Tsutomu Fujimura 1 and Rosa Esteban From the Instituto de Biología Funcional y Genómica, CSIC/Universidad de Salamanca, Salamanca 37007, Spain - PDF

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 16, pp , April 13, by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Cap Snatching of

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 16, pp , April 13, by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Cap Snatching of Yeast L-A Double-stranded RNA Virus Can Operate in Trans and Requires Viral Polymerase Actively Engaging in Transcription * S Received for publication, November 25, 2011, and in revised form, February 15, 2012 Published, JBC Papers in Press, February 24, 2012, DOI /jbc.M Tsutomu Fujimura 1 and Rosa Esteban From the Instituto de Biología Funcional y Genómica, CSIC/Universidad de Salamanca, Salamanca 37007, Spain Background: L-A virus has a novel cap-snatching mechanism to furnish its transcripts with caps. Results: Cap snatching can operate in trans and requires the viral polymerase active in transcription. Conclusion: Coordination between capping and transcription ensures an efficient expression of viral proteins when the polymerase is active. Significance: There is communication between the outer capping site and the inner polymerase across the capsid layer. Eukaryotic mrna bears a cap structure (m 7 GpppX-) at the 5 terminus crucial for efficient translation and stability. The yeast L-A double-stranded RNA virus furnishes its mrna with this structure by a novel cap-snatching mechanism in which the virus transfers an m 7 Gp moiety from host mrna to the diphosphorylated 5 terminus of the viral transcript, thus forming on it an authentic cap structure (referred to as cap0) in the budding yeast. This capping reaction is essential for efficient viral expression. His-154 of the capsid protein Gag is involved in the cap transfer. Here we show that the virus can utilize an externally added viral transcript as acceptor in the capping reaction. The acceptor needs to be 5 diphosphorylated, consistent with the fact that the viral transcript bears diphosphate at the 5 terminus. A 5 triphosphorylated or monophosphorylated transcript does not function as acceptor. N7 methylation at the 5 cap guanine of mrna is essential for cap donor activity. We also demonstrate that the capping reaction requires the viral polymerase actively engaging in transcription. Because the cap-snatching site of Gag is located at the cytoplasmic surface of the virion, whereas Pol is confined inside the virion, the result indicates coordination between the cap-snatching and polymerization sites. This will allow L-A virus to efficiently produce capsid proteins to form new virions when Pol is actively engaging in transcription. The coordination may also minimize the risk of accidental capping of nonviral RNA when Pol is dormant. The 5 cap structure (m 7 GpppX-) is a hallmark of eukaryotic mrna crucial for efficient translation and stability (1 3). In cells and for most viruses, the cap structure is installed on mrna co-transcriptionally by three sequential catalytic reactions (4, 5): removal of the 5 -phosphate by RNA triphosphatase, transfer of GMP from GTP to the diphosphorylated 5 end by guanylyltransferase, and methylation of added GMP by * This work was supported by Grant BFU from the Spanish Ministry of Education and Science and by Fundación Ramón Areces. S This article contains supplemental Fig. S1. 1 To whom correspondence should be addressed: Instituto de Biología Functional y Genómica, Edificio Departamental, Avda. del Campo Charro s/n, Salamanca 37007, Spain. Tel.: ; Fax: ; methyltransferase. Guanylyltransferase forms a covalent bond with GMP through Lys before transferring it to Pol II transcripts. There are some variations from this conventional capping scheme. In vesicular stomatitis virus, L protein covalently binds the 5 monophosphorylated pre-mrna and transfers the bound pre-mrna to GDP (6). Then methylation at the 5 end follows (7). Members of the alphavirus-like superfamily methylate GTP first and then transfer the m 7 Gp moiety to the 5 diphosphate end of viral transcripts (8). In addition to the de novo synthesis of the cap structure, some RNA viruses steal this structure from host mrna and furnish their mrna with the stolen cap (cap snatching). In influenza virus, the trimeric viral polymerase binds host mrna, cleaves the RNA endonucleolytically nt 2 downstream, and utilizes the capped fragment as a primer to synthesize its transcript (9 11). Negative strand RNA viruses and ambiviruses (the Orthomixoviridae, Bunyaviridae, and Arenaviridae families) have been known to use this strategy to furnish their mrna. Recently we have found that the yeast L-A double-stranded RNA virus synthesizes capped transcripts by a novel cap-snatching mechanism (12). The virus only transfers m 7 Gp from host mrna to the diphosphorylated 5 end of the viral transcript, thus conserving the 5 - and -phosphates of the transcript in the triphosphate linkage of the final product (see Fig. 1A). Furthermore, unlike influenza virus, L-A virus utilizes the capsid protein Gag rather than Pol to catalyze the reaction. The totivirus L-A, which infects the yeast Saccharomyces cerevisiae, has a nonsegmented double-stranded RNA genome of 4.6 kb (13). The genome contains two overlapping genes gag and pol that can be decoded into two proteins, Gag (76 kda) and Gag-Pol (170 kda) (14). The latter is made by a 1 ribosomal frameshifting mechanism (15). The genome is packed inside of a 39-nm icosahedral capsid consisting of 60 asymmetric Gag dimmers, in which one or two Gag molecules are replaced by Gag-Pol. The N-terminal Pol region is necessary for genome packaging, although Gag alone is sufficient to form morphologically normal capsids (16). 2 The abbreviation used are: nt, nucleotides; BAP, bacterial alkaline phosphatase; -S-ATP, adenosine 5 -[ -thio]triphosphate; -S-ADP, adenosine 5 -[ -thio]diphosphate. APRIL 13, 2012 VOLUME 287 NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12797 Isolated L-A virions have transcriptase activity and synthesize positive strand transcripts conservatively (17). The transcripts bear diphosphate at their 5 ends (18). When transcription is primed with GMP, the 5 -phosphate of the transcript is derived from the -phosphate of the ATP present in the reaction. It has been known that L-A virions bind the cap structure of host mrna and cleave it to form an m 7 Gp-Gag adduct through His-154 (19, 20). Previously we speculated that the m 7 Gp-Gag adduct is an intermediate of the cap transfer reaction and tested this hypothesis. We demonstrated that L-A virions could transfer m 7 Gp from mrna to the 5 end of the viral transcript, thus forming an authentic cap0 structure on it (12). This activity was impaired by the Arg-154 mutation of Gag. Furthermore, the synthesis of capped viral transcripts in vivo and their expression were greatly compromised by the mutation, indicating the involvement of Gag in the cap-snatching reaction (12). Here we demonstrate that L-A virus can utilize exogenously added viral transcripts as cap acceptors in the cap-snatching reaction. A diphosphate status at the viral 5 end is essential for the acceptor activity. Furthermore, the cap-snatching activity requires the viral polymerase actively engaging in transcription, indicating coordination between the cap-snatching and RNA polymerization sites in the virion. EXPERIMENTAL PROCEDURES Cap-snatching Reaction The standard cap-snatching reaction in trans (25 l) contained 50 mm Tris-HCl, ph 7.5, 5 mm MgCl 2, 0.1 mm EDTA, 20 mm NaCl, 5 mm KCl, 0.5 mm ATP, 0.5 mm m 7 GpppG, 20% PEG 4000, bentonite (4.5 mg/ml), and 32 P- labeled 16-mer ( ,000 cpm). This radioactivity corresponds to pmol of 16-mer when it was 5 end-labeled with [ - 32 P]GTP and 7-fold less when internally labeled with [ - 32 P]UTP. The reaction was started by the addition of L-A virions (25 g of protein), and the mixture was kept at 30 C for min. The products were extracted with phenol, precipitated with ethanol, and separated on an 8 M urea/15% acrylamide gel. The gel was run in 0.5 TBE (21) at 16 V/cm for2h30 min. The products were visualized by autoradiography. The 32 P-cap acceptor was labeled with either [ - 32 P]GTP or [ - 32 P]UTP by L-A virions in a CTP-omitted transcription reaction (18) and purified through an acrylamide gel. The capsnatching reaction in cis is the same as the standard reaction in trans described above, except that m 7 GpppG and the 32 P-labeled 16-mer were replaced by GTP (or GDP) and UTP, 0.5 mm each, and a 32 P-labeled 5-nt cap donor ( cpm; pmol). The reaction was kept at 30 C for 1 h, and the products were analyzed as described above. The 5-nt cap donor was synthesized using the mscript mrna production system (Epicenter) as described (12). Each experiment was carried out at least twice. The variation observed between experiments was less than 10%. SP6 Transcription 5 tri-, di-, and monophosphorylated 16-mers were transcribed from ptf42 by SP6 RNA polymerase using GTP, GDP, and GMP, respectively, as primers. The reaction contained 40 mm Tris-HCl, ph 7.9, 6 mm MgCl 2,2mM spermidine, 10 mm NaCl, 10 mm DTT, 0.5 g of PvuII-digested ptf42, 20 units of RNasin, 20 M [ - 32 P]UTP (20 Ci), 0.5 mm FIGURE 1. A, schematic diagram of L-A cap-snatching mechanism. Gag of L-A virion decaps mrna and forms an intermediate with m 7 Gp through His-154. Then m 7 Gp is transferred to the diphosphorylated 5 end of the emerging viral transcript to form a 5-5 triphosphate linkage (cap snatching in cis). In this work, we show that m 7 Gp can also be transferred to an externally added viral transcript (cap snatching in trans). B, the nucleotide sequence of L-A transcript at the 5 end. The first C appears at position 17. L-A virion synthesizes 16-nt transcript (16-mer) when CTP is omitted from the transcription reaction. The 16-mer contains a single G at the 5 end. ATP, 0.5 mm GTP (or m 7 GTP), GDP (or m 7 GDP) or GMP as primer, and 19 units of SP6 RNA polymerase (Promega). The reaction was kept at 37 C for 1 h. The products were extracted with phenol and purified through a 15% acrylamide gel. ptf42 contains the 5 end sequence of X, a deletion mutant of L-A (22), directly fused to the SP6 promoter. Thus the 16-mer made by SP6 polymerase is identical to the nucleotide sequence of the first 16 nt of L-A. Miscellaneous TLC was done as described (18). L-A virions were purified from yeast strain TF229 (Mata his(3,4) leu2 ski2 2 L-A-HN) as described (23). Radioactive nucleotides were obtained from PerkinElmer Life Sciences. RNA 5 -polyphosphatase was from Epicenter. S1 nuclease and RNasin were from Promega, and bacterial alkaline phosphatase was from Invitrogen. Tobacco acid pyrophosphatase, -S-GTP, -S-GDP, m 7 GTP, and m 7 GDP were from Sigma-Aldrich. m 7 GpppG was from Ambion, and GpppG was from Amersham Biosciences. RESULTS Cap Snatching in Trans Previously we demonstrated that the m 7 Gp moiety from a cap donor was transferred to the 5 end of the L-A viral transcript to form an inverted 5-5 triphosphate linkage (12). In those experiments, L-A transcripts were synthesized by L-A virions and simultaneously used as acceptors for cap transfer (cap snatching in cis). To analyze the transfer reaction in more detail, here we decided to use exogenously added viral transcripts as cap acceptors. Because the first C in the L-A positive strand appears at position 17 (Fig. 1B), the L-A virion synthesizes short transcripts (16-mer) when CTP is omitted from the transcription reaction (18). The 16-mer contains a single G at the 5 end. Thus it can be labeled with [ - 32 P]GTP. The labeled 16-mer was purified from an acrylamide gel and then incubated with L-A virions in a transcription mixture to which the cap analog m 7 GpppG was added as cap donor. As shown in Fig. 2A, the capped product appeared in the first 5-min incubation, and cap transfer was completed within JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 NUMBER 16 APRIL 13, 2012 FIGURE 2. Cap snatching in trans. The 16-nt transcript labeled with [ - 32 P]GTP was used as cap acceptor. A, time course. The incubation times are indicated below the panel. B, dose response of cap snatching to the cap donor m 7 GpppG. The concentrations of the donor used are indicated below the panel. C, BAP resistance of the capped product. The 16-mer after capping reaction (Capped) was treated without ( ) or with BAP ( ) and separated on a denaturing acrylamide gel. The 16-mer without capping reaction (Acceptor) was run in parallel. D, proof of capping. BAP-resistant capped product (Capped) was gel-purified, treated with the enzymes as indicated below the panel, and analyzed on polyethyleneimine-cellulose chromatography with 0.3 M (NH 4 ) 2 SO 4 as solvent. In lane 6, the capped product was treated with tobacco acid pyrophosphatase (TAP) first, then extracted with phenol, and finally digested again with S1 nuclease. Noncapped 16-mer (Acceptor) was also processed in parallel as control. The mobility of nonlabeled nucleotides is indicated at the left. The deduced transfer reaction is shown on the left with S1 and tobacco acid pyrophosphatase cleavage sites. 30 min. The cap-snatching reaction was saturated with the high concentration of m 7 GpppG used (1 mm) (Fig. 2B). We confirmed that the product was 5 -capped (Fig. 2, C and D). The acceptor contains 32 Patthe -position in the 5 diphosphate and thus was susceptible to BAP treatment, whereas the capped product was resistant to the enzyme (Fig. 2C). After BAP treatment, the capped product was purified from the acrylamide gel and analyzed by TLC (Fig. 2D). Nuclease S1 digested the RNA body but could not work on the triphosphate linkage; thus m 7 GpppG was released from the product (lane 4). Tobacco acid pyrophosphatase could hydrolyze anhydrous bonds between and -phosphates and also - and -phosphates of the triphosphate linkage, and the label at the -position was still associated with the RNA body of 16 nt (lane 5). A further treatment with S1 released the label as GMP (lane 6). By contrast, the acceptor released GDP by S1 treatment (lane 2) as demonstrated previously (18). Therefore, these results confirm that the product acquired the cap structure at the 5 end and that the label at the -position of the 5 diphosphate in the acceptor molecule (16- mer) was preserved at the -position of the triphosphate linkage of the product, as expected from the cap-snatching scheme (Fig. 1A) established previously for the reaction in cis (12). The capping reaction required nucleotide triphosphates (Fig. 3A, lane 5). However, ATP alone was sufficient for the reaction (Fig. 3A, lane 4). The addition of EDTA abolished cap transfer (Fig. 3A, lane 6). Nonmethylated cap analog (GpppG) did not function as cap donor (Fig. 3A, lanes 8 and 9). Previously we also showed that in the cap transfer reaction in cis, a 5-nt nonmethylated capped molecule did not work as cap donor (12). These FIGURE 3. Requirements for cap snatching in trans. A, N7 methylation of cap is required for cap donor activity. The cap-snatching reaction was carried out in the presence of 0.5 mm m 7 GpppG or GpppG or in their absence ( )as indicated below the panel, along with NTPs (0.5 mm each) as indicated above the panel. The 16-nt transcript labeled with [ - 32 P]GTP was used as cap acceptor. In lane 6,10mM EDTA was also added to the reaction. In lane 1, the acceptor was incubated in the absence of L-A virions ( ) as control. B, m 7 GpppG is the smallest cap donor. The capping reaction was carried out in the presence of 0.5 mm m 7 GDP (lane 3), m 7 GTP (lane 4), or m 7 GpppG (lane 5)as cap donor. In lane 1, 16-nt transcript (Acceptor) was incubated in the absence of L-A virions ( ) as control. The arrowheads indicate capped products. results, therefore, indicate that N7 methylation is essential for cap donor activity. m 7 GDP or m 7 GTP did not work as cap donor (Fig. 3B, lanes 3 and 4); thus the smallest molecule with donor activity is m 7 GpppG. Cap Acceptor Activity Requires 5 Diphosphate The 16-mer made by L-A virions in the absence of CTP bears diphosphate at the 5 end. To examine the role of the phosphate status at the 5 end in the cap transfer reaction, the 16-mer was treated with RNA 5 polyphosphatase to eliminate the 5 -phosphate (Fig. 4A). Then the 5 monophosphorylated 16-mer was incubated with L-A virions for cap transfer reaction. As shown in Fig. 4B, lane 4, the 5 monophosphorylated molecule did not function as cap acceptor. We also synthesized the 5 tri-, di-, and monophosphorylated 16-mer using SP6 RNA polymerase with GTP, GDP, and GMP, respectively, as primers. Only diphosphorylated 16-mer formed capped products, whereas tri- or monophosphorylated molecules did not function as cap acceptors (Fig. 4C). Methylation at the 5 terminal G did not significantly affect cap acceptor activity in the 5 diphosphorylated molecule (Fig. 4D, lanes 4 and 6). The 5 triphosphorylated 16-mer with the terminal G N7-methylated showed no cap acceptor activity (Fig. 4D, lane 2). Cap Snatching Requires Viral Polymerase Actively Engaging in Transcription We examined the effect of nucleoside triphosphates in the capping reaction and found that only ATP could promote cap snatching. The other three nucleoside triphosphates did not support the reaction (Fig. 5A). There are three possible roles of ATP for cap snatching: (i) ATP may promote the reaction as an effector without being modified, (ii) the APRIL 13, 2012 VOLUME 287 NUMBER 16 JOURNAL OF BIOLOGICAL CHEMISTRY 12799 FIGURE 4. A diphosphate status at the 5 end is required for cap acceptor activity. A, conversion of 5 diphosphate to monophosphate with RNA 5 -polyphosphatase (RPP). A 16-nt transcript labeled with [ - 32 P]GTP was treated with RPP. The product was digested without (None) or with S1 nuclease (S1) and analyzed on polyethyleneimine cellulose with 1 M LiCl as solvent. The 16-nt transcript not treated with RPP ( ) was processed in parallel as control. Below the panel is shown a schematic diagram of the reactions. The asterisk indicates the position of 32 P. B, the 16-nt transcript treated with ( ) or without ( ) RPP was incubated with L-A virions ( ) in the presence of 0.5 mm m 7 GpppG as cap donor. The capped product was separated on a denaturing acrylamide gel. The arrowhead indicates the capped product. C, 16-nt acceptor molecules with 5 tri-, di-, and monophosphate synthesized by SP6 RNA polymerase using GTP, GDP, and GMP, respectively, as primer, were incubated with L-A virions ( ) in the presence of the cap donor m 7 GpppG (0.5 mm). The capped products were separated on a denaturing acrylamide gel. D, 16-nt molecule N7-methylated at the 5 end with 5 tri- or diphosphate made by SP6 RNA polymerase was tested for its cap acceptor activity as described above. Nonmethylated 16-nt molecule with 5 diphosphate made by SP6 RNA polymerase was used as control. The arrowheads indicate the capped products. hydrolysis of ATP to ADP may be required for the reaction, and (iii) because L-A virions can incorporate m 7 GpppG as a primer into transcripts (18), the presence of ATP will allow the virion to synthesize a short transcript (m 7 GpppGAAAAA-OH). This activity may somehow promote the cap-snatching reaction. Here we analyze these possible roles of ATP. -S-ATP could support the cap-snatching reaction (Fig. 5B, lane 4). This ATP analog is generally considered as nonhydrolyzable; thus the result disfavors the role of ATP hydrolysis in the cap-snatching reaction. -S-ATP, however, can be used as substrate for RNA polymerization (24). In fact, L-A virions can utilize this analog as efficiently as ATP for transcription (Fig. 5C, lanes 3 and 4). Thus these results are consistent with the involvement of transcription in the cap-snatching reaction. Interestingly ADP, although less active than ATP, could also promote cap snatching (Fig. 5B, lane 5). Furthermore, ADP also supported transcription, although it is less potent than ATP (Fig. 5C, lanes 3 and 5). These results again point out the importance of transcription in cap transfer. It has been known that L-A virions are FIGURE 5. Cap-snatching reaction r
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