Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM The EMBO Journal (2007) 26, 4566–4575 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM case, particularly for eukaryotic genome replication and Atsushi Kawaguchi and Kyosuke Nagata* transcription. The reconstitution of cell-free transcription Department of Infection Biology, Graduate School of Comprehensive and replication systems of the adenovirus and SV40 genomes, Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan and the identification of host factors by biochemical fractio- nation and complementation have greatly contributed to our By dissecting and reconstituting a cell-free influenza virus knowledge of the fundamental processes of eukaryotic repli- genome replication system, we have purified and identi- cation and transcription. fied the minichromosome maintenance (MCM) complex, The genome of influenza type A viruses consists of eight- which is thought to be a DNA replicative helicase, as one of segmented and single-stranded RNAs of negative polarity. the host factors that regulate the virus genome replication. The viral RNA genome (vRNA) is transcribed into mRNA and MCM interacted with the PA subunit of the viral RNA- replicated through cRNA (full-sized complementary copy of dependent RNA polymerase that is found to be involved in vRNA) to produce a large number of progeny vRNAs in the the replication genetically. The virus genome replication nucleus (reviewed in Engelhardt and Fodor, 2006). In viral was decreased in MCM2 knockdown cells. The viral poly- particles and infected cells, the vRNA exists as ribonucleo- merase appeared to be a nonproductive complex, that is, protein (designated vRNP) complexes with viral RNA-depen- it was capable of initiating replication but produced only dent RNA polymerases consisting of three subunits—PB1, abortive short RNA chains. MCM stimulated de novo- PB2, and PA—and nucleoprotein (NP). PB1 contains the initiated replication reaction by stabilizing a replication conserved motifs characteristic of RNA polymerases and complex during its transition from initiation to elongation. functions as a polymerase catalytic subunit for the sequential Based on the findings, including the result that the MCM- addition of nucleotides to the elongating RNA. PB1 binds to 0 0 mediated RNA replication reaction was competed with the 5 - and 3 -terminal sequences of vRNA and cRNA, which exogenously added RNA, we propose that MCM functions are conserved in all segments and act as cis-acting elements as a scaffold between the nascent RNA chains and the viral for the viral RNA synthesis. Transcription is initiated using polymerase. the oligonucleotide containing the cap-1 structure derived The EMBO Journal (2007) 26, 4566–4575. doi:10.1038/ from cellular pre-mRNAs as a primer. The capped oligonu- sj.emboj.7601881; Published online 11 October 2007 cleotide is generated by the recognition of the cap structure Subject Categories: RNA; microbiology & pathogens by PB2 and endonucleolytic cleavage by PB1. The elongation Keywords: host factor; influenza virus; MCM; replication; of the mRNA chain proceeds until the polymerase reaches a RNA-dependent RNA polymerase polyadenylation signal, which consists of 5–7 U residues located near the 5 -terminal region of the vRNA. In contrast, the genome replication is primer-independent and generates full-length vRNA through cRNA synthesis. Genetic analyses suggest that PA participates in the replication process, Introduction although the precise function of PA is not well established Viruses are intracellular parasites. Since virus genomes are (Sugiura et al, 1975; Ritchey and Palese, 1977). Recently, we considerably small and can thus encode only a limited found that PA is involved in the assembly of a functional number of genes, viruses must use host factors and machi- polymerase (Kawaguchi et al, 2005). It was reported that NP neries to replicate. Therefore, the identification and func- is also important for the replication process (Shapiro and tional characterization of host factors are indispensable to Krug, 1988; Medcalf et al, 1999). However, the precise func- understand the mechanism of viral replication and patho- tion of NP in the replication remains uncertain. genicity, and provide with critical insights into the virus–host Since the replication and regulated transcription of the interaction, which is shaped by unending adaptation and influenza virus genome do not occur only by the influenza eradication between a virus and its host. Viruses often utilize viral components associated with virions, it has been thought critical regulatory processes of the host cell; therefore, studies that some factor(s) present in infected cells is required for the on the processes subverted by viruses have often highlighted regulation of these processes. In fact, it has been reported that cellular regulatory mechanisms. This has indeed been the vRNP interacts with several cellular proteins (Wang et al, 1997; Digard et al, 1999; Huarte et al, 2001; Engelhardt et al, *Corresponding author. Department of Infection Biology, Graduate 2005; Garcia-Robles et al, 2005; Deng et al, 2006). In this School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba regard, by dissecting a cell-free viral RNA synthesis system 305-8575, Japan. Tel.: þ 81 29 853 3233; Fax: þ 81 29 853 3233; mimicking the viral transcription, we have identified RAF-1/ E-mail: [email protected] Hsp90 and RAF-2p48/UAP56/BAT1 as host factors that sti- mulate the viral RNA synthesis. RAF-1/Hsp90 regulates the Received: 12 June 2007; accepted: 18 September 2007; published online: 11 October 2007 assembly of viral RNA polymerase complexes and is also 4566 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata involved in their stabilization during their transfer between templates (Momose et al, 2002; Naito et al, 2007). RAF-2p48/ UAP56/BAT1, which is thought to be involved in the RNA splicing of cellular mRNA, interacts with NP and facilitates the NP–RNA complex formation, thereby stimulating viral RNA synthesis by the viral RNA polymerase (Momose et al, 2001). Interestingly, it was reported that the viral RNA poly- merase interacts with the serine 5-phosphorylated carboxy- terminal domain of the largest subunit of the cellular RNA polymerase II (pol II), which is associated with a pol II transcription initiation complex and plays a role in the recruitment and stimulation of capping enzyme (Chan et al, 2006). However, the host factor(s) involved in the influenza virus genome replication has not been identified yet. Recently, it was suggested that the efficiency of transcription and replication by the viral RNA polymerase is a determinant of both host specificity and pathogenicity, and this may be regulated by the adaptation of the viral RNA polymerase to an unknown host factor(s) (Gabriel et al, 2005, 2007). Here, we reconstituted a cell-free virus genome replication system with virion-associated vRNP and nuclear extracts prepared from uninfected HeLa cells. By biochemical fractio- nation and complementation, we purified Influenza virus REplication Factor (IREF)-1 as a factor that is required for successful virus genome replication. TOF-MS analyses re- Figure 1 Purification of IREF-1. (A) General scheme for purifica- vealed that IREF-1 is a minichromosome maintenance tion of IREF-1. (B) Stimulatory activity of IREF-1 for cell-free virus (MCM) heterohexamer complex consisting of MCM2, 3, 4, genome replication. RNA synthesis was carried out in the absence 5, 6, and 7. MCM proteins were first identified for their roles (lanes 1–4) or presence (lane 5) of ApG dinucleotide primer with 0 (lane 1), 0.5 (lane 2), 1 (lane 3), and 2 (lane 4) ml of purified IREF-1. in plasmid replication or cell cycle progression in yeast (C) Identification of the polarity of newly synthesized RNA by using (Maine et al, 1984; Sinha et al, 1986). It is generally believed, RNase T1 digestion. A band corresponding to segment 8 newly although not completely proven, that MCM functions as a synthesized in the presence of [a- P]UTP was excised from gel, and eukaryotic DNA replication fork helicase. In this report, we digested with RNase T1 (lane 3). Cleaved products were separated and visualized by autoradiography. In vitro synthesized vRNA demonstrate that IREF-1/MCM stimulates virus genome re- (lane 1) and cRNA (lane 2) of segment 8 using T7 RNA polymerase plication by increasing the stability of replicating RNA poly- were also analyzed. merases, which produce only abortive short RNA chains in the absence of IREF-1/MCM. IREF-1/MCM interacted with vRNP through its contact with PA. Biochemical analyses strongly suggest that MCM stabilizes the replicating polymer- Thus, it is reasonably hypothesized that some host protein(s) ase complexes by promoting the interaction between the viral supports the primer-independent virus genome replication. polymerase and nascent cRNA. Taken together with the fact Here, we first examined whether nuclear extracts prepared that virus genome replication was decreased in MCM knock- from uninfected cells promote vRNP to replicate the virus down (KD) cells, it is suggested that IREF-1/MCM is a host genome. This was indeed the case. The primer-independent factor that regulates the influenza virus genome replication. replicative full-sized cRNA synthesis was promoted in the presence of nuclear extracts (see below), suggesting the presence of a host factor(s) involved in virus genome replica- Results tion. We designated such an activity as Influenza virus REplication Factor-1 (IREF-1), and attempted to purify the Purification and characterization of IREF-1 factor(s) responsible for replication by the biochemical frac- The RNA-dependent RNA polymerase of influenza virus tionation of nuclear extracts prepared from uninfected HeLa catalyzes both primer-dependent mRNA synthesis and pri- cells through sequential column chromatography and com- mer-independent virus genome replication in the nuclei of infected cells. It is demonstrated in cell-free RNA synthesis plementation of vRNP for replicative activity (Figure 1A). systems that vRNP isolated from virions catalyzes the primer- IREF-1 promoted the synthesis of full-length viral RNAs in a dose-dependent manner (Figure 1B). Since the influenza dependent RNA synthesis (Plotch and Krug, 1977; Plotch virus genome consists of eight-segmented and single- et al, 1979), which is stimulated by host factors (Momose stranded RNAs, we detected eight segments of newly synthe- et al, 2001; Momose et al, 2002). De novo initiation of cRNA sized RNAs. The electrophoretic mobility of these RNAs synthesis was also observed in a cell-free RNA synthesis system using vRNP or partially purified polymerase fractions synthesized in the presence of IREF-1 was similar to that of as enzyme source (Lee et al, 2002; Vreede and Brownlee, the virus genome RNAs prepared from purified virions (data 2007). However, in the system, only partial fragments of not shown) and that of RNAs synthesized in the presence of the ApG dinucleotide primer complementary to the nucleo- cRNA were detected, but the efficient full-length cRNA synth- tide positions 1 and 2 of the 3 -terminus of vRNA (lane 5, and esis was not demonstrated. Therefore, it is possible that vRNP see Ritchey and Palese, 1977; Honda et al, 1986). could be potentially but not highly active to synthesize cRNA. &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4567 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata To determine the polarity of newly synthesized RNA, the RNA possibly synthesized from segment 8 was excised from the gel, and the isolated RNA was then digested with RNase T1. Since RNase T1 cleaves between the 3 -phosphate group of a guanine ribonucleotide and the 5 -hydroxyl group of the adjacent nucleotide, the digestion of vRNA by RNase T1 yields RNA fragments of 42, 19, and 17 nt with various smaller fragments, while only small fragments are yielded for cRNA (Figure 1C, lanes 1 and 2). The cleavage pattern of the RNA synthesized in the presence of IREF-1 was found to be similar to that of the cRNA (Figure 1C, lane 3). These RNAs were digested with RNase H when hybridized with an oligonucleotide specific for the cRNA polarity (data not shown). Therefore, it is confirmed that the RNA synthesis mediated by IREF-1 generates full-sized cRNA in the absence of any primer. The RNA synthesis level in the presence of IREF-1 varied from segment to segment. The reason for this segment-specific efficiency of RNA synthesis is presently unknown. De novo initiation of the genome replication in the presence of IREF-1 One of the essential features of the RNA synthesized in a primer-independent manner is the presence of a triphosphate moiety at its 5 -end. In order to confirm that newly synthe- Figure 2 Detection of 5 triphosphate end group. (A) Limited elongation assays. Limited elongation assays were carried out in sized cRNA has the 5 -triphosphate end group, we compared the absence (lanes 1 and 2) or presence (lane 3) of ApG with (lane the mobility of the cRNAs synthesized in the presence of 2) or without (lanes 1 and 3) IREF-1. (B) Alkaline phosphatase IREF-1 (Figure 2A, lane 2) with that of ApG-primed products treatment of limited elongation products. Limited elongation pro- (Figure 2A, lane 3) in a limited elongation assay, in which ducts (indicated by asterisks in panel A) synthesized in the presence of ApG (lanes 1 and 2) or IREF-1 (lanes 3 and 4) were excised from UTP is omitted from the reaction mixture and the RNA gel, and treated with (lanes 1 and 3) or without (lanes 2 and 4) polymerase pauses at the first adenine residue on the template alkaline phosphatase (AP). (C) Detection of pppAp using thin-layer (Momose et al, 2002). The expected lengths of limited elonga- chromatography. Twelve nucleotide long of limited elongation tion products are 12 nt for segments 1, 3, and 7; 13 nt for products in the presence of ApG (lanes 1–3) or IREF-1 (lanes 4–6) were excised from gel, and treated with RNase T2 (lanes 1, 2, 4, 5, segments 5 and 8; 14 nt for segment 6; 18 nt for segment 4; and 7) or SV-PDE (lanes 3 and 6). Products were separated through and 19 nt for segment 2. The cRNAs synthesized in the thin-layer chromatography with 1.6 M LiCl, and visualized by presence of IREF-1 migrated differently from the ApG-primed autoradiography. Alkaline phosphatase-treated limited elongation products (Figure 2A, compare lane 2 with lane 3). The short- products (AP; lanes 2 and 4) and RNA synthesized by T7 RNA polymerase in the presence of [g- P]ATP (lane 7) were used as est limited elongation product in the presence of ApG is 12 nt, control products. but the shortest product in the presence of IREF-1 migrated faster than the ApG-primed 12-nt-long product. To examine unprimed RNA synthesis of short RNA of cRNA polarity whether this difference in mobility is due to the absence or by the viral RNA polymerase (Lee et al, 2002; Vreede and presence of 5 -triphosphate, the synthesized RNA bands in- Brownlee, 2007). Thus, IREF-1 may be required for a step(s) dicated by asterisks were eluted from the gel, treated with post the initiation and the early elongation reactions, in Escherichia coli alkaline phosphatase, and then subjected to separation by 15% PAGE containing 8 M urea (Figure 2B). which only short cRNAs are synthesized (Figure 2A, lane Following alkaline phosphatase treatment, the cRNA synthe- 2). Taken altogether, we assumed that the viral RNA poly- merase encounters difficulty after a de novo initiation reac- sized in the presence of IREF-1 migrated to the same position tion, and that IREF-1 resolves this to promote the viral RNA as the ApG-primed products (Figure 2B, lane 4). polymerase to synthesize the de novo-initiated and full-sized Next, we attempted to separate 5 -triphosphate by thin- cRNA (discussed later along with Figures 6 and 7). layer chromatography after digestion with either RNase T2 or snake venom phosphodiesterase (SV-PDE) (Figure 2C). The first and second nucleotides of the cRNA are A and Identification of IREF-1 as MCM, a putative DNA G residues, respectively. Digestion by RNase T2 of replicative helicase 32 32 [a- P]GTP-labeled unprimed cRNA would yield pppA pif Analyses of a Mono Q column fraction with MALDI-TOF MS the 5 -triphosphate is present, whereas SV-PDE yields pG. indicated that human MCM proteins 2, 4, 5, 6, and 7 are We detected pppA p only from the cRNA synthesized in the major components of the IREF-1 fraction (Figure 3A, lanes presence of IREF-1 by RNase T2 digestion (Figure 2C, lane 4). 2–5, and Supplementary Table 1S). MCM possesses the DNA Therefore, it was concluded that IREF-1 promotes de novo- helicase activity and plays important roles in the regulation initiated virus genome replication. of genomic DNA replication (reviewed in Forsburg, 2004). To Note that unprimed cRNAs in limited elongation assays confirm the results obtained from MALDI-TOF MS analyses, were detected even in the absence of IREF-1 and ApG western blotting assays were carried out with rabbit anti- (Figure 2A, lane 1). This is in good agreement with the MCM2–7 antibodies. All MCM2–7 proteins were detected in 4568 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata IREF-1/MCM complex interacts with PA polymerase subunit Because IREF-1/MCM was identified as a stimulatory factor in the cell-free virus genome replication system by using vRNP as the enzyme source, the target(s) of MCM must be one or more of the vRNP components, that is, PB1, PB2, PA, and NP. To determine the viral factor(s) that binds to MCM, we carried out immunoprecipitation assays with cell lysates prepared from cells expressing one of the FLAG- or Myc-tagged viral proteins (Figures 4A and B). MCM2 was immuno- precipitated with the PA subunit of the viral RNA polymerase (Figure 4A, lanes 10–12), but not with other viral proteins (lanes 4–9 and 16–18), suggesting that MCM interacts with the viral RNA polymerase through the contact with PA. To confirm whether PA interacts with MCM2 as a component of the MCM complex, we tried to detect other MCM proteins that co-immunoprecipitated with PA by performing western blotting analyses (Figure 4B). Not only MCM2 but also MCM3, 4, 5, and 7 were immunoprecipitated with PA, demonstrating that PA interacts with the MCM complex. We could not detect MCM6 since an anti-MCM6 antisera with high titer was not available. Next, to examine whether MCM also interacts with viral proteins in infected cells, the lysates were prepared from infected cells after crosslinking with DSP and formaldehyde and subjected to immunoprecipitation assays using an anti- Figure 3 Identification of functional components in IREF-1. (A) MCM2 antibody (Figure 4C). We observed that PA, PB2, and (Left panel) SDS–PAGE analysis. The loaded amounts were adjusted NP co-immunoprecipitated with MCM2. Since Figure 4A to the equal level of the IREF-1 stimulatory activity attained in the reveals that MCM2 does not interact with PB2 and NP cell-free virus genome replication assay. Lane 1, molecular size marker (Bio-Rad); lane 2, uninfected HeLa cell nuclear extracts; when singly expressed (Figure 4A, lanes 7–9 and 16–18) lane 3, 0.2 M KCl eluate from phosphocellulose column; lane 4, and the immunoprecipitation assay using rMCM and micro- 0.25 M (NH ) SO eluate from phenyl Sepharose column; lane 5, 4 2 4 coccal nuclease-treated vRNP also showed that NP does not 0.33 M KCl eluate from Mono Q column (purified IREF-1 fraction). interact with MCM (Supplementary Figure 1S), it is quite The gel was visualized by silver staining assay. (Right panel) Western blotting assay. The IREF-1 fraction of Mono Q column likely that MCM interacts with PA in the vRNP complexes in was separated through SDS–PAGE, and subjected to western blot- infected cells. Further to confirm this, RNA was purified from ting assays with rabbit anti-MCM2, 3, 4, 5, 6, and 7 antibodies. (B) immunoprecipitates and subjected to RT–PCR with primers (Left panel) Purification of recombinant MCM complex from insect specific for the segment 5 vRNA. The virus genome was cells. The details of purification scheme and column chromatogra- phy are described in Supplementary data. Lane 1, molecular size immunoprecipitated with MCM2 (Figure 4D), indicating that marker (Bio-Rad); lane 2, purified IREF-1 fraction from uninfected MCM interacts with vRNP. There is a significant difference in HeLa nuclear extracts (Mono Q fraction); lanes 3–5, recombinant the amount of proteins co-immunoprecipitated with MCM2 MCM heterohexamer complex containing 7.5 (lane 3), 15 (lane 4), between polymerase proteins and NP (Figure 4C). This could 30 ng (lane 5) of MCM2 equivalent. The gel was visualized by silver staining assay. (Right panel) Cell-free virus genome replication be due to the fact that the amount of polymerase proteins in assay. Equal amounts of IREF-1 and rMCM loaded in left panel vRNP was significantly less than that of NP. One NP was were examined in the cell-free virus genome replication assay. Lane shown to bind every 20 nucleotides (Yamanaka et al, 1990). 6, purified IREF-1 fraction (Mono Q fraction); lanes 8–10, rMCM Exactly the same patterns were observed with lysates pre- complex containing 7.5, 15, 30 ng of rMCM2. The assay in lane 7 was carried out in the absence of any proteins. pared in the absence of crosslink reagents (data not shown). However, the amount of immunoprecipitated proteins was considerably less. Therefore, it is possible that MCM interacts with vRNP transiently or catalytically. the IREF-1 fraction (Figure 3A, lanes 6–12). To examine whether MCM proteins are responsible for the IREF-1 activity, Involvement of MCM in influenza virus genome unprimed RNA synthesis was performed in the presence replication in infected cells of a recombinant MCM heterohexamer complex (rMCM) Since each MCM gene is essential for the cellular DNA (Figure 3B). The rMCM was purified from insect cells expres- replication, we cannot establish MCM gene knockout cells. sing MCM proteins by recombinant baculovirus infection It was reported that the nuclear transport of MCM complex is (Figure 3B, lanes 3–5). Since MCM 4, 5, and 7 were fused interdependent among MCM proteins (Pasion and Forsburg, with histidine-tag, the mobility of these proteins was slightly 1999). Therefore, using siRNA-mediated gene silencing of the different from that of authentic MCM proteins. Figure 3B MCM genes, we tried to examine whether MCM functions in shows that rMCM and authentic IREF-1 stimulate virus the influenza virus genome replication in cultured cells genome replication up to comparable levels (Figure 3B, (Figure 5A). After 60 h post transfection of duplex RNA lanes 6–10). These results indicate that MCM is responsible oligonucleotides corresponding to the MCM2 and MCM3 for IREF-1 activity. genes, the expression level of MCM2 and MCM3 proteins in &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4569 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata Figure 4 MCM complex interacts with PA polymerase subunit. (A) The interaction of MCM2 with singly expressed viral proteins. HeLa cells were mock-transfected (lanes 1–3 and 13–15) or transfected with each plasmid encoding PB1-FLAG (lanes 4–6), PB2-FLAG (lanes 7–9), PA- FLAG (lanes 10–12), and NP-Myc (lanes 16–18). After 24 h post transfection, cell lysates were prepared and subjected to immunoprecipitation assays in the absence of antibody (lanes 2, 5, 8, 11, 14, and 17) or presence of either mouse anti-FLAG (lanes 3, 6, 9, and 12) or anti-Myc antibody (lanes 15 and 18). Immunoprecipitated proteins were separated, and then visualized by western blotting assays with goat anti-MCM2 and rabbit anti-PB1, PB2, PA, and NP antibodies. (B) The interaction of MCM proteins with singly expressed PA. HeLa cells expressing PA (lanes 4–6) were lysed and subjected to immunoprecipitation assays in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of anti-FLAG antibody. Lanes 1–3 represent mock experiments. Western blotting analyses were performed with rabbit anti-MCM2, 3, 4, 5, 7, and PA antibodies. (C) The interaction between MCM2 and viral proteins in infected cells. HeLa cells were infected with PR/8/34 influenza virus at an MOI ¼ 10. After 8 h post infection, cell lysates were prepared and subjected to immunoprecipitation assays with either goat control IgG (lanes 2 and 5) or anti-MCM2 antibody (lanes 3 and 6). Immunoprecipitated proteins were detected by western blotting analyses with rabbit anti-PA, PB2, NP, and MCM2 antibodies. (D) The interaction between MCM2 and the virus genome in infected cells. vRNAs immunoprecipitated as described in (C) were purified, and then semiquantitatively analyzed by RT–PCR with primers specific for segment 5 vRNA. To quantitatively evaluate, 0.5% equivalents of mock-treated sample (lane 1) and 0.05, 0.15, and 0.5% equivalents of infected sample (lanes 2–4) were also subjected to RT–PCR. transfected HeLa cells decreased to 20% of the cells trans- ing vRNP in MCM KD cells was similar to that in control cells fected with random sequence siRNA (Figure 5A, lanes 1 and (Figure 5C). These results indicate that MCM is involved 2). FACS analysis showed that the cell proliferation rate of predominantly in virus genome replication in infected cells. MCM protein knockdown (KD) cells was found largely un- These observations were confirmed by a plasmid-based changed under this condition (data not shown). MCM pro- replication system (Supplementary Figure 3S). teins are expressed in excess over a number of replication It could be expected that the reduction of the vRNA origins (Lei et al, 1996; Mahbubani et al, 1997; Edwards et al, template causes concomitant reduction in mRNA. It was 2002), and the normal replication rate is maintained even reported that the level of mRNA synthesis was not completely when the number of MCM is reduced to 1–2 per origin decreased by the defect of vRNA amplification (Vreede et al, (Mahbubani et al, 1997; Edwards et al, 2002; Cortez et al, 2004; Kawaguchi et al, 2005; Hara et al, 2006). This was 2004; Snyder et al, 2005). It is possible that KD of MCM also the case in the plasmid-based replication system proteins may cause some level of the G1 arrest and thereby (Supplementary Figure 3S-D). The level of mRNA synthesis affect the virus replication. We confirmed that the synthesis remained unchanged at different levels of vRNA synthesis. level of cRNA and viral mRNA in G1 phase-synchronized The amount of vRNA responsible for mRNA synthesis could cells was similar to that in S phase cells (Supplementary be only a small portion of replicated vRNA. Figure 2S). Note that the viral genome replication in MCM KD cells Quantitative RT–PCR assays with primer sets specific for was not abolished completely. This could be due to the fact segment 5 cRNA and NP mRNA showed that the level of that the rest of MCM proteins in KD HeLa cells is enough for cRNA synthesis in MCM KD HeLa cells reduce to 70% of that the viral genome replication since MCM proteins are highly in control cells (Figure 5B, left panel, Po0.01). In contrast, abundant in a cell (Lei et al, 1996; Mahbubani et al, 1997; there was no significant difference in the level of mRNA Edwards et al, 2002). Then, we used human normal fibro- synthesis between control and MCM KD HeLa cells blast WI-38 cells since the amount of MCM2-7 proteins is (Figure 5B, right panel, P40.05). To evaluate the viral found to be 10 times less in WI-38 cells than in HeLa cells transcription activity independent of the virus genome repli- (Figure 5A, lanes 1 and 3, and see Ishimi et al, 2003). The cation, we examined the level of viral transcription from expression level of the MCM proteins in MCM KD WI-38 cells infecting vRNP using cycloheximide (CHX), a potent protein decreased to 30% of that in control cells (Figure 5A, lanes 3 synthesis inhibitor (Figure 5C). It is shown that CHX sup- and 4). The synthesis level of cRNA in MCM KD WI-38 cells presses viral protein synthesis and thereby leads to degrada- reduced to 40% of that in control cells (Figure 5D, left panel). tion of replicated virus genome RNA but not viral mRNA In contrast, the synthesis level of mRNA did not differ since newly vRNP formation was repressed (Vreede et al, between control and MCM KD WI-38 cells (Figure 5D, right 2004). As expected, the level of mRNA synthesis from infect- panels). 4570 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata Figure 5 Involvement of MCM in influenza virus genome replication in infected cells. (A) Expression level of MCM2 and MCM3 in MCM knockdown (KD) cells. HeLa (lanes 1 and 2) and WI-38 cells (lanes 3 and 4) were transfected with random siRNA (lanes 1 and 3) or both MCM2 and MCM3 siRNAs (lanes 2 and 4). After 60 h post transfection, the amount of MCM2, MCM3, and b-actin proteins were determined by western blotting assays. (B) The level of virus genome replication in MCM KD HeLa cells. At 60 h post transfection of random or MCM siRNA, cells were infected with influenza virus at an MOI ¼ 10. At 8 h post infection, total RNAs were purified, and then subjected to real-time quantitative RT–PCR with primer sets specific for segment 5 cRNA (left panel, Po0.01) and NP mRNA (right panel, P40.05). The details of the method employed are described in Supplementary data. (C) The level of viral mRNA synthesis in MCM KD HeLa cells in the presence of cycloheximide. Control and MCM KD HeLa cells were infected with influenza virus in the presence of 100 mg/ml cycloheximide at an MOI ¼ 10 for 8 h. The real-time quantitative RT–PCR assays were carried out with a primer set specific for NP mRNA. (D) The level of virus genome replication in MCM KD WI-38 cells. Control and MCM KD WI-38 cells were infected with influenza virus at an MOI ¼ 10 for 8 h. The real-time quantitative RT–PCR assays were carried out with primer sets specific for segment 5 cRNA (left panel) and NP mRNA (right panel). b-actin mRNA was used as an internal control for the whole procedure. IREF-1/MCM stabilizes RNA polymerase elongation upper panel), whereas exactly the same amounts of the vRNA complex template were immunoprecipitated in the absence and As shown in Figures 1B and 2A, it is likely that IREF-1/MCM presence of MCM (Figure 6A, lower panel). These results does not enhance the frequency of the replication initiation, indicate that MCM stabilizes the elongation complex possibly but rather makes a nonproductive viral RNA polymerase to by preventing the release of nascent cRNA from RNA poly- override the step for abortive synthesis. Both eukaryotic and merase complexes. bacterial DNA-dependent RNA polymerases proceed to the If the nascent cRNA tends to be dissociated from the productive stage by passing through the abortive transcrip- elongation complex in the absence of MCM, full-length tion stage, during which the polymerase releases short nas- cRNA would not be synthesized even after allowing the cent RNA chains. After the synthesis of an approximately 10- elongation reaction to restart from limited elongation by the nt-long RNA, the RNA synthesizing complex transits into a addition of MCM and nucleotides absent in the limited stable transcription elongation form. This transition from the elongation assays. To examine whether MCM acts in the initiation step, including a complex possibly undergoing initiation or the elongation process of genome replication, abortive synthesis to the stable elongation step, is one of unprimed limited elongation assays were performed to the important steps in the regulation of RNA synthesis by synthesize 2-nt- or 12-nt-long nascent cRNA from the seg- RNA polymerases. ment 7 vRNA in the absence of either CTP or UTP (Figures 6B To clarify the role of MCM in virus genome replication, we and C). After the unprimed limited elongation, elongation examined the effect of MCM on this transition. The amount of reactions were restarted by the addition of either CTP and the elongation complex should reflect the stability of the UTP or UTP, and MCM was also added either before or after elongation complex. To assess the stability of the elongation the limited elongation. We detected the full-length cRNA from complex, we tried to measure the amount of the nascent the limited elongation assays synthesizing 2-nt-long RNA cRNA (Figure 6A, upper panel) and vRNA template irrespective of the presence or absence of MCM during the (Figure 6A, lower panel) associated with the RNA polymerase limited elongation reaction (Figure 6C, lanes 1–3). In con- complexes stalled on the vRNA template in limited elongation trast, the full-length cRNA was synthesized by restarting the assays by immunoprecipitation using an anti-PB2 antibody. limited elongation assay for 12-nt-long RNA in the presence The [a- P]GTP-labeled nascent cRNAs were detected by of MCM, but not in the course of the addition of MCM after autoradiography, whereas the vRNA templates were sub- the limited elongation (Figure 6C, lanes 4–7). The hairpin- jected to semiquantitative RT–PCR with primers specific for loop and double-stranded promoter regions, which act as the segment 5 vRNA. The level of immunoprecipitated nas- cis-elements essential for the interaction with the viral RNA cent cRNA was increased by the addition of MCM (Figure 6A, polymerase, are located between nucleotide positions 1–12 of &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4571 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata Figure 6 MCM complex stabilizes RNA polymerase elongation complex. (A) Immunoprecipitation of replication elongation complex. After the limited elongation reaction performed in the absence (lanes 1–3 and 7–11) or presence (lanes 4–6 and 12–14) of rMCM, viral polymerase complexes were immunoprecipitated without (lanes 2, 5, 10, and 13) or with (lanes 3, 6, 11, and 14) anti-PB2 antibody. Immunoprecipitated [a- P]GTP-labeled nascent cRNAs were separated and visualized by autoradiography (upper panel). Immunoprecipitated vRNA templates were semiquantitatively analyzed by RT–PCR as described in the legend of Figure 4 (lower panel). Inputs (2 (lane 7), 6 (lane 8), and 20% (lanes 9 and 12) equivalents) were also analyzed by RT–PCR. (B) The structure of the influenza virus segment 7 vRNA promoter. The figure is modified from one in Crow et al (2004). (C) MCM complex functions during transition from initiation to elongation. Limited elongation reactions were performed in the absence of CTP (lanes 1–3) or UTP (lanes 4–7) without (lanes 1, 3, 4, and 6) or with (lanes 2, 5, and 7) rMCM. After 1 h incubation, elongation reactions were restarted by the addition of CTP and UTP for lanes 1–3 and UTP for lanes 4–6. For lanes 3 and 6, rMCM was added at the restart of elongation reaction. (D) MCM functions as a scaffold between nascent cRNA and viral polymerase complexes. Limited elongation assays were performed with (lanes 1, and 3–5) or without (lane 2) rMCM. The reaction mixture was 0 0 preincubated for 10 min in the absence (lane 1) or presence of 20 pmol of RNA-AG (5 -AGGGGAAAGGAGAAG-3 , lanes 2–4) and/or 1 nmol 0 0 RNA-CC (5 -AGGGGAAAGGAGACC-3 , lanes 4 and 5). (E) ATPase dependency of the IREF-1 activity. The cell-free virus genome replication assays were performed in the presence of ATP (lanes 1 and 3) or ATPgS (lanes 2 and 4) without (lanes 1and 2) or with (lanes 3 and 4) rMCM. the 3 -terminal region of vRNA (Figure 6B). It is possible that which could be utilized as the ApG primer. By adding RNA- the viral RNA polymerase remains at and begins escaping AG, limited elongation products of 25, 26, 27, 31, and 32 nt from the promoter under the limited elongation condition were synthesized as expected (Figure 6D, lane 2). MCM synthesizing 2-nt- and 12-nt-long RNAs, respectively. It is stimulated RNA-AG primed RNA synthesis (Figure 6D, lane quite likely that the RNA polymerase associated with the 3). Furthermore, the effect of MCM was quenched by the nascent 12-nt-long RNA synthesized in the presence of MCM addition of RNA-CC, which is 15-mer RNA with the 3 - can override the abortive destiny. Further, to avoid the terminal CpC and thus does not serve as a primer for RNA abortive RNA synthesis, MCM is required prior to the synth- synthesis (Figure 6D, lanes 4 and 5). It could be interpreted esis of 12-nt-long RNA and possibly after the synthesis of that RNA-CC competes with RNA-AG for binding to MCM. 2-nt-long RNA. The same results were obtained for other These results indicate that RNA-AG is recruited to the viral segments (data not shown). Altogether, these findings sug- RNA polymerase by the interaction with MCM. Thus, it is gest that MCM is involved in the stabilization of the elonga- highly possible that MCM stabilizes the replication elongation tion complex during the transition from the initiation to the complex through scaffolding between nascent cRNA and the productive elongation process around the promoter. viral RNA polymerase to make the polymerase competent for It is possible that MCM functions as a scaffold protein full-length cRNA synthesis. It should be noted that MCM did between nascent cRNA and vRNP possibly through the PA not facilitate the unprimed limited elongation reactions subunit of the viral RNA polymerase since nascent cRNA (Figure 2A). It is possible that the length of nascent cRNA tends to be released from the viral RNA polymerase in the in unprimed limited elongation reactions may not be enough absence of MCM (Figure 6A). To test this hypothesis, we for binding with MCM. examined whether a 15-nt-long RNA containing ApG at its 3 - Biochemical studies have shown that MCM possesses an terminus (RNA-AG) is recruited to the RNA polymerase by ATP-dependent DNA helicase activity (You et al, 1999). MCM (Figure 6D). Limited elongation assays were performed Therefore, it is speculated that MCM acts as an RNA helicase in the presence of 15 nt RNA-AG, the 3 -terminal ApG of in virus genome replication. However, full-length cRNA 4572 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata synthesis occurred in the presence of ATPgS, which is a nonhydrolyzable analog of ATP but can be a substrate for the chain elongation of RNA synthesis (Figure 6E, compare lane 3 with lane 4). Discussion We have identified IREF-1 as a factor that stimulates the cell- free influenza virus genome replication activity. IREF-1 was found to be identical to the MCM complex consisting of MCM2, 3, 4, 5, 6, and 7. By the addition of IREF-1/MCM, the full-length cRNA was synthesized from each virus gen- ome segment. The RNA synthesis level in the presence of Figure 7 A proposed model. MCM stabilizes replication elongation MCM differed from segment to segment (Figure 1B, lanes complexes through scaffolding between nascent cRNA and viral 2–4). However, the amounts of replicated cRNAs are almost RNA polymerase complexes during its transition from initiation to elongation to allow viral RNA polymerase complexes to synthesize equal among eight segments in infected cells (Hatada et al, full-length cRNA. 1989). Therefore, it is possible that another host factor(s) is required for the quantitative control of the virus genome replication. However, this issue is still an open question. Previous reports showed that NP is important for virus genome replication (Shapiro and Krug, 1988; Medcalf et al, (data not shown). We speculate that another host and/or viral factor(s) other than MCM may be required for the 1999). NP is required for elongation of RNA synthesis (Honda switching mechanism of initiation reactions. et al, 1988). Moreover, the in vitro cRNA synthesis using A universal characteristic feature of RNA polymerases is infected cell extracts as an enzyme source depends on a supply of non-RNP-associated NP (Shapiro and Krug, the regulated conversion from an initiating form that holds 1988). The latter finding may be interpreted as that NP nascent RNA weakly to an elongating form that holds RNA functions in co-transcriptional encapsidation of nascent tightly during RNA synthesis. This transition occurs during promoter escape, and involves a complex series of molecular RNA for prevention of discrete RNA synthesis. Therefore, it transformations in both prokaryotic and eukaryotic DNA- is possible that MCM and NP free of vRNA act in a similar dependent RNA polymerases (Zawel et al, 1995; Hsu, fashion and cooperatively facilitate the virus genome replica- 2002). TFIIB and TFIIE, the general transcription factors for tion. Further, it was reported that minimal cRNA synthesis occurs even in the absence of newly synthesized NP (Vreede the cellular RNA polymerase II (pol II), are released from the et al, 2004). Thus, it is speculated that MCM activates and elongation complex by the synthesis of an approximately 10- nt-long nascent RNA (Zawel et al, 1995). The nascent RNA guarantees the virus genome replication from incoming vRNP forms a 9 bp RNA:DNA hybrid in the elongation complex at immediate early phases of infection, where newly synthe- (Gnatt et al, 2001), which is thought to be a primary stability sized NP is absent. determinant for the elongation complex (Kireeva et al, 2000). MCM interacted with the viral RNA polymerase through its contact with PA (Figure 4). Previous genetic analyses sug- It is speculated that the hybridization between template DNA gested that PA participates in the replication process, and nascent RNA chains with lengths less than 9 bp is less although its precise role had not been well established effective for the stabilization of the elongating complex. Our results showed that the influenza viral RNA polymerase (Sugiura et al, 1975; Ritchey and Palese, 1977; Kawaguchi catalyzes the initiation reaction, but cannot synthesize the et al, 2005). Recently, it has been reported that the influenza full-length cRNA because the nascent cRNA was dissociated virus vRNP interacts with nucleosomes (Garcia-Robles et al, from the elongation complex in the absence of MCM 2005). Both vRNP and NP that is free of RNA are capable of binding to core histones in vitro. It has also been reported (Figure 6). In contrast, in the presence of MCM, the elonga- that the viral RNA polymerase interact with the phospho- tion complex was stabilized and converted to the form competent for the processive RNA synthesis since MCM serine 5 form of the largest subunit of pol II (Engelhardt et al, may act as a scaffold between nascent cRNA and the viral 2005). MCM also interacts with pol II holoenzyme in addition RNA polymerase without its helicase activity (Figures 6 and to DNA replication origins in nucleosomes (Yankulov et al, 7). It is possible that the association of nascent RNA with the 1999). Based on these, we tentatively hypothesize that vRNP binds to chromatin where MCM is present while interacting elongation complex could be stabilized by MCM through its with chromatin proteins such as pol II and recruits MCM binding to nascent RNA. On this line, it was proposed that PA increases the interaction between PB1 and RNA (Lee et al, through the contact with PA. 2002). PA is UV-crosslinked to the 5 -terminal promoter of A functional form of the viral polymerase for the transcrip- vRNA (Fodor et al, 1994). Thus, it is also possible that MCM tion and replication is thought to be a ternary complex modulates the interaction between the viral polymerase and consisting of PB1, PB2, and PA (Fodor et al, 2002; Gastaminza et al, 2003). However, the molecular basis of the promoter through its contact with PA. the switching mechanism between transcription and replica- It was reported that MCM is associated with pol II- tion is unknown. In infected cells, MCM is involved predo- mediated transcription. Antibodies against MCM2 inhibit pol II transcription in Xenopus oocytes (Yankulov et al, minantly in virus genome replication, but not transcription 1999); the interaction between MCM5 and the activation (Figure 5). MCM did not inhibit initiation of capped RNA- domain of STAT1a is essential for the expression of IFN-g primed mRNA synthesis in a cell-free RNA synthesis system &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4573 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata in the absence of UTP, and RNA products were separated through responsive genes (Snyder et al, 2005). The finding reported 15% PAGE containing 8 M urea. here may provide useful information to further understand the mechanism of cellular transcription regulated by MCM. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Materials and methods Cell-free virus genome replication system Acknowledgements vRNP was prepared from purified influenza A/PR/8/34 virus as previously described (Shimizu et al, 1994). A cell-free virus genome We thank F Momose (Kitasato University), Y Ishimi (Ibaraki replication was carried out at 301C for 90 min in a final volume of University), H Nojima (Osaka University), and N Yabuta (Osaka 25 ml containing 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl , University) for the generous gifts of pCAGGS-NP Myc (FM), UW31- 50 mM KCl, 1.5 mM dithiothreitol, 500 mM each ATP, CTP, and hMCM4(His)-6, UW31-hMCM2-7(His), and pAcAB4-hMCM3-5(His) UTP, 25 mMGTP,5 mCi of [a- P]GTP (3000 Ci/mmol), 8 U of RNase (YI), anti-MCM proteins antibodies (HN and NY). We also thank J inhibitor, 25 mg of actinomycin D/ml, and vRNP (50 ng of NP Yanagisawa (University of Tsukuba) for his help for MALDI-TOF MS equivalents) in the presence or absence of host factor fraction or analyses. This research was supported in part by a grant-in-aid from purified proteins. RNA products were purified, subjected to 4% the Ministry of Education, Culture, Sports, Science, and Technology PAGE in the presence of 8 M urea, and visualized by autoradio- of Japan (to KN) and Research Fellowships of the Japanese Society graphy. For limited elongation assay, RNA synthesis was performed for the Promotion of Science (JSPS) (to AK). References Chan AY, Vreede FT, Smith M, Engelhardt OG, Fodor E (2006) virus RNA polymerase play a critical role in protein stability, Influenza virus inhibits RNA polymerase II elongation. Virology endonuclease activity, cap binding, and virion RNA promoter 351: 210–217 binding. J Virol 80: 7789–7798 Cortez D, Glick G, Elledge SJ (2004) Minichromosome maintenance Hatada E, Hasegawa M, Mukaigawa J, Shimizu K, Fukuda R (1989) proteins are direct targets of the ATM and ATR checkpoint Control of influenza virus gene expression: quantitative analysis kinases. Proc Natl Acad Sci USA 101: 10078–10083 of each viral RNA species in infected cells. J Biochem (Tokyo) 105: Crow M, Deng T, Addley M, Brownlee GG (2004) Mutational 537–546 analysis of the influenza virus cRNA promoter and identification Honda A, Mizumoto K, Ishihama A (1986) RNA polymerase of of nucleotides critical for replication. J Virol 78: 6263–6270 influenza virus. Dinucleotide-primed initiation of transcription at Deng T, Engelhardt OG, Thomas B, Akoulitchev AV, Brownlee GG, specific positions on viral RNA. J Biol Chem 261: 5987–5991 Fodor E (2006) Role of ran binding protein 5 in nuclear import Honda A, Ueda K, Nagata K, Ishihama A (1988) RNA polymerase of and assembly of the influenza virus RNA polymerase complex. influenza virus: role of NP in RNA chain elongation. J Biochem J Virol 80: 11911–11919 (Tokyo) 104: 1021–1026 Digard P, Elton D, Bishop K, Medcalf E, Weeds A, Pope B (1999) Hsu LM (2002) Promoter clearance and escape in prokaryotes. Modulation of nuclear localization of the influenza virus nucleopro- Biochim Biophys Acta 1577: 191–207 tein through interaction with actin filaments. JVirol 73: 2222–2231 Huarte M, Sanz-Ezquerro JJ, Roncal F, Ortin J, Nieto A (2001) PA Edwards MC, Tutter AV, Cvetic C, Gilbert CH, Prokhorova TA, subunit from influenza virus polymerase complex interacts with a Walter JC (2002) MCM2–7 complexes bind chromatin in a cellular protein with homology to a family of transcriptional distributed pattern surrounding the origin recognition complex activators. J Virol 75: 8597–8604 in Xenopus egg extracts. J Biol Chem 277: 33049–33057 Ishimi Y, Okayasu I, Kato C, Kwon HJ, Kimura H, Yamada K, Song Engelhardt OG, Fodor E (2006) Functional association between viral SY (2003) Enhanced expression of Mcm proteins in cancer cells and cellular transcription during influenza virus infection. Rev derived from uterine cervix. Eur J Biochem 270: 1089–1101 Med Virol 16: 329–345 Kawaguchi A, Naito T, Nagata K (2005) Involvement of influenza Engelhardt OG, Smith M, Fodor E (2005) Association of the influ- virus PA subunit in assembly of functional RNA polymerase enza A virus RNA-dependent RNA polymerase with cellular RNA complexes. J Virol 79: 732–744 polymerase II. J Virol 79: 5812–5818 Kireeva ML, Komissarova N, Waugh DS, Kashlev M (2000) The Fodor E, Crow M, Mingay LJ, Deng T, Sharps J, Fechter P, Brownlee 8-nucleotide-long RNA:DNA hybrid is a primary stability deter- GG (2002) A single amino acid mutation in the PA subunit of the minant of the RNA polymerase II elongation complex. J Biol influenza virus RNA polymerase inhibits endonucleolytic clea- Chem 275: 6530–6536 vage of capped RNAs. J Virol 76: 8989–9001 Lee MT, Bishop K, Medcalf L, Elton D, Digard P, Tiley L (2002) Fodor E, Pritlove DC, Brownlee GG (1994) The influenza virus Definition of the minimal viral components required for the panhandle is involved in the initiation of transcription. J Virol 68: initiation of unprimed RNA synthesis by influenza virus RNA 4092–4096 polymerase. Nucleic Acids Res 30: 429–438 Forsburg SL (2004) Eukaryotic MCM proteins: beyond replication Lei M, Kawasaki Y, Tye BK (1996) Physical interactions among Mcm initiation. Microbiol Mol Biol Rev 68: 109–131 proteins and effects of Mcm dosage on DNA replication in Gabriel G, Abram M, Keiner B, Wagner R, Klenk HD, Stech J (2007) Saccharomyces cerevisiae. Mol Cell Biol 16: 5081–5090 Differential polymerase activity in avian and Mammalian cells Mahbubani HM, Chong JP, Chevalier S, Thommes P, Blow JJ (1997) determines host range of influenza virus. J Virol 81: 9601–9604 Cell cycle regulation of the replication licensing system: involve- Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J (2005) The ment of a Cdk-dependent inhibitor. J Cell Biol 136: 125–135 viral polymerase mediates adaptation of an avian influenza virus Maine GT, Sinha P, Tye BK (1984) Mutants of S. cerevisiae to a mammalian host. Proc Natl Acad Sci USA 102: 18590–18595 defective in the maintenance of minichromosomes. Genetics Garcia-Robles I, Akarsu H, Muller CW, Ruigrok RW, Baudin F (2005) 106: 365–385 Interaction of influenza virus proteins with nucleosomes. Medcalf L, Poole E, Elton D, Digard P (1999) Temperature-sensitive Virology 332: 329–336 lesions in two influenza A viruses defective for replicative tran- Gastaminza P, Perales B, Falcon AM, Ortin J (2003) Mutations in the scription disrupt RNA binding by the nucleoprotein. J Virol 73: N-terminal region of influenza virus PB2 protein affect virus RNA 7349–7356 replication but not transcription. J Virol 77: 5098–5108 Momose F, Basler CF, O’Neill RE, Iwamatsu A, Palese P, Nagata K Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD (2001) (2001) Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 in- Structural basis of transcription: an RNA polymerase II elonga- teracts with the influenza virus nucleoprotein and enhances viral tion complex at 3.3 A resolution. Science 292: 1876–1882 RNA synthesis. J Virol 75: 1899–1908 Hara K, Schmidt FI, Crow M, Brownlee GG (2006) Amino acid Momose F, Naito T, Yano K, Sugimoto S, Morikawa Y, Nagata K residues in the N-terminal region of the PA subunit of influenza A (2002) Identification of Hsp90 as a stimulatory host factor 4574 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata involved in influenza virus RNA synthesis. J Biol Chem 277: Sugiura A, Ueda M, Tobita K, Enomoto C (1975) Further isolation and characterization of temperature-sensitive mutants of influen- 45306–45314 Naito T, Momose F, Kawaguchi A, Nagata K (2007) Involvement of za virus. Virology 65: 363–373 Hsp90 in assembly and nuclear import of influenza virus RNA Vreede FT, Brownlee GG (2007) Influenza virion-derived viral polymerase subunits. J Virol 81: 1339–1349 ribonucleoproteins synthesize both mRNA and cRNA in vitro. Pasion SG, Forsburg SL (1999) Nuclear localization of J Virol 81: 2196–2204 Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM com- Vreede FT, Jung TE, Brownlee GG (2004) Model suggesting that plex assembly. Mol Biol Cell 10: 4043–4057 replication of influenza virus is regulated by stabilization of Plotch SJ, Bouloy M, Krug RM (1979) Transfer of 5 -terminal cap of replicative intermediates. J Virol 78: 9568–9572 globin mRNA to influenza viral complementary RNA during Wang P, Palese P, O’Neill RE (1997) The NPI-1/NPI-3 (karyopherin transcription in vitro. Proc Natl Acad Sci USA 76: 1618–1622 alpha) binding site on the influenza a virus nucleoprotein NP Plotch SJ, Krug RM (1977) Influenza virion transcriptase: synthesis is a nonconventional nuclear localization signal. J Virol 71: in vitro of large, polyadenylic acid-containing complementary 1850–1856 RNA. J Virol 21: 24–34 Yamanaka K, Ishihama A, Nagata K (1990) Reconstitution of Ritchey MB, Palese P (1977) Identification of the defective genes in influenza virus RNA–nucleoprotein complexes structurally re- three mutant groups of influenza virus. J Virol 21: 1196–1204 sembling native viral ribonucleoprotein cores. J Biol Chem 265: Shapiro GI, Krug RM (1988) Influenza virus RNA replication in 11151–11155 vitro: synthesis of viral template RNAs and virion RNAs in the Yankulov K, Todorov I, Romanowski P, Licatalosi D, Cilli K, absence of an added primer. J Virol 62: 2285–2290 McCracken S, Laskey R, Bentley DL (1999) MCM proteins are Shimizu K, Handa H, Nakada S, Nagata K (1994) Regulation of associated with RNA polymerase II holoenzyme. Mol Cell Biol 19: influenza virus RNA polymerase activity by cellular and viral 6154–6163 factors. Nucleic Acids Res 22: 5047–5053 You Z, Komamura Y, Ishimi Y (1999) Biochemical analysis of the Sinha P, Chang V, Tye BK (1986) A mutant that affects the function of intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity. Mol Cell Biol autonomously replicating sequences in yeast. J Mol Biol 192: 805–814 19: 8003–8015 Snyder M, He W, Zhang JJ (2005) The DNA replication factor MCM5 Zawel L, Kumar KP, Reinberg D (1995) Recycling of the general is essential for Stat1-mediated transcriptional activation. Proc transcription factors during RNA polymerase II transcription. Natl Acad Sci USA 102: 14539–14544 Genes Dev 9: 1479–1490 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4575 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM

Kawaguchi, Atsushi; Nagata, Kyosuke
The EMBO Journal , Volume 26 (21) – Oct 31, 2007
Download PDF
10 pages

Loading next page...
 
/lp/springer-journals/de-novo-replication-of-the-influenza-virus-rna-genome-is-regulated-by-0XPrLyNPa7

References (50)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 2007
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1038/sj.emboj.7601881
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal (2007) 26, 4566–4575 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL De novo replication of the influenza virus RNA genome is regulated by DNA replicative helicase, MCM case, particularly for eukaryotic genome replication and Atsushi Kawaguchi and Kyosuke Nagata* transcription. The reconstitution of cell-free transcription Department of Infection Biology, Graduate School of Comprehensive and replication systems of the adenovirus and SV40 genomes, Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan and the identification of host factors by biochemical fractio- nation and complementation have greatly contributed to our By dissecting and reconstituting a cell-free influenza virus knowledge of the fundamental processes of eukaryotic repli- genome replication system, we have purified and identi- cation and transcription. fied the minichromosome maintenance (MCM) complex, The genome of influenza type A viruses consists of eight- which is thought to be a DNA replicative helicase, as one of segmented and single-stranded RNAs of negative polarity. the host factors that regulate the virus genome replication. The viral RNA genome (vRNA) is transcribed into mRNA and MCM interacted with the PA subunit of the viral RNA- replicated through cRNA (full-sized complementary copy of dependent RNA polymerase that is found to be involved in vRNA) to produce a large number of progeny vRNAs in the the replication genetically. The virus genome replication nucleus (reviewed in Engelhardt and Fodor, 2006). In viral was decreased in MCM2 knockdown cells. The viral poly- particles and infected cells, the vRNA exists as ribonucleo- merase appeared to be a nonproductive complex, that is, protein (designated vRNP) complexes with viral RNA-depen- it was capable of initiating replication but produced only dent RNA polymerases consisting of three subunits—PB1, abortive short RNA chains. MCM stimulated de novo- PB2, and PA—and nucleoprotein (NP). PB1 contains the initiated replication reaction by stabilizing a replication conserved motifs characteristic of RNA polymerases and complex during its transition from initiation to elongation. functions as a polymerase catalytic subunit for the sequential Based on the findings, including the result that the MCM- addition of nucleotides to the elongating RNA. PB1 binds to 0 0 mediated RNA replication reaction was competed with the 5 - and 3 -terminal sequences of vRNA and cRNA, which exogenously added RNA, we propose that MCM functions are conserved in all segments and act as cis-acting elements as a scaffold between the nascent RNA chains and the viral for the viral RNA synthesis. Transcription is initiated using polymerase. the oligonucleotide containing the cap-1 structure derived The EMBO Journal (2007) 26, 4566–4575. doi:10.1038/ from cellular pre-mRNAs as a primer. The capped oligonu- sj.emboj.7601881; Published online 11 October 2007 cleotide is generated by the recognition of the cap structure Subject Categories: RNA; microbiology & pathogens by PB2 and endonucleolytic cleavage by PB1. The elongation Keywords: host factor; influenza virus; MCM; replication; of the mRNA chain proceeds until the polymerase reaches a RNA-dependent RNA polymerase polyadenylation signal, which consists of 5–7 U residues located near the 5 -terminal region of the vRNA. In contrast, the genome replication is primer-independent and generates full-length vRNA through cRNA synthesis. Genetic analyses suggest that PA participates in the replication process, Introduction although the precise function of PA is not well established Viruses are intracellular parasites. Since virus genomes are (Sugiura et al, 1975; Ritchey and Palese, 1977). Recently, we considerably small and can thus encode only a limited found that PA is involved in the assembly of a functional number of genes, viruses must use host factors and machi- polymerase (Kawaguchi et al, 2005). It was reported that NP neries to replicate. Therefore, the identification and func- is also important for the replication process (Shapiro and tional characterization of host factors are indispensable to Krug, 1988; Medcalf et al, 1999). However, the precise func- understand the mechanism of viral replication and patho- tion of NP in the replication remains uncertain. genicity, and provide with critical insights into the virus–host Since the replication and regulated transcription of the interaction, which is shaped by unending adaptation and influenza virus genome do not occur only by the influenza eradication between a virus and its host. Viruses often utilize viral components associated with virions, it has been thought critical regulatory processes of the host cell; therefore, studies that some factor(s) present in infected cells is required for the on the processes subverted by viruses have often highlighted regulation of these processes. In fact, it has been reported that cellular regulatory mechanisms. This has indeed been the vRNP interacts with several cellular proteins (Wang et al, 1997; Digard et al, 1999; Huarte et al, 2001; Engelhardt et al, *Corresponding author. Department of Infection Biology, Graduate 2005; Garcia-Robles et al, 2005; Deng et al, 2006). In this School of Comprehensive Human Sciences and Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba regard, by dissecting a cell-free viral RNA synthesis system 305-8575, Japan. Tel.: þ 81 29 853 3233; Fax: þ 81 29 853 3233; mimicking the viral transcription, we have identified RAF-1/ E-mail: [email protected] Hsp90 and RAF-2p48/UAP56/BAT1 as host factors that sti- mulate the viral RNA synthesis. RAF-1/Hsp90 regulates the Received: 12 June 2007; accepted: 18 September 2007; published online: 11 October 2007 assembly of viral RNA polymerase complexes and is also 4566 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata involved in their stabilization during their transfer between templates (Momose et al, 2002; Naito et al, 2007). RAF-2p48/ UAP56/BAT1, which is thought to be involved in the RNA splicing of cellular mRNA, interacts with NP and facilitates the NP–RNA complex formation, thereby stimulating viral RNA synthesis by the viral RNA polymerase (Momose et al, 2001). Interestingly, it was reported that the viral RNA poly- merase interacts with the serine 5-phosphorylated carboxy- terminal domain of the largest subunit of the cellular RNA polymerase II (pol II), which is associated with a pol II transcription initiation complex and plays a role in the recruitment and stimulation of capping enzyme (Chan et al, 2006). However, the host factor(s) involved in the influenza virus genome replication has not been identified yet. Recently, it was suggested that the efficiency of transcription and replication by the viral RNA polymerase is a determinant of both host specificity and pathogenicity, and this may be regulated by the adaptation of the viral RNA polymerase to an unknown host factor(s) (Gabriel et al, 2005, 2007). Here, we reconstituted a cell-free virus genome replication system with virion-associated vRNP and nuclear extracts prepared from uninfected HeLa cells. By biochemical fractio- nation and complementation, we purified Influenza virus REplication Factor (IREF)-1 as a factor that is required for successful virus genome replication. TOF-MS analyses re- Figure 1 Purification of IREF-1. (A) General scheme for purifica- vealed that IREF-1 is a minichromosome maintenance tion of IREF-1. (B) Stimulatory activity of IREF-1 for cell-free virus (MCM) heterohexamer complex consisting of MCM2, 3, 4, genome replication. RNA synthesis was carried out in the absence 5, 6, and 7. MCM proteins were first identified for their roles (lanes 1–4) or presence (lane 5) of ApG dinucleotide primer with 0 (lane 1), 0.5 (lane 2), 1 (lane 3), and 2 (lane 4) ml of purified IREF-1. in plasmid replication or cell cycle progression in yeast (C) Identification of the polarity of newly synthesized RNA by using (Maine et al, 1984; Sinha et al, 1986). It is generally believed, RNase T1 digestion. A band corresponding to segment 8 newly although not completely proven, that MCM functions as a synthesized in the presence of [a- P]UTP was excised from gel, and eukaryotic DNA replication fork helicase. In this report, we digested with RNase T1 (lane 3). Cleaved products were separated and visualized by autoradiography. In vitro synthesized vRNA demonstrate that IREF-1/MCM stimulates virus genome re- (lane 1) and cRNA (lane 2) of segment 8 using T7 RNA polymerase plication by increasing the stability of replicating RNA poly- were also analyzed. merases, which produce only abortive short RNA chains in the absence of IREF-1/MCM. IREF-1/MCM interacted with vRNP through its contact with PA. Biochemical analyses strongly suggest that MCM stabilizes the replicating polymer- Thus, it is reasonably hypothesized that some host protein(s) ase complexes by promoting the interaction between the viral supports the primer-independent virus genome replication. polymerase and nascent cRNA. Taken together with the fact Here, we first examined whether nuclear extracts prepared that virus genome replication was decreased in MCM knock- from uninfected cells promote vRNP to replicate the virus down (KD) cells, it is suggested that IREF-1/MCM is a host genome. This was indeed the case. The primer-independent factor that regulates the influenza virus genome replication. replicative full-sized cRNA synthesis was promoted in the presence of nuclear extracts (see below), suggesting the presence of a host factor(s) involved in virus genome replica- Results tion. We designated such an activity as Influenza virus REplication Factor-1 (IREF-1), and attempted to purify the Purification and characterization of IREF-1 factor(s) responsible for replication by the biochemical frac- The RNA-dependent RNA polymerase of influenza virus tionation of nuclear extracts prepared from uninfected HeLa catalyzes both primer-dependent mRNA synthesis and pri- cells through sequential column chromatography and com- mer-independent virus genome replication in the nuclei of infected cells. It is demonstrated in cell-free RNA synthesis plementation of vRNP for replicative activity (Figure 1A). systems that vRNP isolated from virions catalyzes the primer- IREF-1 promoted the synthesis of full-length viral RNAs in a dose-dependent manner (Figure 1B). Since the influenza dependent RNA synthesis (Plotch and Krug, 1977; Plotch virus genome consists of eight-segmented and single- et al, 1979), which is stimulated by host factors (Momose stranded RNAs, we detected eight segments of newly synthe- et al, 2001; Momose et al, 2002). De novo initiation of cRNA sized RNAs. The electrophoretic mobility of these RNAs synthesis was also observed in a cell-free RNA synthesis system using vRNP or partially purified polymerase fractions synthesized in the presence of IREF-1 was similar to that of as enzyme source (Lee et al, 2002; Vreede and Brownlee, the virus genome RNAs prepared from purified virions (data 2007). However, in the system, only partial fragments of not shown) and that of RNAs synthesized in the presence of the ApG dinucleotide primer complementary to the nucleo- cRNA were detected, but the efficient full-length cRNA synth- tide positions 1 and 2 of the 3 -terminus of vRNA (lane 5, and esis was not demonstrated. Therefore, it is possible that vRNP see Ritchey and Palese, 1977; Honda et al, 1986). could be potentially but not highly active to synthesize cRNA. &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4567 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata To determine the polarity of newly synthesized RNA, the RNA possibly synthesized from segment 8 was excised from the gel, and the isolated RNA was then digested with RNase T1. Since RNase T1 cleaves between the 3 -phosphate group of a guanine ribonucleotide and the 5 -hydroxyl group of the adjacent nucleotide, the digestion of vRNA by RNase T1 yields RNA fragments of 42, 19, and 17 nt with various smaller fragments, while only small fragments are yielded for cRNA (Figure 1C, lanes 1 and 2). The cleavage pattern of the RNA synthesized in the presence of IREF-1 was found to be similar to that of the cRNA (Figure 1C, lane 3). These RNAs were digested with RNase H when hybridized with an oligonucleotide specific for the cRNA polarity (data not shown). Therefore, it is confirmed that the RNA synthesis mediated by IREF-1 generates full-sized cRNA in the absence of any primer. The RNA synthesis level in the presence of IREF-1 varied from segment to segment. The reason for this segment-specific efficiency of RNA synthesis is presently unknown. De novo initiation of the genome replication in the presence of IREF-1 One of the essential features of the RNA synthesized in a primer-independent manner is the presence of a triphosphate moiety at its 5 -end. In order to confirm that newly synthe- Figure 2 Detection of 5 triphosphate end group. (A) Limited elongation assays. Limited elongation assays were carried out in sized cRNA has the 5 -triphosphate end group, we compared the absence (lanes 1 and 2) or presence (lane 3) of ApG with (lane the mobility of the cRNAs synthesized in the presence of 2) or without (lanes 1 and 3) IREF-1. (B) Alkaline phosphatase IREF-1 (Figure 2A, lane 2) with that of ApG-primed products treatment of limited elongation products. Limited elongation pro- (Figure 2A, lane 3) in a limited elongation assay, in which ducts (indicated by asterisks in panel A) synthesized in the presence of ApG (lanes 1 and 2) or IREF-1 (lanes 3 and 4) were excised from UTP is omitted from the reaction mixture and the RNA gel, and treated with (lanes 1 and 3) or without (lanes 2 and 4) polymerase pauses at the first adenine residue on the template alkaline phosphatase (AP). (C) Detection of pppAp using thin-layer (Momose et al, 2002). The expected lengths of limited elonga- chromatography. Twelve nucleotide long of limited elongation tion products are 12 nt for segments 1, 3, and 7; 13 nt for products in the presence of ApG (lanes 1–3) or IREF-1 (lanes 4–6) were excised from gel, and treated with RNase T2 (lanes 1, 2, 4, 5, segments 5 and 8; 14 nt for segment 6; 18 nt for segment 4; and 7) or SV-PDE (lanes 3 and 6). Products were separated through and 19 nt for segment 2. The cRNAs synthesized in the thin-layer chromatography with 1.6 M LiCl, and visualized by presence of IREF-1 migrated differently from the ApG-primed autoradiography. Alkaline phosphatase-treated limited elongation products (Figure 2A, compare lane 2 with lane 3). The short- products (AP; lanes 2 and 4) and RNA synthesized by T7 RNA polymerase in the presence of [g- P]ATP (lane 7) were used as est limited elongation product in the presence of ApG is 12 nt, control products. but the shortest product in the presence of IREF-1 migrated faster than the ApG-primed 12-nt-long product. To examine unprimed RNA synthesis of short RNA of cRNA polarity whether this difference in mobility is due to the absence or by the viral RNA polymerase (Lee et al, 2002; Vreede and presence of 5 -triphosphate, the synthesized RNA bands in- Brownlee, 2007). Thus, IREF-1 may be required for a step(s) dicated by asterisks were eluted from the gel, treated with post the initiation and the early elongation reactions, in Escherichia coli alkaline phosphatase, and then subjected to separation by 15% PAGE containing 8 M urea (Figure 2B). which only short cRNAs are synthesized (Figure 2A, lane Following alkaline phosphatase treatment, the cRNA synthe- 2). Taken altogether, we assumed that the viral RNA poly- merase encounters difficulty after a de novo initiation reac- sized in the presence of IREF-1 migrated to the same position tion, and that IREF-1 resolves this to promote the viral RNA as the ApG-primed products (Figure 2B, lane 4). polymerase to synthesize the de novo-initiated and full-sized Next, we attempted to separate 5 -triphosphate by thin- cRNA (discussed later along with Figures 6 and 7). layer chromatography after digestion with either RNase T2 or snake venom phosphodiesterase (SV-PDE) (Figure 2C). The first and second nucleotides of the cRNA are A and Identification of IREF-1 as MCM, a putative DNA G residues, respectively. Digestion by RNase T2 of replicative helicase 32 32 [a- P]GTP-labeled unprimed cRNA would yield pppA pif Analyses of a Mono Q column fraction with MALDI-TOF MS the 5 -triphosphate is present, whereas SV-PDE yields pG. indicated that human MCM proteins 2, 4, 5, 6, and 7 are We detected pppA p only from the cRNA synthesized in the major components of the IREF-1 fraction (Figure 3A, lanes presence of IREF-1 by RNase T2 digestion (Figure 2C, lane 4). 2–5, and Supplementary Table 1S). MCM possesses the DNA Therefore, it was concluded that IREF-1 promotes de novo- helicase activity and plays important roles in the regulation initiated virus genome replication. of genomic DNA replication (reviewed in Forsburg, 2004). To Note that unprimed cRNAs in limited elongation assays confirm the results obtained from MALDI-TOF MS analyses, were detected even in the absence of IREF-1 and ApG western blotting assays were carried out with rabbit anti- (Figure 2A, lane 1). This is in good agreement with the MCM2–7 antibodies. All MCM2–7 proteins were detected in 4568 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata IREF-1/MCM complex interacts with PA polymerase subunit Because IREF-1/MCM was identified as a stimulatory factor in the cell-free virus genome replication system by using vRNP as the enzyme source, the target(s) of MCM must be one or more of the vRNP components, that is, PB1, PB2, PA, and NP. To determine the viral factor(s) that binds to MCM, we carried out immunoprecipitation assays with cell lysates prepared from cells expressing one of the FLAG- or Myc-tagged viral proteins (Figures 4A and B). MCM2 was immuno- precipitated with the PA subunit of the viral RNA polymerase (Figure 4A, lanes 10–12), but not with other viral proteins (lanes 4–9 and 16–18), suggesting that MCM interacts with the viral RNA polymerase through the contact with PA. To confirm whether PA interacts with MCM2 as a component of the MCM complex, we tried to detect other MCM proteins that co-immunoprecipitated with PA by performing western blotting analyses (Figure 4B). Not only MCM2 but also MCM3, 4, 5, and 7 were immunoprecipitated with PA, demonstrating that PA interacts with the MCM complex. We could not detect MCM6 since an anti-MCM6 antisera with high titer was not available. Next, to examine whether MCM also interacts with viral proteins in infected cells, the lysates were prepared from infected cells after crosslinking with DSP and formaldehyde and subjected to immunoprecipitation assays using an anti- Figure 3 Identification of functional components in IREF-1. (A) MCM2 antibody (Figure 4C). We observed that PA, PB2, and (Left panel) SDS–PAGE analysis. The loaded amounts were adjusted NP co-immunoprecipitated with MCM2. Since Figure 4A to the equal level of the IREF-1 stimulatory activity attained in the reveals that MCM2 does not interact with PB2 and NP cell-free virus genome replication assay. Lane 1, molecular size marker (Bio-Rad); lane 2, uninfected HeLa cell nuclear extracts; when singly expressed (Figure 4A, lanes 7–9 and 16–18) lane 3, 0.2 M KCl eluate from phosphocellulose column; lane 4, and the immunoprecipitation assay using rMCM and micro- 0.25 M (NH ) SO eluate from phenyl Sepharose column; lane 5, 4 2 4 coccal nuclease-treated vRNP also showed that NP does not 0.33 M KCl eluate from Mono Q column (purified IREF-1 fraction). interact with MCM (Supplementary Figure 1S), it is quite The gel was visualized by silver staining assay. (Right panel) Western blotting assay. The IREF-1 fraction of Mono Q column likely that MCM interacts with PA in the vRNP complexes in was separated through SDS–PAGE, and subjected to western blot- infected cells. Further to confirm this, RNA was purified from ting assays with rabbit anti-MCM2, 3, 4, 5, 6, and 7 antibodies. (B) immunoprecipitates and subjected to RT–PCR with primers (Left panel) Purification of recombinant MCM complex from insect specific for the segment 5 vRNA. The virus genome was cells. The details of purification scheme and column chromatogra- phy are described in Supplementary data. Lane 1, molecular size immunoprecipitated with MCM2 (Figure 4D), indicating that marker (Bio-Rad); lane 2, purified IREF-1 fraction from uninfected MCM interacts with vRNP. There is a significant difference in HeLa nuclear extracts (Mono Q fraction); lanes 3–5, recombinant the amount of proteins co-immunoprecipitated with MCM2 MCM heterohexamer complex containing 7.5 (lane 3), 15 (lane 4), between polymerase proteins and NP (Figure 4C). This could 30 ng (lane 5) of MCM2 equivalent. The gel was visualized by silver staining assay. (Right panel) Cell-free virus genome replication be due to the fact that the amount of polymerase proteins in assay. Equal amounts of IREF-1 and rMCM loaded in left panel vRNP was significantly less than that of NP. One NP was were examined in the cell-free virus genome replication assay. Lane shown to bind every 20 nucleotides (Yamanaka et al, 1990). 6, purified IREF-1 fraction (Mono Q fraction); lanes 8–10, rMCM Exactly the same patterns were observed with lysates pre- complex containing 7.5, 15, 30 ng of rMCM2. The assay in lane 7 was carried out in the absence of any proteins. pared in the absence of crosslink reagents (data not shown). However, the amount of immunoprecipitated proteins was considerably less. Therefore, it is possible that MCM interacts with vRNP transiently or catalytically. the IREF-1 fraction (Figure 3A, lanes 6–12). To examine whether MCM proteins are responsible for the IREF-1 activity, Involvement of MCM in influenza virus genome unprimed RNA synthesis was performed in the presence replication in infected cells of a recombinant MCM heterohexamer complex (rMCM) Since each MCM gene is essential for the cellular DNA (Figure 3B). The rMCM was purified from insect cells expres- replication, we cannot establish MCM gene knockout cells. sing MCM proteins by recombinant baculovirus infection It was reported that the nuclear transport of MCM complex is (Figure 3B, lanes 3–5). Since MCM 4, 5, and 7 were fused interdependent among MCM proteins (Pasion and Forsburg, with histidine-tag, the mobility of these proteins was slightly 1999). Therefore, using siRNA-mediated gene silencing of the different from that of authentic MCM proteins. Figure 3B MCM genes, we tried to examine whether MCM functions in shows that rMCM and authentic IREF-1 stimulate virus the influenza virus genome replication in cultured cells genome replication up to comparable levels (Figure 3B, (Figure 5A). After 60 h post transfection of duplex RNA lanes 6–10). These results indicate that MCM is responsible oligonucleotides corresponding to the MCM2 and MCM3 for IREF-1 activity. genes, the expression level of MCM2 and MCM3 proteins in &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4569 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata Figure 4 MCM complex interacts with PA polymerase subunit. (A) The interaction of MCM2 with singly expressed viral proteins. HeLa cells were mock-transfected (lanes 1–3 and 13–15) or transfected with each plasmid encoding PB1-FLAG (lanes 4–6), PB2-FLAG (lanes 7–9), PA- FLAG (lanes 10–12), and NP-Myc (lanes 16–18). After 24 h post transfection, cell lysates were prepared and subjected to immunoprecipitation assays in the absence of antibody (lanes 2, 5, 8, 11, 14, and 17) or presence of either mouse anti-FLAG (lanes 3, 6, 9, and 12) or anti-Myc antibody (lanes 15 and 18). Immunoprecipitated proteins were separated, and then visualized by western blotting assays with goat anti-MCM2 and rabbit anti-PB1, PB2, PA, and NP antibodies. (B) The interaction of MCM proteins with singly expressed PA. HeLa cells expressing PA (lanes 4–6) were lysed and subjected to immunoprecipitation assays in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of anti-FLAG antibody. Lanes 1–3 represent mock experiments. Western blotting analyses were performed with rabbit anti-MCM2, 3, 4, 5, 7, and PA antibodies. (C) The interaction between MCM2 and viral proteins in infected cells. HeLa cells were infected with PR/8/34 influenza virus at an MOI ¼ 10. After 8 h post infection, cell lysates were prepared and subjected to immunoprecipitation assays with either goat control IgG (lanes 2 and 5) or anti-MCM2 antibody (lanes 3 and 6). Immunoprecipitated proteins were detected by western blotting analyses with rabbit anti-PA, PB2, NP, and MCM2 antibodies. (D) The interaction between MCM2 and the virus genome in infected cells. vRNAs immunoprecipitated as described in (C) were purified, and then semiquantitatively analyzed by RT–PCR with primers specific for segment 5 vRNA. To quantitatively evaluate, 0.5% equivalents of mock-treated sample (lane 1) and 0.05, 0.15, and 0.5% equivalents of infected sample (lanes 2–4) were also subjected to RT–PCR. transfected HeLa cells decreased to 20% of the cells trans- ing vRNP in MCM KD cells was similar to that in control cells fected with random sequence siRNA (Figure 5A, lanes 1 and (Figure 5C). These results indicate that MCM is involved 2). FACS analysis showed that the cell proliferation rate of predominantly in virus genome replication in infected cells. MCM protein knockdown (KD) cells was found largely un- These observations were confirmed by a plasmid-based changed under this condition (data not shown). MCM pro- replication system (Supplementary Figure 3S). teins are expressed in excess over a number of replication It could be expected that the reduction of the vRNA origins (Lei et al, 1996; Mahbubani et al, 1997; Edwards et al, template causes concomitant reduction in mRNA. It was 2002), and the normal replication rate is maintained even reported that the level of mRNA synthesis was not completely when the number of MCM is reduced to 1–2 per origin decreased by the defect of vRNA amplification (Vreede et al, (Mahbubani et al, 1997; Edwards et al, 2002; Cortez et al, 2004; Kawaguchi et al, 2005; Hara et al, 2006). This was 2004; Snyder et al, 2005). It is possible that KD of MCM also the case in the plasmid-based replication system proteins may cause some level of the G1 arrest and thereby (Supplementary Figure 3S-D). The level of mRNA synthesis affect the virus replication. We confirmed that the synthesis remained unchanged at different levels of vRNA synthesis. level of cRNA and viral mRNA in G1 phase-synchronized The amount of vRNA responsible for mRNA synthesis could cells was similar to that in S phase cells (Supplementary be only a small portion of replicated vRNA. Figure 2S). Note that the viral genome replication in MCM KD cells Quantitative RT–PCR assays with primer sets specific for was not abolished completely. This could be due to the fact segment 5 cRNA and NP mRNA showed that the level of that the rest of MCM proteins in KD HeLa cells is enough for cRNA synthesis in MCM KD HeLa cells reduce to 70% of that the viral genome replication since MCM proteins are highly in control cells (Figure 5B, left panel, Po0.01). In contrast, abundant in a cell (Lei et al, 1996; Mahbubani et al, 1997; there was no significant difference in the level of mRNA Edwards et al, 2002). Then, we used human normal fibro- synthesis between control and MCM KD HeLa cells blast WI-38 cells since the amount of MCM2-7 proteins is (Figure 5B, right panel, P40.05). To evaluate the viral found to be 10 times less in WI-38 cells than in HeLa cells transcription activity independent of the virus genome repli- (Figure 5A, lanes 1 and 3, and see Ishimi et al, 2003). The cation, we examined the level of viral transcription from expression level of the MCM proteins in MCM KD WI-38 cells infecting vRNP using cycloheximide (CHX), a potent protein decreased to 30% of that in control cells (Figure 5A, lanes 3 synthesis inhibitor (Figure 5C). It is shown that CHX sup- and 4). The synthesis level of cRNA in MCM KD WI-38 cells presses viral protein synthesis and thereby leads to degrada- reduced to 40% of that in control cells (Figure 5D, left panel). tion of replicated virus genome RNA but not viral mRNA In contrast, the synthesis level of mRNA did not differ since newly vRNP formation was repressed (Vreede et al, between control and MCM KD WI-38 cells (Figure 5D, right 2004). As expected, the level of mRNA synthesis from infect- panels). 4570 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata Figure 5 Involvement of MCM in influenza virus genome replication in infected cells. (A) Expression level of MCM2 and MCM3 in MCM knockdown (KD) cells. HeLa (lanes 1 and 2) and WI-38 cells (lanes 3 and 4) were transfected with random siRNA (lanes 1 and 3) or both MCM2 and MCM3 siRNAs (lanes 2 and 4). After 60 h post transfection, the amount of MCM2, MCM3, and b-actin proteins were determined by western blotting assays. (B) The level of virus genome replication in MCM KD HeLa cells. At 60 h post transfection of random or MCM siRNA, cells were infected with influenza virus at an MOI ¼ 10. At 8 h post infection, total RNAs were purified, and then subjected to real-time quantitative RT–PCR with primer sets specific for segment 5 cRNA (left panel, Po0.01) and NP mRNA (right panel, P40.05). The details of the method employed are described in Supplementary data. (C) The level of viral mRNA synthesis in MCM KD HeLa cells in the presence of cycloheximide. Control and MCM KD HeLa cells were infected with influenza virus in the presence of 100 mg/ml cycloheximide at an MOI ¼ 10 for 8 h. The real-time quantitative RT–PCR assays were carried out with a primer set specific for NP mRNA. (D) The level of virus genome replication in MCM KD WI-38 cells. Control and MCM KD WI-38 cells were infected with influenza virus at an MOI ¼ 10 for 8 h. The real-time quantitative RT–PCR assays were carried out with primer sets specific for segment 5 cRNA (left panel) and NP mRNA (right panel). b-actin mRNA was used as an internal control for the whole procedure. IREF-1/MCM stabilizes RNA polymerase elongation upper panel), whereas exactly the same amounts of the vRNA complex template were immunoprecipitated in the absence and As shown in Figures 1B and 2A, it is likely that IREF-1/MCM presence of MCM (Figure 6A, lower panel). These results does not enhance the frequency of the replication initiation, indicate that MCM stabilizes the elongation complex possibly but rather makes a nonproductive viral RNA polymerase to by preventing the release of nascent cRNA from RNA poly- override the step for abortive synthesis. Both eukaryotic and merase complexes. bacterial DNA-dependent RNA polymerases proceed to the If the nascent cRNA tends to be dissociated from the productive stage by passing through the abortive transcrip- elongation complex in the absence of MCM, full-length tion stage, during which the polymerase releases short nas- cRNA would not be synthesized even after allowing the cent RNA chains. After the synthesis of an approximately 10- elongation reaction to restart from limited elongation by the nt-long RNA, the RNA synthesizing complex transits into a addition of MCM and nucleotides absent in the limited stable transcription elongation form. This transition from the elongation assays. To examine whether MCM acts in the initiation step, including a complex possibly undergoing initiation or the elongation process of genome replication, abortive synthesis to the stable elongation step, is one of unprimed limited elongation assays were performed to the important steps in the regulation of RNA synthesis by synthesize 2-nt- or 12-nt-long nascent cRNA from the seg- RNA polymerases. ment 7 vRNA in the absence of either CTP or UTP (Figures 6B To clarify the role of MCM in virus genome replication, we and C). After the unprimed limited elongation, elongation examined the effect of MCM on this transition. The amount of reactions were restarted by the addition of either CTP and the elongation complex should reflect the stability of the UTP or UTP, and MCM was also added either before or after elongation complex. To assess the stability of the elongation the limited elongation. We detected the full-length cRNA from complex, we tried to measure the amount of the nascent the limited elongation assays synthesizing 2-nt-long RNA cRNA (Figure 6A, upper panel) and vRNA template irrespective of the presence or absence of MCM during the (Figure 6A, lower panel) associated with the RNA polymerase limited elongation reaction (Figure 6C, lanes 1–3). In con- complexes stalled on the vRNA template in limited elongation trast, the full-length cRNA was synthesized by restarting the assays by immunoprecipitation using an anti-PB2 antibody. limited elongation assay for 12-nt-long RNA in the presence The [a- P]GTP-labeled nascent cRNAs were detected by of MCM, but not in the course of the addition of MCM after autoradiography, whereas the vRNA templates were sub- the limited elongation (Figure 6C, lanes 4–7). The hairpin- jected to semiquantitative RT–PCR with primers specific for loop and double-stranded promoter regions, which act as the segment 5 vRNA. The level of immunoprecipitated nas- cis-elements essential for the interaction with the viral RNA cent cRNA was increased by the addition of MCM (Figure 6A, polymerase, are located between nucleotide positions 1–12 of &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4571 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata Figure 6 MCM complex stabilizes RNA polymerase elongation complex. (A) Immunoprecipitation of replication elongation complex. After the limited elongation reaction performed in the absence (lanes 1–3 and 7–11) or presence (lanes 4–6 and 12–14) of rMCM, viral polymerase complexes were immunoprecipitated without (lanes 2, 5, 10, and 13) or with (lanes 3, 6, 11, and 14) anti-PB2 antibody. Immunoprecipitated [a- P]GTP-labeled nascent cRNAs were separated and visualized by autoradiography (upper panel). Immunoprecipitated vRNA templates were semiquantitatively analyzed by RT–PCR as described in the legend of Figure 4 (lower panel). Inputs (2 (lane 7), 6 (lane 8), and 20% (lanes 9 and 12) equivalents) were also analyzed by RT–PCR. (B) The structure of the influenza virus segment 7 vRNA promoter. The figure is modified from one in Crow et al (2004). (C) MCM complex functions during transition from initiation to elongation. Limited elongation reactions were performed in the absence of CTP (lanes 1–3) or UTP (lanes 4–7) without (lanes 1, 3, 4, and 6) or with (lanes 2, 5, and 7) rMCM. After 1 h incubation, elongation reactions were restarted by the addition of CTP and UTP for lanes 1–3 and UTP for lanes 4–6. For lanes 3 and 6, rMCM was added at the restart of elongation reaction. (D) MCM functions as a scaffold between nascent cRNA and viral polymerase complexes. Limited elongation assays were performed with (lanes 1, and 3–5) or without (lane 2) rMCM. The reaction mixture was 0 0 preincubated for 10 min in the absence (lane 1) or presence of 20 pmol of RNA-AG (5 -AGGGGAAAGGAGAAG-3 , lanes 2–4) and/or 1 nmol 0 0 RNA-CC (5 -AGGGGAAAGGAGACC-3 , lanes 4 and 5). (E) ATPase dependency of the IREF-1 activity. The cell-free virus genome replication assays were performed in the presence of ATP (lanes 1 and 3) or ATPgS (lanes 2 and 4) without (lanes 1and 2) or with (lanes 3 and 4) rMCM. the 3 -terminal region of vRNA (Figure 6B). It is possible that which could be utilized as the ApG primer. By adding RNA- the viral RNA polymerase remains at and begins escaping AG, limited elongation products of 25, 26, 27, 31, and 32 nt from the promoter under the limited elongation condition were synthesized as expected (Figure 6D, lane 2). MCM synthesizing 2-nt- and 12-nt-long RNAs, respectively. It is stimulated RNA-AG primed RNA synthesis (Figure 6D, lane quite likely that the RNA polymerase associated with the 3). Furthermore, the effect of MCM was quenched by the nascent 12-nt-long RNA synthesized in the presence of MCM addition of RNA-CC, which is 15-mer RNA with the 3 - can override the abortive destiny. Further, to avoid the terminal CpC and thus does not serve as a primer for RNA abortive RNA synthesis, MCM is required prior to the synth- synthesis (Figure 6D, lanes 4 and 5). It could be interpreted esis of 12-nt-long RNA and possibly after the synthesis of that RNA-CC competes with RNA-AG for binding to MCM. 2-nt-long RNA. The same results were obtained for other These results indicate that RNA-AG is recruited to the viral segments (data not shown). Altogether, these findings sug- RNA polymerase by the interaction with MCM. Thus, it is gest that MCM is involved in the stabilization of the elonga- highly possible that MCM stabilizes the replication elongation tion complex during the transition from the initiation to the complex through scaffolding between nascent cRNA and the productive elongation process around the promoter. viral RNA polymerase to make the polymerase competent for It is possible that MCM functions as a scaffold protein full-length cRNA synthesis. It should be noted that MCM did between nascent cRNA and vRNP possibly through the PA not facilitate the unprimed limited elongation reactions subunit of the viral RNA polymerase since nascent cRNA (Figure 2A). It is possible that the length of nascent cRNA tends to be released from the viral RNA polymerase in the in unprimed limited elongation reactions may not be enough absence of MCM (Figure 6A). To test this hypothesis, we for binding with MCM. examined whether a 15-nt-long RNA containing ApG at its 3 - Biochemical studies have shown that MCM possesses an terminus (RNA-AG) is recruited to the RNA polymerase by ATP-dependent DNA helicase activity (You et al, 1999). MCM (Figure 6D). Limited elongation assays were performed Therefore, it is speculated that MCM acts as an RNA helicase in the presence of 15 nt RNA-AG, the 3 -terminal ApG of in virus genome replication. However, full-length cRNA 4572 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata synthesis occurred in the presence of ATPgS, which is a nonhydrolyzable analog of ATP but can be a substrate for the chain elongation of RNA synthesis (Figure 6E, compare lane 3 with lane 4). Discussion We have identified IREF-1 as a factor that stimulates the cell- free influenza virus genome replication activity. IREF-1 was found to be identical to the MCM complex consisting of MCM2, 3, 4, 5, 6, and 7. By the addition of IREF-1/MCM, the full-length cRNA was synthesized from each virus gen- ome segment. The RNA synthesis level in the presence of Figure 7 A proposed model. MCM stabilizes replication elongation MCM differed from segment to segment (Figure 1B, lanes complexes through scaffolding between nascent cRNA and viral 2–4). However, the amounts of replicated cRNAs are almost RNA polymerase complexes during its transition from initiation to elongation to allow viral RNA polymerase complexes to synthesize equal among eight segments in infected cells (Hatada et al, full-length cRNA. 1989). Therefore, it is possible that another host factor(s) is required for the quantitative control of the virus genome replication. However, this issue is still an open question. Previous reports showed that NP is important for virus genome replication (Shapiro and Krug, 1988; Medcalf et al, (data not shown). We speculate that another host and/or viral factor(s) other than MCM may be required for the 1999). NP is required for elongation of RNA synthesis (Honda switching mechanism of initiation reactions. et al, 1988). Moreover, the in vitro cRNA synthesis using A universal characteristic feature of RNA polymerases is infected cell extracts as an enzyme source depends on a supply of non-RNP-associated NP (Shapiro and Krug, the regulated conversion from an initiating form that holds 1988). The latter finding may be interpreted as that NP nascent RNA weakly to an elongating form that holds RNA functions in co-transcriptional encapsidation of nascent tightly during RNA synthesis. This transition occurs during promoter escape, and involves a complex series of molecular RNA for prevention of discrete RNA synthesis. Therefore, it transformations in both prokaryotic and eukaryotic DNA- is possible that MCM and NP free of vRNA act in a similar dependent RNA polymerases (Zawel et al, 1995; Hsu, fashion and cooperatively facilitate the virus genome replica- 2002). TFIIB and TFIIE, the general transcription factors for tion. Further, it was reported that minimal cRNA synthesis occurs even in the absence of newly synthesized NP (Vreede the cellular RNA polymerase II (pol II), are released from the et al, 2004). Thus, it is speculated that MCM activates and elongation complex by the synthesis of an approximately 10- nt-long nascent RNA (Zawel et al, 1995). The nascent RNA guarantees the virus genome replication from incoming vRNP forms a 9 bp RNA:DNA hybrid in the elongation complex at immediate early phases of infection, where newly synthe- (Gnatt et al, 2001), which is thought to be a primary stability sized NP is absent. determinant for the elongation complex (Kireeva et al, 2000). MCM interacted with the viral RNA polymerase through its contact with PA (Figure 4). Previous genetic analyses sug- It is speculated that the hybridization between template DNA gested that PA participates in the replication process, and nascent RNA chains with lengths less than 9 bp is less although its precise role had not been well established effective for the stabilization of the elongating complex. Our results showed that the influenza viral RNA polymerase (Sugiura et al, 1975; Ritchey and Palese, 1977; Kawaguchi catalyzes the initiation reaction, but cannot synthesize the et al, 2005). Recently, it has been reported that the influenza full-length cRNA because the nascent cRNA was dissociated virus vRNP interacts with nucleosomes (Garcia-Robles et al, from the elongation complex in the absence of MCM 2005). Both vRNP and NP that is free of RNA are capable of binding to core histones in vitro. It has also been reported (Figure 6). In contrast, in the presence of MCM, the elonga- that the viral RNA polymerase interact with the phospho- tion complex was stabilized and converted to the form competent for the processive RNA synthesis since MCM serine 5 form of the largest subunit of pol II (Engelhardt et al, may act as a scaffold between nascent cRNA and the viral 2005). MCM also interacts with pol II holoenzyme in addition RNA polymerase without its helicase activity (Figures 6 and to DNA replication origins in nucleosomes (Yankulov et al, 7). It is possible that the association of nascent RNA with the 1999). Based on these, we tentatively hypothesize that vRNP binds to chromatin where MCM is present while interacting elongation complex could be stabilized by MCM through its with chromatin proteins such as pol II and recruits MCM binding to nascent RNA. On this line, it was proposed that PA increases the interaction between PB1 and RNA (Lee et al, through the contact with PA. 2002). PA is UV-crosslinked to the 5 -terminal promoter of A functional form of the viral polymerase for the transcrip- vRNA (Fodor et al, 1994). Thus, it is also possible that MCM tion and replication is thought to be a ternary complex modulates the interaction between the viral polymerase and consisting of PB1, PB2, and PA (Fodor et al, 2002; Gastaminza et al, 2003). However, the molecular basis of the promoter through its contact with PA. the switching mechanism between transcription and replica- It was reported that MCM is associated with pol II- tion is unknown. In infected cells, MCM is involved predo- mediated transcription. Antibodies against MCM2 inhibit pol II transcription in Xenopus oocytes (Yankulov et al, minantly in virus genome replication, but not transcription 1999); the interaction between MCM5 and the activation (Figure 5). MCM did not inhibit initiation of capped RNA- domain of STAT1a is essential for the expression of IFN-g primed mRNA synthesis in a cell-free RNA synthesis system &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4573 | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata in the absence of UTP, and RNA products were separated through responsive genes (Snyder et al, 2005). The finding reported 15% PAGE containing 8 M urea. here may provide useful information to further understand the mechanism of cellular transcription regulated by MCM. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Materials and methods Cell-free virus genome replication system Acknowledgements vRNP was prepared from purified influenza A/PR/8/34 virus as previously described (Shimizu et al, 1994). A cell-free virus genome We thank F Momose (Kitasato University), Y Ishimi (Ibaraki replication was carried out at 301C for 90 min in a final volume of University), H Nojima (Osaka University), and N Yabuta (Osaka 25 ml containing 50 mM HEPES-NaOH (pH 7.9), 3 mM MgCl , University) for the generous gifts of pCAGGS-NP Myc (FM), UW31- 50 mM KCl, 1.5 mM dithiothreitol, 500 mM each ATP, CTP, and hMCM4(His)-6, UW31-hMCM2-7(His), and pAcAB4-hMCM3-5(His) UTP, 25 mMGTP,5 mCi of [a- P]GTP (3000 Ci/mmol), 8 U of RNase (YI), anti-MCM proteins antibodies (HN and NY). We also thank J inhibitor, 25 mg of actinomycin D/ml, and vRNP (50 ng of NP Yanagisawa (University of Tsukuba) for his help for MALDI-TOF MS equivalents) in the presence or absence of host factor fraction or analyses. This research was supported in part by a grant-in-aid from purified proteins. RNA products were purified, subjected to 4% the Ministry of Education, Culture, Sports, Science, and Technology PAGE in the presence of 8 M urea, and visualized by autoradio- of Japan (to KN) and Research Fellowships of the Japanese Society graphy. For limited elongation assay, RNA synthesis was performed for the Promotion of Science (JSPS) (to AK). References Chan AY, Vreede FT, Smith M, Engelhardt OG, Fodor E (2006) virus RNA polymerase play a critical role in protein stability, Influenza virus inhibits RNA polymerase II elongation. Virology endonuclease activity, cap binding, and virion RNA promoter 351: 210–217 binding. J Virol 80: 7789–7798 Cortez D, Glick G, Elledge SJ (2004) Minichromosome maintenance Hatada E, Hasegawa M, Mukaigawa J, Shimizu K, Fukuda R (1989) proteins are direct targets of the ATM and ATR checkpoint Control of influenza virus gene expression: quantitative analysis kinases. Proc Natl Acad Sci USA 101: 10078–10083 of each viral RNA species in infected cells. J Biochem (Tokyo) 105: Crow M, Deng T, Addley M, Brownlee GG (2004) Mutational 537–546 analysis of the influenza virus cRNA promoter and identification Honda A, Mizumoto K, Ishihama A (1986) RNA polymerase of of nucleotides critical for replication. J Virol 78: 6263–6270 influenza virus. Dinucleotide-primed initiation of transcription at Deng T, Engelhardt OG, Thomas B, Akoulitchev AV, Brownlee GG, specific positions on viral RNA. J Biol Chem 261: 5987–5991 Fodor E (2006) Role of ran binding protein 5 in nuclear import Honda A, Ueda K, Nagata K, Ishihama A (1988) RNA polymerase of and assembly of the influenza virus RNA polymerase complex. influenza virus: role of NP in RNA chain elongation. J Biochem J Virol 80: 11911–11919 (Tokyo) 104: 1021–1026 Digard P, Elton D, Bishop K, Medcalf E, Weeds A, Pope B (1999) Hsu LM (2002) Promoter clearance and escape in prokaryotes. Modulation of nuclear localization of the influenza virus nucleopro- Biochim Biophys Acta 1577: 191–207 tein through interaction with actin filaments. JVirol 73: 2222–2231 Huarte M, Sanz-Ezquerro JJ, Roncal F, Ortin J, Nieto A (2001) PA Edwards MC, Tutter AV, Cvetic C, Gilbert CH, Prokhorova TA, subunit from influenza virus polymerase complex interacts with a Walter JC (2002) MCM2–7 complexes bind chromatin in a cellular protein with homology to a family of transcriptional distributed pattern surrounding the origin recognition complex activators. J Virol 75: 8597–8604 in Xenopus egg extracts. J Biol Chem 277: 33049–33057 Ishimi Y, Okayasu I, Kato C, Kwon HJ, Kimura H, Yamada K, Song Engelhardt OG, Fodor E (2006) Functional association between viral SY (2003) Enhanced expression of Mcm proteins in cancer cells and cellular transcription during influenza virus infection. Rev derived from uterine cervix. Eur J Biochem 270: 1089–1101 Med Virol 16: 329–345 Kawaguchi A, Naito T, Nagata K (2005) Involvement of influenza Engelhardt OG, Smith M, Fodor E (2005) Association of the influ- virus PA subunit in assembly of functional RNA polymerase enza A virus RNA-dependent RNA polymerase with cellular RNA complexes. J Virol 79: 732–744 polymerase II. J Virol 79: 5812–5818 Kireeva ML, Komissarova N, Waugh DS, Kashlev M (2000) The Fodor E, Crow M, Mingay LJ, Deng T, Sharps J, Fechter P, Brownlee 8-nucleotide-long RNA:DNA hybrid is a primary stability deter- GG (2002) A single amino acid mutation in the PA subunit of the minant of the RNA polymerase II elongation complex. J Biol influenza virus RNA polymerase inhibits endonucleolytic clea- Chem 275: 6530–6536 vage of capped RNAs. J Virol 76: 8989–9001 Lee MT, Bishop K, Medcalf L, Elton D, Digard P, Tiley L (2002) Fodor E, Pritlove DC, Brownlee GG (1994) The influenza virus Definition of the minimal viral components required for the panhandle is involved in the initiation of transcription. J Virol 68: initiation of unprimed RNA synthesis by influenza virus RNA 4092–4096 polymerase. Nucleic Acids Res 30: 429–438 Forsburg SL (2004) Eukaryotic MCM proteins: beyond replication Lei M, Kawasaki Y, Tye BK (1996) Physical interactions among Mcm initiation. Microbiol Mol Biol Rev 68: 109–131 proteins and effects of Mcm dosage on DNA replication in Gabriel G, Abram M, Keiner B, Wagner R, Klenk HD, Stech J (2007) Saccharomyces cerevisiae. Mol Cell Biol 16: 5081–5090 Differential polymerase activity in avian and Mammalian cells Mahbubani HM, Chong JP, Chevalier S, Thommes P, Blow JJ (1997) determines host range of influenza virus. J Virol 81: 9601–9604 Cell cycle regulation of the replication licensing system: involve- Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J (2005) The ment of a Cdk-dependent inhibitor. J Cell Biol 136: 125–135 viral polymerase mediates adaptation of an avian influenza virus Maine GT, Sinha P, Tye BK (1984) Mutants of S. cerevisiae to a mammalian host. Proc Natl Acad Sci USA 102: 18590–18595 defective in the maintenance of minichromosomes. Genetics Garcia-Robles I, Akarsu H, Muller CW, Ruigrok RW, Baudin F (2005) 106: 365–385 Interaction of influenza virus proteins with nucleosomes. Medcalf L, Poole E, Elton D, Digard P (1999) Temperature-sensitive Virology 332: 329–336 lesions in two influenza A viruses defective for replicative tran- Gastaminza P, Perales B, Falcon AM, Ortin J (2003) Mutations in the scription disrupt RNA binding by the nucleoprotein. J Virol 73: N-terminal region of influenza virus PB2 protein affect virus RNA 7349–7356 replication but not transcription. J Virol 77: 5098–5108 Momose F, Basler CF, O’Neill RE, Iwamatsu A, Palese P, Nagata K Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD (2001) (2001) Cellular splicing factor RAF-2p48/NPI-5/BAT1/UAP56 in- Structural basis of transcription: an RNA polymerase II elonga- teracts with the influenza virus nucleoprotein and enhances viral tion complex at 3.3 A resolution. Science 292: 1876–1882 RNA synthesis. J Virol 75: 1899–1908 Hara K, Schmidt FI, Crow M, Brownlee GG (2006) Amino acid Momose F, Naito T, Yano K, Sugimoto S, Morikawa Y, Nagata K residues in the N-terminal region of the PA subunit of influenza A (2002) Identification of Hsp90 as a stimulatory host factor 4574 The EMBO Journal VOL 26 NO 21 2007 &2007 European Molecular Biology Organization | | MCM stimulates influenza virus genome replication A Kawaguchi and K Nagata involved in influenza virus RNA synthesis. J Biol Chem 277: Sugiura A, Ueda M, Tobita K, Enomoto C (1975) Further isolation and characterization of temperature-sensitive mutants of influen- 45306–45314 Naito T, Momose F, Kawaguchi A, Nagata K (2007) Involvement of za virus. Virology 65: 363–373 Hsp90 in assembly and nuclear import of influenza virus RNA Vreede FT, Brownlee GG (2007) Influenza virion-derived viral polymerase subunits. J Virol 81: 1339–1349 ribonucleoproteins synthesize both mRNA and cRNA in vitro. Pasion SG, Forsburg SL (1999) Nuclear localization of J Virol 81: 2196–2204 Schizosaccharomyces pombe Mcm2/Cdc19p requires MCM com- Vreede FT, Jung TE, Brownlee GG (2004) Model suggesting that plex assembly. Mol Biol Cell 10: 4043–4057 replication of influenza virus is regulated by stabilization of Plotch SJ, Bouloy M, Krug RM (1979) Transfer of 5 -terminal cap of replicative intermediates. J Virol 78: 9568–9572 globin mRNA to influenza viral complementary RNA during Wang P, Palese P, O’Neill RE (1997) The NPI-1/NPI-3 (karyopherin transcription in vitro. Proc Natl Acad Sci USA 76: 1618–1622 alpha) binding site on the influenza a virus nucleoprotein NP Plotch SJ, Krug RM (1977) Influenza virion transcriptase: synthesis is a nonconventional nuclear localization signal. J Virol 71: in vitro of large, polyadenylic acid-containing complementary 1850–1856 RNA. J Virol 21: 24–34 Yamanaka K, Ishihama A, Nagata K (1990) Reconstitution of Ritchey MB, Palese P (1977) Identification of the defective genes in influenza virus RNA–nucleoprotein complexes structurally re- three mutant groups of influenza virus. J Virol 21: 1196–1204 sembling native viral ribonucleoprotein cores. J Biol Chem 265: Shapiro GI, Krug RM (1988) Influenza virus RNA replication in 11151–11155 vitro: synthesis of viral template RNAs and virion RNAs in the Yankulov K, Todorov I, Romanowski P, Licatalosi D, Cilli K, absence of an added primer. J Virol 62: 2285–2290 McCracken S, Laskey R, Bentley DL (1999) MCM proteins are Shimizu K, Handa H, Nakada S, Nagata K (1994) Regulation of associated with RNA polymerase II holoenzyme. Mol Cell Biol 19: influenza virus RNA polymerase activity by cellular and viral 6154–6163 factors. Nucleic Acids Res 22: 5047–5053 You Z, Komamura Y, Ishimi Y (1999) Biochemical analysis of the Sinha P, Chang V, Tye BK (1986) A mutant that affects the function of intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity. Mol Cell Biol autonomously replicating sequences in yeast. J Mol Biol 192: 805–814 19: 8003–8015 Snyder M, He W, Zhang JJ (2005) The DNA replication factor MCM5 Zawel L, Kumar KP, Reinberg D (1995) Recycling of the general is essential for Stat1-mediated transcriptional activation. Proc transcription factors during RNA polymerase II transcription. Natl Acad Sci USA 102: 14539–14544 Genes Dev 9: 1479–1490 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 21 2007 4575 | |

Journal

The EMBO Journal – Springer Journals

Published: Oct 31, 2007

Keywords: host factor; influenza virus; MCM; replication; RNA‐dependent RNA polymerase

There are no references for this article.