb2tap, and pcdna-pa protein expression plasmids for the three polymerase subunits of influenza A/Turkey/England/5092/91 virus polymerase were provided by Ruth Harvey. RNA oligonucleotides. RNA oligonucleotides (Dharmacon) for the vrna promoter were 5 AGUAGAAACAAGGCC 3 and 5 GGCCUGCUUUUGCU 3 and for the crna promoter were 5 AGCAAAAGCAGGCC 3 and 5 GGCCUUGUUUCUACU 3. They contained the conserved sequence (underlined) plus two additional residues. Mutant oligonucleotides based on the above vrna and crna promoters were also from Dharmacon. Preparation of partially purified recombinant influenza virus RNA polymerase. 293T cells were transfected with plasmids (see above) using Lipofectamine After 48 h the cells were lysed and TAP-tagged influenza virus polymerase (3P) complex with or without NP was purified by affinity purification with immunoglobulin G-Sepharose, and released by tobacco etch virus protease as before (9). Partially purified proteins in tobacco etch virus protease cleavage buffer (0.15 M NaCl, 0.01 M HEPES, ph 8.0, 0.1% NP-40, 1 mm dithiothreitol, 10% glycerol) were stored at 20 C in 35% glycerol. Partially purified Histagged influenza virus polymerase, used in the UV cross-linking competition assay, was prepared as described previously (8). In vitro crna replication assay. We performed 3- l reactions with 1.5 l partially purified 3P/NP and 0.7 M synthetic model crna promoter (an equimolar mixture of the 5 and 3 strands) in the presence of 5 mm MgCl 2,1 mm dithiothreitol, 1 mm ATP, 0.5 mm GTP, 0.5 mm UTP, 0.1 M [ - 32 P]CTP ( 3,000 Ci/mmol, Amersham), and 1 U/ l RNasin (Promega). Where indicated, 0.5 mm ApG (Sigma) was added to the reaction. Reactions were incubated at 30 C overnight for convenience, although significant activity was obtained within 1 to 3 h. An equal volume of formamide/bromophenol blue/edta was added and the mixture was heated at 95 C for 5 min. An aliquot, usually 4 l, was analyzed by 25% polyacrylamide gel electrophoresis (PAGE) in 1 Tris-borate-EDTA and 6 M urea. Autoradiography was performed with Kodak Biomax MS films and Trans screen HE intensifying screens at 80 C. UV cross-linking assay. UV cross-linking assays were performed as described previously (8). Dinucleotide replication initiation assay and extension to trinucleotide assay. We performed 3- l incubations with (normally) 1.5 l polymerase, 0.02 M [ - 32 P]GTP ( 3,000 Ci/mmol, Amersham) in the presence of 5 mm MgCl 2,1 U/ l RNasin (Promega). and 1 mm dithiothreitol with the addition of 1 mm ATP and 0.7 M model vrna (an equimolar mixture of the 5 and 3 strands) or 0.7 M model crna (an equimolar mixture of the 5 and 3 strands) promoters. To assay for extension to trinucleotides, either 1 mm UTP or 1 mm CTP, as specified in the individual experiments, were added. In the transplant experiment (see Fig. 3), the 5 strand was added first and incubated for 10 min at 30 C prior to addition of the 3 strand to avoid self-annealing between the two strands. In the template-switching experiment (Fig. 6), a preincubation of either the vrna or the crna promoter with polymerase was performed at 30 C for 10 min before mixing. Incubations were at 30 C for various times (usually 1 to 3 h) up to 16 h. Formamide/bromophenol blue/edta (10 l) was added and the mixture was heated at 95 C for 2 min. Analysis, usually of a 5 l aliquot, was by 25% PAGE in 1 Tris-borate-EDTA and 6 M urea, followed by autoradiography. Gel elution, enzymatic digestion, and analysis of oligoribonucleotides. The excised band of gel containing oligoribonucleotides 5 residues in length was rinsed for 1 min in 1 ml deionized water and then crushed in 0.5 ml deionized water and shaken overnight at 4 C. After centrifugation at 10,000 g to remove gel particles, the supernatant was freeze-dried. Finally, the lyophilized sample was dissolved in a minimum volume (5 to 10 l) of deionized water. T 2 RNase (Sigma), which degrades RNA to give 3 -phosphorylated mononucleotides, was used essentially as recommended by the manufacturers. After digestion, 0.5 l10 mm ATP was normally added to the sample (usually 3 to 5 l) prior to loading onto a polyethyleneimine-cellulose (Merck PEI-cellulose F) thin layer, which was developed for 4 cm above the origin in 0.5 M ammonium sulfate, and then further developed in 0.7 M ammonium sulfate. The mobility of pppa 32 p on PEI-cellulose thin-layer chromatography (TLC) was deduced because the product (pa 32 p) of its further digestion with tobacco acid pyrophosphatase comigrated (R F 0.50) with unlabeled, marker pap (Sigma) (results not shown). The mobility of pp 32 pgp on PEI-cellulose TLC was deduced from T 2 RNase digestion of pp 32 pgpu, which had been characterized as possessing a pppg end group because it gave rise to a product, after P1 nuclease digestion, that comigrated with GTP on PEI-cellulose TLC (results not shown). Oligonucleotides 5 residues were gel-eluted in 0.5 M ammonium acetate and ethanol precipitated by standard methods. Calf intestinal phosphatase (Roche), tobacco acid pyrophosphatase (Epicenter) and polynucleotide kinase (Roche) treatments were carried out for 1 h at 37 C in the supplied buffer. Digested products were analyzed by 25% PAGE as above. Primer extension assay. Primer extension assays were performed as described previously (8). Terminal sequencing of viral RNA. Sequencing of the 5 and 3 termini of viral RNA transcripts was carried out essentially as described previously (43). Briefly, total cellular RNA from transfected 293T cells was extracted with Trizol (Invitrogen) and treated with TAP (Epicenter) prior to being reextracted with Trizol. The RNA was then circularized with T4 RNA ligase (Epicenter). cdna copies of the ligated 3 and 5 termini were synthesized by reverse transcription (8) with separate vrna- or crna-specific primers. The cdna was amplified by PCR using Taq polymerase (Promega) and both vrna- and crna-specific primers, cloned using the TOPO TA cloning kit (Invitrogen), and sequenced. RESULTS Mutational analysis of the 3 end of the influenza virus crna promoter using a primer-independent in vitro replication assay. An ApG-primed replication assay has been used previously to study the sequence requirements of the 3 end of the influenza virus crna promoter for RNA replication with nonrecombinant RNA polymerase (40). However, the exact nature of this assay, whether it mimics replication or transcription, was not clear. Here we developed an in vitro primer-

3 VOL. 80, 2006 INFLUENZA VIRUS REPLICATION 2339 FIG. 1. Mutational analysis of the influenza virus model crna promoter using a primer-independent replication assay, an ApGprimed transcription assay, and a UV cross-linking assay in vitro. (A) Autoradiograph showing the full-length unprimed or ApG-primed transcription product (TP) synthesized by partially purified 3P/NP complex on a wild-type (lane 1) or mutant (lanes 3 to 11) model crna promoter, as indicated, in the presence of 1 mm ATP, 0.5 mm GTP, 0.5 mm UTP, and 0.1 M [ 32 P]CTP, and analyzed on 25% PAGE in 6 M urea. The negative control (lane 2) represents replication on the wild-type (WT) crna promoter template in the absence of ATP. The weak signal in lane 11 has a slightly slower mobility due to the different sequence. Additional bands presumably representing early termination products ( ) or transcripts with a single nontemplated nucleotide extension (asterisk) are indicated. The origin of the band labeled with an arrow head is unknown. (B) Autoradiograph of variously phosphorylated forms of full-length in vitro crna replication products separated on 25% PAGE in 6 M urea. The 15-nucleotide transcription products obtained by ApG-primed or unprimed in vitro replication as described in (A) were gel-eluted and left untreated (lanes 1 and 4) or treated with either calf intestinal phosphatase (CIP) (lanes 2 and 3), tobacco acid pyrophosphatase (TAP) (lane 5), or polynucleotide kinase (PNK) (lane 6) as indicated. (C) Detailed analysis of polymerase binding of the model crna promoter mutants 4U3A and 5C3A by a UV cross-linking competition assay. Lanes 1, 5, and 9, cross-linking to the wild-type 32 P-labeled 3 end of crna in the presence of excess unlabeled 5 strand. Lanes 2, 6, and 10, in the presence of 0.01 M wild-type and mutant competitors. Lanes 3, 7, and 11, in the presence of 0.1 M wild-type and mutant competitors. Lanes 4, 8, and 12, in the presence of 5 M wild-type and mutant competitors. independent replication assay based on the incorporation of [ - 32 P]CTP (0.1 M) into a 15-nucleotide transcript of a model crna promoter (Fig. 2A) in the presence of 0.5 mm GTP and UTP and 1 mm ATP. Partially purified recombinant influenza virus RNA polymerase isolated from transiently transfected 293T cells was used as a source of enzyme (5). Figure 1A (lane 1, upper panel) shows that a 15-nucleotide full-length transcript was synthesized from the model wild-type crna promoter. The essential feature of an authentic primer-independent replication product is the presence of a triphosphate moiety at its 5 end. In order to confirm that unprimed transcripts had a 5 triphosphate end group, we compared the mobilities of the 15-nucleotide unprimed transcript with the ApG-primed transcript, variously treated with calf intestinal phosphatase, tobacco acid pyrophosphatase, or polynucleotide kinase on a 25% PAGE urea gel (Fig. 1B). The 15-nucleotide unprimed transcripts (5 pppa) migrated faster than the ApG-primed transcripts (5 OH A) (Fig. 1B, compare lane 4 with 1 and 2). Unprimed transcripts treated with calf intestinal phosphatase (5 pppa35 OH A), comigrated with the ApG-primed transcripts (5 OH A) (Fig. 1B, compare lanes 3 with 1). When the unprimed transcripts were treated with tobacco acid pyrophosphatase (5 pppa35 pa), they comigrated with the polynucleotide kinase-treated ApG-primed transcripts (5 OH A35 pa) (Fig. 1B, compare lanes 5 and 6). In addition, a faint band migrated slightly faster than the unprimed transcripts in lane 4. This faint band is likely to be a 5 diphosphate-ended transcript since it had an intermediate mobility between the 5 p- and 5 ppp-ended transcripts. Its origin is uncertain, but it may be due to a contaminant nucleotidyl pyrophosphatase in our enzyme preparation. Therefore, a majority of transcripts had a 5 triphosphate end group. Having validated the authenticity of this primer-independent transcription assay, we compared the effects of point mutations at positions 1 to 9 from the 3 end of the 3 strand of the model crna promoter in this primer-independent replication assay (Fig. 1A, upper panel) with an ApG-primed transcription assay (Fig. 1A, lower panel) under the same conditions. In the unprimed replication assay, point mutants of the promoter at positions 1, 6, 7, and 8 showed transcription activities similar to the wild type (Fig. 1A, upper panel, compare lanes 3 and 8 to 10 with lane 1). Lower yields were obtained with the mutant at position 3, and only residual levels were obtained for the mutant at position 9. Interestingly, however, no products were detected with the mutants at positions 2 and 4 to 5. In the ApG-primed transcription assay, however, significant activity was observed with the mutants at positions 3 to 4 and 6 to 8 (Fig. 1A, lower panel, compare lanes 5 to 6 and 8 to 10 with lane 1), lower activities were observed with the mutants at positions 1 and 5, residual activity was observed with the mutant at position 9, but no activity was observed with the mutant at position 2. The most obvious difference between the results of the two assays is at positions 4 and 5. Mutations here interfered with unprimed transcription completely, but not with the ApG-primed transcription (Fig. 1A, compare lanes 6 and 7 in the upper and lower panels). The polymerase binding properties of these two mutants had been previously studied in a UV cross-linking competition assay where it was shown that both mutants compete with the wild-type model crna promoter (8). A more detailed UV cross-linking competition analysis of these two mutants is shown here (Fig. 1C), confirming that the 4U3A and 5C3A mutants were not significantly different from the wild-type model crna promoter in competing with cross-linking of a radiolabeled wild-type promoter to the subunits (PB1, PB2, and PA) of the polymerase (Fig. 1C, compare lanes 5 to 8 and 9 to 12 with lanes 1 to 4). Thus, we excluded the possibility that the absence of any replication products observed for the mu-

4 2340 DENG ET AL. J. VIROL. tants at positions 4 and 5 of the 3 strand of the model crna promoter in the primer-independent replication assay (Fig. 1A, upper panel) was due to their weak binding to the polymerase. pppapg is copied from nucleotides at positions 4 and 5 of the 3 end of the crna promoter in contrast with positions 1 and 2 of the vrna promoter. Influenza virus ribonucleoprotein isolated from virions is known to synthesize the dinucleotide pppapg in vitro by copying the terminal 3 OH U-C 5 residues of the vrna template under conditions where only ATP and GTP are present (28). Here we have adapted this dinucleotide synthesis assay to investigate initial events in replication (i.e., synthesis of pppapg) in the absence of added primer with recombinant influenza virus RNA polymerase and model vrna and crna promoters (Fig. 2A and B). Wild-type and mutant promoters were used to test whether the observed lack of replication (Fig. 1A) was caused by events early in replication. We found that mutations at positions 4 and 5 of the 3 strand of the model crna promoter (Fig. 2C, lanes 6 to 7) inhibited pppapg synthesis dramatically, whereas mutants at positions 1 and 2 (Fig. 2C, lanes 3 and 4) showed activity similar to that of the wild type. By contrast, mutants at positions 1 and 2 of the model vrna promoter (Fig. 2D, lanes 3 to 4) were inactive in pppapg synthesis. These results indicated that the vrna and crna promoters have distinctive sites of pppapg synthesis i.e., the 3 OH U-C 5 at positions 1 to 2 of the 3 end of the vrna promoter, but the 3 U-C 5 at positions 4 to 5, from the 3 end of the crna promoter. In addition, mutations elsewhere within the promoters can influence pppapg synthesis activity (Fig. 2C and D). In the case of the crna promoter, mutants at positions 6, 7 and 8 (Fig. 2C, lanes 8, 9, and 10) retained significant pppapg synthesis activity, while the mutant at position 9 showed only residual activity (Fig. 2C, lane 11). The dinucleotide synthesized from the position 3 (A3U) mutant (Fig. 2C, lane 5) showed a slightly slower mobility and was found to consist of both pppapg and pppgpa by T 2 RNase digestion (results not shown). In the case of the vrna promoter, mutants at positions 4 to 6 and 8 showed weak activity (Fig. 2D, lanes 6 to 8 and 10), although mutants at positions 7 and 9 were inactive (Fig. 2D, lanes 9 and 11 to 12). Interestingly, the mutant at position 3 (G3A) was more active than the wild type (Fig. 2D, compare lanes 2 with 5). In summary, the pattern of pppapg synthesis observed in the mutagenesis screening (Fig. 2C and D) differs significantly between the two promoters. This further suggests that the RNA polymerase initiates pppapg synthesis on the vrna and crna promoters in different ways. We next asked if a model vrna promoter, mutated at position 5 to a C residue, thereby generating a 3 U-C 5 sequence at positions 4 and 5, could allow pppapg synthesis at positions 4 and 5. To do this we had to introduce an additional mutation at position 1 (U3A) to block any possibility of terminal initiation. Figure 2E shows that the double mutant (5U3C, 1U3A) (lane 4) was inactive in pppapg synthesis, while the 5U3C mutant (lane 3) still synthesized pppapg as we had observed with 5U3A mutant in Fig. 2D (lane 7). This suggested that simply mutating position 5 U3C in the vrna promoter does not permit the synthesis of pppapg internally. Overall, these results indicate that intrinsic differences in the FIG. 2. Identification of nucleotide residues required for pppapg synthesis in the 3 strand of both the crna and vrna promoters. (A) Influenza virus model crna promoter in the corkscrew configuration (B) Influenza virus model vrna promoter in the corkscrew configuration. The prime notation is used to identify nucleotides of the 5 end of the promoter. (C) Autoradiograph showing pppapg synthesized by 3P with the wild-type (cwt) or mutant 3 crna strand, as indicated, in the presence of the wild-type 5 crna strand. (D) Autoradiograph showing pppapg synthesized by 3P with the wild-type (vwt) or mutant 3 vrna strand, as indicated, in the presence of the wild-type 5 vrna strand. (E) Autoradiograph showing pppapg synthesized by 3P with the wild-type (vwt) or mutant (single or double) 3 vrna strand, as indicated, in the presence of the wild-type 5 vrna strand. ve, 3 crna or vrna promoter strands alone. *p, 32 P. vrna and crna promoters may control the site of pppapg synthesis. Absence of a hinge in the 5 strand and presence of a hinge and identity of the nucleotide at position 3 in the 3 strand of the crna promoter are the major determinants of internal pppapg synthesis. The crna promoter is complementary to the vrna promoter, and a very similar corkscrew secondary structure model has been proposed for both promoters (2, 8, 12) (Fig. 2A and B). Nevertheless, there are obvious differences between the two promoters. First, the lengths of the 5 and 3 arms differ due to the presence or absence of a hinge, an extra nucleotide located between the base-paired duplex region and the hairpin-loop structure (Fig. 2A and B). In the case

5 VOL. 80, 2006 INFLUENZA VIRUS REPLICATION 2341 of the 5 arm of the promoters, a hinge A residue is present in the vrna promoter but is absent in the crna promoter. In the case of the 3 arm of the promoters, a hinge U residue is present in the crna promoter but is absent in the vrna promoter. Second, the identities of the 3 to 8 base pair and the nucleotide at position 5 of the hairpin loop structures of both 5 and 3 arms vary. Specifically, a C-G base pair at positions 3 and 8 and an A at position 5 in the 5 hairpin loop of crna are replaced by a U-A base pair and a G at the corresponding positions of vrna. In the 3 hairpin loop, an A-U base pair at positions 3 and 8 and acatposition 5 of crna are replaced by G-C and U, respectively, in vrna (Fig. 2A and B). In order to test which of these sequences or presumed secondary structural elements in crna are responsible for internal pppapg synthesis, a series of model 5 -strand and 3 strand promoter mutants that transplant the crna-like elements one by one into the vrna promoter were designed (Fig. 3A and C). In addition, a mutation was introduced at position 1 (U3A) of the 3 strand to exclude any possibility of terminal pppapg synthesis (Fig. 2D, lane 3). The 5 -arm vrna promoter mutants transplanted with different 5 -strand crna-like elements were first tested in combination with the 3 arm of the crna promoter with the additional 1U3A mutation (see above) to evaluate which elements of the 5 arm were needed for internal initiation on the 3 arm of crna. Figure 3B shows that no internal pppapg synthesis occurred when the nucleotide 5 A of crna replaced 5 G of vrna (lane 3), and only residual activity was obtained when the 3 C-8 G base pair of crna replaced the 3 U-8 A base pair of vrna (lane 4). However, significant activity was detected when 10 A was deleted from vrna (lane 5). No further significant increase in activity was observed if this 10 A deletion was combined with either the 5 A element (lane 6) or the 3 C-8 G element (lane 7). This result suggests that the absence of a 10A hinge in the 5 arm of the crna promoter is the key structural element controlling internal pppapg synthesis. Second, we tested which 3 -arm crna elements were required for internal pppapg synthesis by again transplanting crna elements into the vrna promoter. In this case, the residue at position 5 (a U residue) of the vrna promoter had to be mutated to a C residue to allow pppapg transcription from positions 4 and 5. The 1(U3A) mutation was also used throughout to block pppapg synthesis from nucleotides 1 and 2. The crna elements 3A and 8U were treated separately since point mutants at these positions showed significant activity (Fig. 2C). These elements and the 10U hinge were then transplanted individually or in different combinations into the 3 arm of the modified vrna backbone and tested in combination with the 5 arm of the crna promoter for pppapg synthesis (Fig. 3C). No significant activity was obtained with the mutants bearing either the 3A or the 8U, or both the 3A and 8U crna elements (Fig. 3D, lanes 6, 7, and 8), whereas residual activity was observed when the 10U hinge was inserted (lane 9). However, when the 10U hinge was introduced in combination with the 3A element, wild-type activity was observed (Fig. 3D, compare lanes 10 and 2). In contrast, only partial activity was obtained when the 10U hinge was introduced in combination with the 8U element (Fig. 3D, lane 11). As expected, wild-type activity was obtained when all three elements (3A, 8U, and 10U) were present (Fig. 3D, lane 12). These results suggest that the combination of the 3A element and the 10U hinge in the 3 end of the crna promoter is required for full internal pppapg synthesis activity. Overall, we concluded that the absence of the hinge in the 5 strand and the presence of the hinge and the identity of the nucleotide at position 3 in the 3 strand of the crna promoter are the major structural elements that determine internal pppapg synthesis. Internally synthesized pppapg anneals to position 1 and 2 of the 3 strand of the crna promoter for extension into trinucleotides in a template-directed manner. Theoretically, to maintain the integrity of the viral genome, internally synthesized pppapg from nucleotides 4 and 5 of the 3 strand of the crna promoter would have to realign to nucleotides 1 and 2 for elongation. To test this model, we performed the pppapg synthesis reaction in the presence of UTP in addition to ATP and radiolabeled GTP. Under these conditions, we observed pppapg and pppgpu synthesis (see Discussion) (Fig. 4A and B, band A) as well as elongation to a trinucleotide, although elongation was inefficient (Fig. 4A, lane 2). This trinucleotide (Fig. 4B, band E) was characterized as pppapgpx by gel elution and T 2 RNase digestion, followed by PEI-cellulose TLC. The identity of X was deduced as a U residue because no trinucleotide product was observed when the UTP was omitted or CTP was added instead (results not shown). However, we failed to observe elongation to either a 5-nucleotide product, pppagaaa, derived from residues 4 to 8 of the crna promoter (Fig. 2A) or an 8-nucleotide product, pppaguagaaa, derived from residues 1 to 8 (results not shown). This failure, and the low yield of the trinucleotide pppapgpu, could be due to the low concentration of GTP used in this reaction. To further test the properties of the crna promoter required for realignment, we studied the 1U3A and 2C3A mutations, together with the 4U3A and 5C3A mutants as controls. The previous result that mutations at positions 4 and 5 inhibited pppapg synthesis was confirmed (Fig. 4A, lanes 5 and 6), whereas pppgpu (Fig. 4B, band D) was the major product synthesized from the 4U3A mutant. Lanes 3 and 4 showed that the 1U3A mutation but not the 2C3A mutation allows trinucleotide pppapgpu synthesis. The presence of pppapgpu, derived from the wild type and the mutant at position 1 suggested that pppapg, internally synthesized from residues 4 and 5 of the model crna promoter, was able to realign with wild-type residues 1 and 2 of the promoter and extend by one residue, albeit inefficiently. The 5 pppa end group of band F derived from the 1U3A mutant (Fig. 4A, band F), as confirmed by T 2 RNase digestion and PEI-cellulose TLC (Fig. 4B), shows that the trinucleotide transcript had effectively corrected the 1U3A mutation of the crna template by realignment. This result further suggests that base-pairing between the first nucleotide of pppapg and the terminal residue of the 3 end of the crna promoter template is not critical for realignment. On the other hand, no trinucleotide was observed from the mutant at position 2 (Fig. 4A, lane 4), suggesting that base-pairing between the second nucleotide of pppapg and the C residue at position 2 of the 3 end of the model crna promoter template is essential and sufficient for extension in vitro. Based on these results, we

6 2342 DENG ET AL. J. VIROL. FIG. 3. Identification of sequence requirements in the 5 and 3 arms of the crna promoter for internal pppapg synthesis by a transplant experiment. (A) Model wild-type (WT) or mutant 5 vrna strands with the 5 crna-like elements transplanted one by one. The transplanted 5 crna elements are boxed. 10 D, hinge A deleted in the 5 strand. (B) Autoradiograph showing internal pppapg synthesis activity with the 5 mutants shown in A in combination with the 3 crna promoter strand containing a mutation at position 1(U3A). (C) Model wild-type (WT) or mutant 3 vrna strands with the 3 crna-like elements transplanted one by one. The transplanted 3 crna elements and a mutation at position 1 are boxed. 10I, hinge U inserted in the 3 strand. (D) Autoradiograph showing internal pppapg synthesis activity with the 5 wild-type crna promoter in combination with the 3 mutants shown in C. Note that the promoter mutant used in lane 12 differs in sequence only at position 1 from the wild-type crna promoter used in lane 2. ve, 3 strand of the promoter alone. The position of pppapg is shown. *p, 32 P. conclude that the influenza virus crna promoter might use an internal initiation and realignment mechanism to initiate viral RNA synthesis. Mutated or deleted nucleotide at position 1 of the 3 end of the crna promoter is corrected to the wild-type nucleotide in a minireplicon system. As was shown in Fig. 4A (lane 3, band F) above, a wild-type trinucleotide transcript had been generated in vitro from a crna template mutated at position 1. In vivo, it has been shown that a mutation at position 1 of the 3 end of the crna promoter was compatible with the synthesis of significant levels of mrna, vrna and crna from a CAT reporter (8). Thus, it was of interest to test whether the 1U3A mutant previously studied in vivo was corrected to the wildtype sequence.

7 VOL. 80, 2006 INFLUENZA VIRUS REPLICATION 2343 FIG. 4. Internally synthesized pppapg from influenza virus crna can realign to terminal residues for extension into a trinucleotide in a template-directed manner. (A) Autoradiograph showing the trinucleotide pppapgpu and dinucleotides synthesized in the presence of 1 mm UTP, 1 mm ATP, and 0.02 M [ - 32 P]GTP with wild-type (WT) or mutant 3 crna strand in the presence of the 5 crna strand. The positions of the dinucleotides and pppapgpu are shown. (B) Characterization of bands A to F by treatment with T 2 RNase and PEIcellulose TLC. The positions of pppa*p and pp*pgp are shown. ve, 3 crna strand alone. *p, 32 P. Two additional mutants with a 1U3C or a deleted residue 1 (1 U) were generated and, together with the previously isolated 1U3A mutant, were tested in a primer extension assay (8). This assay measures the steady-state levels of vrna, mrna and crna in a minireplicon system in which 293T cells were transiently transfected with 4 protein expression plasmids encoding the three subunits of influenza virus RNA polymerase and the nucleoprotein, together with a ppoliccat plasmid which encodes a CAT reporter gene flanked by the influenza virus crna promoter (8). Figure 5A shows that templates with a mutated or deleted residue 1 were replicated (vrna and crna) and transcribed (mrna) to wild-type levels (Fig. 5A, lanes 1, 3, 5, and 7). As controls, the background levels of unamplified polymerase I (POLI) transcripts of the wild-type and the three mutant templates (Fig. 5A, lanes 2, 4, 6, and 8) were obtained by transfection of an active-site polymerase mutant (replacing wildtype PB1 with mutant PB1-D445A/D446A, abbreviated 3P- ASM) (46). Sequencing of the RNA (see materials and methods) showed that all three mutants (i.e., the two mutated and the one deleted nucleotide at position 1) had reverted to the wild-type sequence (Fig. 5B). pppapg derived from the crna promoter can be released and realigned onto a vrna promoter in vitro. According to our model, pppapg initiates internally and is extended by realignment onto terminal residues of the crna template (cisrealignment). It was of interest to establish whether pppapg synthesized internally on a crna template could dissociate and realign to nucleotides 1 and 2 of a vrna template for extension (trans-realignment). To test this, we designed an in vitro template-switching experiment in which a crna promoter mutant, which allows pppapg synthesis internally but does not allow extension to a trinucleotide, is mixed with a vrna promoter mutant, which does not allow pppapg synthesis terminally but would allow extension to a trinucleotide if pppapg were available. Thus, if the pppapg dissociated from the crna promoter mutant, it would be available for the vrna promoter mutant to use for extension into trinucleotide. If, however, the pppapg remained associated with the crna promoter mutant, no extension to a trinucleotide would occur. From previous mutational analyses of the crna and vrna promoters (Fig. 2C and D), the 3 -strand crna promoter mutant 2 (C3A) and the 3 -strand vrna promoter mutant 1 (U3A) fit these criteria. Also, by taking advantage of the different mobilities of pppapgpc (Fig. 6A, lane 4) directed by a model vrna template and pppapgpu (Fig. 6A, lane 1) directed by a model crna template, we could distinguish whether the template of the synthesized trinucleotides was the vrna or the crna promoter. As controls, we used model wild-type crna and vrna in the presence of either UTP or CTP to synthesize trinucleotides as markers (Fig. 6A, lanes 1 to 4). As expected, pppapgpu (band D) was only synthesized from the crna template and only in the presence of UTP (lane 1). Similarly, pppapgpc (Fig. 6A, band E) was only synthesized from the vrna template and only in presence of CTP (Fig. 6A, lane 4). The crna and vrna promoter mutants mentioned above were also included as further controls to confirm that the 2C3A crna mutant can generate pppapg internally but cannot be extended into a trinucleotide in the presence of either UTP or CTP (Fig. 6A, lanes 5 and 6), and the 1U3A vrna promoter mutant cannot generate pppapg or trinucleotides (Fig. 6A, lanes 7 and 8). The test for template switching was carried out by mixing the model crna promoter mutant (2C3A) and the model vrna promoter mutant (1U3A). The result showed that the trinucleotide pppapgpc (Fig. 6A, band F) was only observed when CTP, but not UTP, was provided in the reaction (Fig. 6A, compare lanes 9 and 10). This suggested that pppapg synthesized by the crna promoter mutant (2C3A) had realigned to the vrna promoter (1U3A) and extended into the trinucleotide pppapgpc. We were also able to confirm that template switching occurred when the wild-type crna promoter was mixed with the mutant vrna promoter (1U3A) (Fig. 6A, lanes 11 to 12). In this case, both pppapgpu (band G), which was synthesized from the wild-type crna promoter template, and pppapgpc, (band H), which was synthesized by template switching of pppapg from the wild-type crna promoter to the vrna promoter mutant, were observed. The pppa end group of these trinucleotides (bands D to H) was confirmed by T 2 RNase digestion and comparison with T 2 RNase-digested dinucleotides on PEI-cellulose TLC (Fig. 6B). Overall, we

8 2344 DENG ET AL. J. VIROL. Downloaded from FIG. 5. Mutations or deletion of the 3 -terminal residue of the crna template are corrected to wild type in an in vivo minireplicon system. (A) Autoradiograph showing primer extension analysis of virus-like CAT RNA transcribed in an in vivo minireplicon system. 293T cells were transfected with four protein expression plasmids encoding the three subunits of the influenza virus RNA polymerase (either wild-type [3P] or inactive mutant [3P-ASM]) and the nucleoprotein (NP), together with a ppoliccat plasmid encoding a CAT reporter gene flanked by either wild-type (WT) or mutant influenza virus crna promoter sequences as indicated. RNA was extracted at 41 h posttransfection and analyzed by primer extension with vrna- or crna-specific primers. (B) Sample sequence traces of cdna amplified by crna- or vrna-specific RT-PCR from virus-like CAT RNA described in A. Extracted RNA was TAP treated and circularized prior to RT-PCR amplification across the junction and cloning of the amplicon. The arrows indicate the mutated 3 -terminal residue (1U3A complement) of the template crna (left) and the corrected wild-type 5 -terminal residue (5 A) of the replicated vrna (right). on June 13, 2018 by guest conclude that pppapg synthesized internally from the crna template can be realigned onto a crna or a vrna template for elongation. DISCUSSION De novo RNA synthesis is a common strategy used by viral RNA-dependent RNA polymerases for viral RNA replication to maintain the integrity of the viral genome (25, 45). However, the mechanisms for primer-independent initiation vary among different RNA viruses (25, 45). The aim of this work was to study the mechanism by which influenza virus polymerase initiates replication on its vrna and crna promoters. Here, we have developed two in vitro primer-independent RNA synthesis assays with recombinant influenza virus RNA

9 VOL. 80, 2006 INFLUENZA VIRUS REPLICATION 2345 FIG. 6. Internally synthesized pppapg derived from the crna promoter can realign to a vrna template for elongation in vitro. (A) Autoradiograph showing pppapg synthesized internally on the crna promoter and realigned onto 3 -terminal residues of the vrna promoter for extension into a trinucleotide by partially purified influenza A/Turkey/England polymerase. Either 1 mm CTP or 1 mm UTP was added to ATP and radiolabeled GTP as indicated for extension to the third nucleotide on either a vrna or a crna promoter. The synthesized di- or trinucleotides are shown on the left. The solid circle on the right marks an unknown band. (B) Characterization of bands A to H by treatment with T 2 RNase and PEI-cellulose TLC. The positions of pppa*p and pp*pgp are shown. *p, 32 P. polymerase and model promoters. The first assays influenza virus crna promoter-specific replication by primer-independent transcription of a 15-nucleotide template. The transcripts were shown to possess 5 -triphosphate end groups, demonstrating the authenticity of the assay. By mutagenesis screening, we found that point mutations at positions 2, 4, 5, and 9 from the 3 end of the crna promoter largely inhibited replication in this assay. In contrast, ApG-primed transcription was only inhibited by mutations at positions 2 and 9, with partial inhibition at position 5 (Fig. 1A). This suggested that pppapg might be synthesized from residues 4 and 5. We therefore investigated the site of de novo pppapg synthesis by a second assay, an in vitro replication initiation assay. We found that the nucleotides at positions 4 and 5 from the 3 end of the crna promoter were important for directing pppapg synthesis in contrast with the nucleotides at positions 1 and 2 at the 3 end of the vrna promoter (Fig. 2). As residues 4 and 5 from the 3 end of the crna promoter are 3 U-C 5, we hypothesized that pppapg may be templated internally from these residues and then realigned to the terminal 3 OH U-C 5 for elongation. Accordingly, we showed that pppapgpu was synthesized from the wild-type crna promoter and the mutant at position 1, but not from the mutant at position 2 (Fig. 4), suggesting that internally synthesized pppapg can realign onto positions 1 and 2 for extension into trinucleotide pppapgpu in a template-directed manner (Fig. 4). This further suggests that base-pairing of pppapg with the C residue at position 2 alone was sufficient for realignment of pppapg and elongation, and that a mutation at position 1 of the template would be corrected to the wild-type base in the transcript. This realignment would explain why mutations at positions 2, 4, and 5 in the model crna failed to synthesize a 15 nucleotide unprimed transcript (Fig. 1). These data are also consistent with the previous unexplained in vivo observations that nucleotides at positions 4 and 5 and the identity of the 2 to 9 base pair of the 3 hairpin loop were crucial for crna promoter activity (8, 33). In addition, we also showed here that a mutated or deleted residue at position 1 at the 3 end of the crna promoter in a minireplicon system can revert to a wild-type sequence in vivo (Fig. 5). Therefore, taking the in vitro and in vivo data together, we conclude that the influenza virus polymerase uses an internal initiation and realignment mechanism for de novo RNA synthesis on the crna promoter. This contrasts with a simpler terminal initiation mechanism used by the polymerase on the vrna promoter.

10 2346 DENG ET AL. J. VIROL. To investigate which structural and sequence properties of the crna promoter were required for internal pppapg initiation, we performed a transplant experiment (Fig. 3). This experiment analyzed the extent of internal pppapg synthesis after transplanting crna-like promoter elements into the model vrna promoter one at a time. The results suggested that internal pppapg synthesis was mainly determined by the absence of a hinge in the 5 strand and the presence of a hinge and the identity of the nucleotide at position 3 in the 3 strand of the crna promoter. These results indicate that the spatial arrangement between the 5 - or3 -strand hairpin-loop and the duplex region in the corkscrew configuration are important determinants controlling whether internal or terminal pppapg synthesis occurs on the vrna and crna promoters. Differences in the hinge residues between the vrna and crna promoters have also been found to be essential for endonuclease cleavage activity (31) and to act as the nuclear export signals for selective packaging of vrna rather than crna into the progeny virions (44). It has previously been suggested that the PB1 subunit of the polymerase might adopt a different conformation upon binding the crna and vrna promoters (20). Differences in vrna and crna promoter structures, in particular in hinge residues, could influence their binding properties to the influenza virus RNA polymerase, thereby altering the site of initiation of pppapg synthesis. Specifically, we propose that there might be differences in the precise binding of the vrna and crna promoters, so as to initially prevent access of incoming nucleotides to positions 1 and 2 of the crna promoter. Such access, however, would be required for positions 1 and 2 of the vrna promoter. Proof of this hypothesis must await the successful crystallization and X-ray analysis of the influenza virus polymerase. To further understand the mechanism of internal initiation and realignment, an in vitro template-switching experiment was performed to test whether internally synthesized pppapg, instead of realigning to the residues 1 and 2 of a crna promoter (cis-realignment), could realign to the terminal nucleotides of a vrna template (trans-realignment). The results (Fig. 6) support the trans-realignment model in vitro. However, as we have not been able to design an experiment to unambiguously prove trans-alignment in vivo, we can only speculate that both cis- and trans-realignment might occur in vivo. A question that arose from this work was the significance (if any) of pppgpu synthesized from the model crna promoter (Fig. 4 and 6, bands A) and pppgpc synthesized from the model vrna promoter (Fig. 6, band C), in addition to pppapg. The potential extension of pppgpu and pppgpc (complementary to residues 2 and 3 of the crna and vrna promoter, respectively) would result in replication products lacking a 5 A end. Because the 5 A ends of both vrna and crna are required for polymerase binding, replication products lacking the 5 A residue would probably not be used efficiently in subsequent rounds of transcription and replication (8, 16, 40). It is possible, however, that pppgpu and pppgpc may not form full-length transcripts as efficiently as pppapg, since pppapg was preferred over pppgpu or pppgpc for elongation to a trinucleotide under our experimental conditions (Fig. 4 and 6). Thus, the significance of the observed abortive synthesis of the dinucleotides pppgpu and pppgpc remains uncertain. Other potential explanations of our data should be considered. For example, the in vivo data for reversion to wild type of a mutated or deleted residue at position 1 at the 3 end of the crna promoter (Fig. 5) could, theoretically, be explained by a model similar to that proposed for the correction of the 3 -end nucleotide of hepatitis C virus RNA (6). These authors proposed that ATP might be preferentially selected over other nucleotides and might be prebound to the RNA polymerase for initiation in a non-template-directed manner. However, the inhibitory effect of a mutation at position 1 from the 3 end of the vrna promoter for in vitro pppapg synthesis (Fig. 2), and for in vivo activity (11) would argue against this possibility. In addition, it might be argued that our model is based, in part, on the synthesis of abortive transcripts and not on full-length replication. Abortive synthesis of short oligonucleotides has been previously observed for influenza virus (42), and is, in fact, common for both prokaryotic and eukaryotic RNA- and DNA-dependent RNA polymerases (34). These abortive transcripts, predominantly di- or trinucleotides, can then be used by polymerases to initiate new rounds of RNA synthesis. Such transcripts may significantly improve the efficiency of the RNA synthesis by circumventing the first rate-limiting nucleotidyl transfer reaction (23, 36). Therefore, we believe that the internal initiation and realignment model best fits our in vitro and in vivo data. An internal initiation and realignment mechanism is well known for many viral RNA and DNA polymerases although the mechanisms vary in detail. In the case of protein-primed transcription, as used by poliovirus RNA polymerase, the VPg primer is uridylylated from an internal loop of the template. The VPgpUpU subsequently anneals to terminal residues for positive-stranded RNA synthesis (39) Bacteriophage 29 DNA polymerase, on the other hand, uses a sliding-back mechanism, in which protein-primed transcription starts at residue 2 and then slides back to residue 1 (35). In the case of de novo RNA synthesis, the RNA polymerases of some viruses, e.g., bunya-, arena-, and nairoviruses, generate oligonucleotides internally from the 3 end of their templates for terminal realignment. However, the mechanism differs from the prime and realign mechanism proposed here because a nontemplated C residue is added (17 19, 24, 35). Prime and realign models have been proposed as the mechanism for repair of the 3 ends of the viral templates since the 3 ends are vulnerable to degradation by host 3 exonucleases (29). In such 3 -end repair models, as reported for turnip crinkle virus, short abortive oligonucleotides serve as a patch to repair one or more nucleotide deletions at the 3 end (19, 21, 25, 37). In the case of the influenza virus RNA polymerase, our in vitro (Fig. 4) and in vivo (Fig. 5) results suggest that a mutation at position 1 of the 3 strand of the crna promoter could revert to the wild-type sequence by the internal initiation and realignment mechanism suggested here. It is also possible that a mutation at position 1 of the vrna promoter could be corrected to the wild-type sequence by internal initiation on the crna promoter, followed by trans-realignment onto the vrna promoter. If so, initiation of pppapg from the crna promoter may have a selective advantage because it would allow mutations at the 3 end of either crna or vrna to be repaired. This mechanism could also correct transcripts to which a nontemplated nucleotide was added at its 3 end (15).