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Cap Snatching in Yeast L-BC Double-Stranded RNA Totivirus *

Tsutomu Fujimura1 and Rosa Esteban1 1From the Instituto de Biología Funcional y Genómica. CSIC/Universidad de Salamanca, Spain

*Running title: Cap snatching in L-BC virus

To whom correspondence should be addressed: Dr. Tsutomu Fujimura, Instituto de Biología Functional y Genómica Lab P2.1. Zacarías González 2, Salamanca 37007, Spain. Tel. 34 923294901, Fax. 34 923224876, E-mail: tfujimura@usal.es Keywords: mRNA synthesis, transcriptase, RNA virus, cap snatching, yeast, Gag --------------------------------------------------------------------------------------------------------------------------- Background: L-A totivirus furnishes its transcript by a novel cap-snatching mechanism. Results: L-BC virus transfers m7Gp from mRNA to the diphosphorylated 5’ end of its transcript to form a cap structure. Conclusion: L-BC totivirus also can perform cap snatching. Significance: The novel cap-snatching mechanism is shared among fungal totiviruses. ABSTRACT

Yeast L-A double-stranded RNA virus furnishes its transcript with a 5’ cap structure by a novel cap-snatching mechanism in which m7Gp from a host mRNA cap structure is transferred to the 5’ diphosphate terminus of the viral transcript. His154 of the coat protein Gag forms an m7Gp-adduct and the H154R mutation abolishes both m7Gp-adduct formation and cap snatching. Here we show that L-BC, another totivirus closely related to L-A, also synthesizes 5’ diphosphorylated transcripts and transfers m7Gp from mRNA to the 5’ termini of the transcripts. L-BC Gag also covalently binds to the cap structure and the mutation H156R, which corresponds to H154R of L-A Gag, abolishes cap-adduct formation. Cap snatching of L-BC virus is very similar to that of L-A: N7 methylation of the mRNA cap is essential for cap donor activity and only 5’ diphosphorylated RNA is used as cap acceptor. L-BC cap snatching is also activated by viral transcription. Furthermore, both viruses require Mg++ and Mn++ for cap snatching. These cations are not only required for transcription activation but also directly involved in the cap transfer process. These findings support our previous proposal that the cap-snatching mechanism of L-A virus is shared by fungal totiviruses closely related to L-A. Interestingly, L-A and L-BC viruses accept either viral transcript as

cap acceptor in vitro. Because L-A and L-BC viruses cohabit in many yeast strains, it raises the possibility that their cohabitation in the same host may be beneficial for their mutual cap acquisition

INTRODUCTION

Eukaryotic mRNA is capped at the 5’

end by a 7-methyl GMP moiety via an inverted 5’-5’ triphosphate linkage (m7GpppN-) (1). The cap structure has important roles in promoting the splicing of pre-mRNAs in the nucleus, transport of mRNA to the cytoplasm, and its translation and stability in the cytoplasm (2-6). In eukaryotic cells and for many viruses the cap structure is installed onto the 5’ end of PolII transcripts through three enzymatic reactions (7, 8). The 5’ γ-phosphate at the 5’ end of the transcript is removed by RNA triphosphatase. Then Gp is transferred from GTP to the 5’ end of the transcript by a guanylyltransferase thus forming a trisphosphate linkage. During the transfer Gp is covalently attached to the guanylyltransferase through Lys as an intermediate. Finally, a methyl group from S-adenosyl methionine is transferred to N7 of G by a methyltransferase.

Influenza virus, instead of synthesizing the cap structure de novo, steals the structure from host mRNAs (cap snatching) (9). The trimeric viral polymerase binds host mRNAs, cleaves the RNA endonucleolytically 10-13 nt downstream, and uses the capped fragment as a primer to synthesize its transcript (10). Recently we found a novel cap-snatching mechanism in the yeast Saccharomyces cerevisiae totivirus L-A (11). L-A virus has a non-segmented double-stranded RNA (dsRNA)1 genome of 4.6 kb (12). The genome contains two overlapping genes, gag and pol that are decoded as Gag (76 kDa) and Gag-Pol (170 kDa) (13, 14). Gag-Pol is

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.490953The latest version is at JBC Papers in Press. Published on July 3, 2013 as Manuscript M113.490953

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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made by a -1 ribosomal frameshifting (15), a mechanism similar to that of retroviruses. The genome is packed inside of a 39-nm icosahedral capsid consisting of 60 asymmetric Gag dimers (16, 17), in which one or two Gag molecules are replaced by Gag-Pol. The L-A virion synthesizes the positive strand transcript conservatively (18), and the newly made transcript is released from the virion. The transcript has diphosphate at the 5’ end (19). Totivirus L-BC has a dsRNA genome of 4.6 Kb and is closely related to L-A. Like L-A virus, L-BC has two overlapping genes, gag and pol (20). L-A and L-BC are independent replicons, and they reside stably in the same cells. L-A requires MAK3, MAK10 and MAK31, the N-acethyltransferase C (NatC) encoding genes (21 22), but L-BC does not, and the clo1 mutation that results in the loss of L-BC does not affect L-A (23). Two decades ago, Sonenberg and colleagues (24) found that L-A Gag as well as L-BC Gag covalently bound mRNA. Subsequent studies indicated that the m7Gp moiety of the cap structure was attached to His154 of L-A Gag (25). A mutant L-A Gag (H154R) failed to form the cap-adduct (25). We have shown recently that L-A virus, in the presence of polyethylene glycol that causes crowding of macromolecules in the aqueous solution (26), transfers m7Gp from mRNA to the 5’ end of L-A transcript, thus forming a cap structure at the 5’ terminus (11) (Fig. 1A). The α- and β-phosphates of the triphosphate linkage are derived from the 5’ terminus of the L-A transcript and the γ-phosphate from mRNA. The H154R mutation abolishes cap snatching, indicating that the ability of Gag to form a cap-adduct is essential for the cap-transfer reaction (11). L-A virus can form a cap structure not only on an ongoing L-A transcript (cap snatching in cis) but also on an externally added L-A transcript (cap snatching in trans) (27). L-A cap snatching requires the N7 methylation of the mRNA cap structure for cap donor activity and the 5’ diphosphate of L-A transcript for cap acceptor activity. The reaction is activated by transcription (27). The coupling between transcription and cap snatching may ensure the efficient capping of L-A transcript.

Fungal totiviruses (including L-BC) closely related to L-A share His154 and other amino acid residues of L-A Gag important for cap binding, at comparative positions in their coat proteins (Fig. 1B). We proposed that these viruses also acquire a cap structure on their transcripts by a mechanism similar to that of L-A

(11). In this work we demonstrate that the L-BC transcript has diphosphate at its 5’ end, and that like L-A, L-BC virus can install a cap structure on it by the cap-snatching mechanism. Thus these findings support our proposal. Interestingly, L-A and L-BC viruses can utilize either viral transcript as cap acceptor in vitro. Because these viruses cohabit in many yeast strains, it raises the possibility that their cohabitation is beneficial for their mutual cap acquisition.

EXPERIMENTAL PROCEDURES

L-BC Virus Preparation- L-BC virus was prepared from stationary phase cells of strain RE458 (Matα ski2-2 L-A-o, L-BC). The purification procedure was similar to that for L-A virus (28), with modifications. Cells grown in 2 l of YPAD (2% peptone, 1% yeast extract, 2% glucose and 0.04% adenine) for 3 days at 28 ºC were harvested, washed once with deionized water, suspended in one volume/g of cells of buffer B (0.1 M Tris-HCl pH 7.5, 1.4 M sorbitol, 1% 2-mercapethanol, and 2 mg/ml of zymolyase 20T (Seikagaku biobusiness corporation)), and incubated at 30 ºC for 45 min. All subsequent steps were carried out at 0-4 ºC. The spheroplasts were collected, suspended in buffer A (50 mM Tris-HCl pH 7.5, 0.2 M NaCl, 2.5 mM EDTA and 1 mM spermidine), and lysed by a single passage through a French pressure cell (14,000 p.s.i.). Cell debris was removed by centrifugation (18,500 xg, 20 min). To the supernatant, ¼ of volume of 25% PEG 4000 and 2.5 M NaCl was added. 30 min later the suspension was centrifuged again (18,500 xg 10 min). The pellet was washed once with 50 ml of buffer A containing 5% PEG 4000 and an additional 0.5 M NaCl. The pellet was dissolved in buffer A. The density of the solution was adjusted to 1.36 g/ml by addition of CsCl, and viral particles were banded by centrifugation at 34,000 rpm for 15 h in a Beckman 60Ti rotor. Fractions containing the virions were pooled and dialyzed against buffer A containing 10% (v/v) glycerol for 2 h. Finally, L-BC virions were concentrated by centrifugation at 65,000 rpm for 1 h in a Beckman TLA 100.3 rotor, suspended in 200 µl of buffer A containing 20% (v/v) glycerol, and kept at -70 ºC before use.

Cap-snatching Reactions- The standard cap-snatching reaction in trans (25 µl) contains 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 20 mM NaCl, 5 mM KCl, 0.1 mM EDTA, 0.5 mM

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m7GpppG as cap donor, 4.5 mg/ml bentonite, 20% PEG and 0.5 mM ATP, 32P-labeled cap acceptor (either 16-nt L-A or 8-nt L-BC transcript, 5,000-10,000 cpm) and L-A or L-BC virions. The reaction was carried out at 30 ºC for 20 min for L-BC virions, or for 30 min for L-A. After phenol extraction, the products were analyzed on a urea-containing polyacrylamide gel as described previously (27). The cap-snatching reaction in cis is the same as the standard reaction in trans, except that m7GpppG and the 32P-labeled cap acceptor were replaced by GTP and UTP, 0.5 mM each, and a 32P-labeled 6-nt cap donor (3,000-5,000 cpm). We used the 6-nt m7Gp*ppGGGCGp (labeled with 32P, indicated by the asterisk) instead of m7GpppGGGCp (27) because of its ease in handling. The reaction was kept at 30 ºC for 1 h, and the products were analyzed as described above. The amount of L-BC virions used was 13 µg of protein per reaction, approximately half of that of L-A (25 µg), except that in Fig. 5, we used less L-BC particles (7 µg of protein).

Cap-adduct Formation- The standard reaction mixture (25 µl) contains 50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 20 mM NaCl, 5 mM KCl, 0.1 mM EDTA, 3.6 mg/ml bentonite, 20% PEG 4000, 5,000 cpm of the 6-nt cap donor (0.4 pmol) and L-A or L-BC virions (10 µg of protein). The reaction was carried out at 30 ºC for 20 min. After removing bentonite by brief centrifugation proteins were analyzed by SDS-PAGE.

Expression of L-BC Gag- The open reading frame of L-BC Gag was amplified from L-BC dsRNA by RT-PCR and inserted into the pI2-based vector under the PGK1 promoter (29). The L-BC Gag expressed has an extra 14 amino acid sequence derived from the vector at the C-terminus (-GGRHRGGAPILNQS). L-BC Gag with H156R mutation was generated by site-directed mutagenesis. Strain 2927 (Mata ura3 trp1 his3 ski2-2, L-BC-o, 20S RNA) transformed with the vector containing the wild type or mutant L-BC Gag was grown overnight at 28 ºC in H-trp synthetic medium (30). The cells were harvested from 3 ml of the culture, washed once with lysis buffer (20 mM Tris-HCl pH 7.5, 0.1 M NaCl) and suspended in 100 µl of lysis buffer. The cells were broken with glass beads by vortex mixing (15 s, 10 times). After removing cell debris and unbroken cells in an Eppendorf centrifuge at maximum speed (13,000 rpm) for 10 min at 4 ºC, the lysates (approximately 20 µg

of protein) were incubated in the reaction mixture for cap-adduct formation described above and proteins were analyzed by SDS-PAGE.

Miscellaneous- TLC was done as described previously (19). L-A virions were purified from yeast strain TF229 (Mata his(3,4) leu2 ski2-2, L-A-HN) as described (28). SP6 transcription was carried out as described in (27). Site-directed mutagenesis was done as described (31). Radioactive nucleotides were obtained from PerkinElmer Life Sciences. S1 nuclease was from Promega. Tobacco acid pyrophosphatase (TAP) was from Epicentre. Bacterial alkaline phosphatase (BAP) was from Invitrogen. γ-S-ATP, m7GpppG and GpppG were from Sigma-Aldrich, Ambion, and Amersham Biosciences, respectively.

RESULTS

Cap-adduct Formation by L-BC Gag- L-BC virions were isolated and purified from an L-A virus-free strain. The purification procedure was essentially the same as that of L-A virus. Due to the fragility and the low copy number of L-BC virus, however, we adopted several measures, including milder conditions for CsCl gradient centrifugation and the addition of a final high-speed centrifugation step to concentrate purified virions as described in Experimental procedures. The L-BC preparations are free from L-A virus as confirmed by Northern hybridization (Fig. 2A). L-BC Gag ran as a 75-kDa polypeptide and moved slightly faster than L-A Gag in SDS gels (Fig. 2B, left panel) as reported previously (24). When the virions were incubated with a 32P-labeled 6-nt cap donor, L-BC Gag formed a cap-adduct (Fig. 2B, right panel) as demonstrated previously (24).

His156 of L-BC Gag is Essential for Cap-adduct Formation- In L-A virus Gag covalently binds to the m7Gp moiety of the cap structure through His154. This residue corresponds to His156 in L-BC Gag (Fig. 1B). We expressed from vectors L-BC Gag with the WT sequence or with the mutation H156R and tested whether His156 is involved in cap-adduct formation. Crude lysates were incubated with the labeled cap donor and the proteins were separated through an SDS gel. As shown in Fig. 2C, WT Gag formed the cap-adduct while H156R Gag failed to do so. Thus these results indicate that His156 of L-BC Gag is essential for cap-adduct formation. Although L-BC virus

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cannot be generated from L-BC cDNA expression vectors yet, it is now possible to perform reverse genetics to analyze cap-adduct formation in L-BC Gag.

L-BC Transcript has Diphosphate at the 5’ End- In the 5’ terminus, the L-BC positive strand possesses the first C at position 9 and the second one at position 16 (Fig. 3C). When L-BC virions were incubated in a CTP-omitted transcription mixture containing [α-32P]GTP, the virions made an 8-nt transcript as a major product and a minor 15-nt transcript (Fig. 3A). The 8-nt transcript was isolated from the gel, digested with S1 nuclease and then analyzed on TLC. The majority of the label was released as GDP and a minority as GTP (Fig. 3B). Because the 8-nt transcript has a single G at the 5’ terminus, the result indicates that the L-BC transcript has diphosphate at its 5’ terminus. It is likely that the minor production of the 15-nt transcript is due to the presence of a trace amount of cytidine nucleotide in the viral preparation. We were, however, unable to eliminate it from the preparation by repeated dialysis or washing with additional centrifugation.

Cap Snatching in cis- Previously we demonstrated that L-A virus could transfer m7Gp* (labeled with 32P, indicated by the asterisk) from a cap donor to an on-going transcript in a CTP-omitted transcription reaction resulting in the production of a 17-nt 5’ capped product (cap-snatching in cis) (Fig. 4A) (11). In the same reaction conditions, L-BC transferred the label into the minor 15-nt transcript thus producing a 16-nt product (Fig. 4A). Even though the 8-nt transcript was the major product in transcription it did not incorporate the label efficiently. To confirm that the product was 5’-capped, we isolated the 16-nt product from the gel and treated it with S1 or TAP. As shown in Fig. 4B, S1 and TAP digestions produced m7Gp*ppG and m7Gp*, respectively. Because S1 does not work on the triphosphate linkage of the cap structure and TAP cleaves anhydrous bonds between α− and β−, and also β− and γ−phosphates of the triphosphate linkage, these results indicate that, like cap snatching in L-A virus, L-BC virus also can transfer the m7Gp moiety from the cap donor to the diphosphorylated 5’ terminus of the transcripts, thus forming a cap structure at the 5’ ends (Fig. 1A).

Cap Snatching in trans- L-A virus can transfer m7Gp from the cap analog m7GpppG to

an externally added L-A transcript (cap acceptor) to form a cap structure at the 5’ terminus (cap snatching in trans) (27). We tested whether L-BC virus also can perform cap snatching in trans. The 8-nt L-BC transcript was isolated and then incubated with L-BC virions in the presence of m7GpppG. As shown in Fig, 5, lane 6, L-BC virions converted a part of the transcript to a slower-moving band. The same band was also produced in a reaction with L-A virus (Fig. 5, lane 5). Furthermore, when the 16-nt L-A transcript was incubated with L-BC virus, L-BC converted a part of it to a slower moving band (Fig. 5, lane 3), which comigrated with the 17-nt capped species produced by L-A virus (Fig. 5, lane 2). These results suggest firstly that L-BC virus can perform cap snatching in trans, and secondly that both L-A and L-BC viruses can use either viral transcript as cap acceptor. Because the 16-nt L-A transcript can be produced with ease (see Fig. 3A), hereafter we use this L-A transcript as acceptor for cap snatching in trans with L-BC virus. We confirmed cap snatching in trans by L-BC virus as shown in Fig. 6. The label located at the 5’ α−phosphate of the 16-nt L-A transcript (acceptor) can be removed by BAP (Fig. 6A, lane 2) while the label in the 17-nt product generated by L-BC virus became resistant to the treatment due to its internalization (Fig. 6A, lane 4). BAP is a phosphomonoesterase that hydrolyzes 5’ and 3’ phosphates from RNA and DNA. We isolated the BAP-resistant 17-nt transcript from the gel. S1 treatment of the 17-nt product generated m7GpppG, thus confirming the formation of cap structure at the 5’ end (Fig. 6B, lane 4). The 17-nt product treated with TAP remained at the origin (Fig. 6B, lane 5) because the remaining 16-nt RNA body was intact. Further S1 treatment generated GMP (Fig. 6B, lane 6). These results thus indicate that the label was located at the α-phosphate of the triphosphate linkage in the cap structure.

Characterization of L-BC Cap-snatching in trans- Cap-snatching activity of L-BC virus has characteristics very similar to those of L-A virus. Like in the case of L-A, L-BC virus could utilize m7GpppG but not GpppG as the cap donor (Fig. 7B), indicating that N7 methylation is essential for cap donor activity. The reaction was completed within 20 min of incubation (Fig. 7A). L-A cap snatching requires ATP and none of the other three nucleotides can replace ATP for the reaction (27). In addition to ATP, L-A cap

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snatching in trans required a primer for transcription, either a guanine nucleotide or the cap analog m7GpppG, resulting in the synthesis of 5’ XAAAAA-OH (X: primer). This led to the conclusion that the cap transfer process is activated by viral transcription. In accordance, a non-hydrolysable ATP analog γ-S-ATP can replace ATP for cap snatching, since the viral polymerase efficiently utilizes this analog for transcription (27). As shown in Fig. 7C, L-BC cap snatching also requires ATP, and none of the other three nucleotides could support the L-BC cap-snatching reaction (Fig. 7D). Furthermore, γ-S-ATP could replace ATP for the reaction (Fig. 7C). These results suggest that even the synthesis of the small fragment m7GpppGAA-OH by transcription is sufficient to activate L-BC cap snatching. The addition of EDTA inhibited the cap snatching of L-BC virus (Fig. 7D) indicating that the reaction requires divalent cations. Mg++ and Mn++ could support both cap snatching (Fig. 8A and B) and transcription (Fig. 8C) in L-A and L-BC viruses, although Mn++ is less active than Mg++ in transcription. These results raise the possibility that the divalent cations support cap snatching indirectly through the activation of transcription. We further analyzed the effects of divalent cations on cap-adduct formation by L-BC Gag. As shown in Fig. 8D, cap-adduct formation also required Mg++ and Mn++. Because this reaction is closely related with the cap transfer process and proceeds in the absence of ATP (without transcription activation), we conclude that these cations are directly involved in the cap transfer reaction itself.

Specificity for Cap Acceptor- Previously we demonstrated that L-A virus accepted 5’ diphosphorylated RNA but not tri- or monophosphorylated molecules as cap acceptors in the cap-snatching reaction (27). Here we synthesized 16-nt L-A 5’ terminal RNAs using SP6 RNA polymerase from an L-A cDNA fused to the SP6 promoter. Using GTP, GDP and GMP as a primer, we generated RNAs with tri-, di-, and monophosphate, respectively, at their 5’ termini. Like L-A virus, L-BC admitted only 5’ diphosphorylated RNA as cap acceptor (Fig. 9A). As demonstrated in Fig. 5, L-A virus accepted not only its 16-nt transcript but also the 8-nt L-BC transcript as cap acceptor. It indicates that the L-A sequence from 9 to 16 is not essential for cap acceptor activity for L-A virus. L-BC virus also used not only its 8-nt transcript but also the 16-nt L-A transcript as cap acceptor. In the first 8 nucleotides, L-A and L-BC transcripts

share 1-GAA-3 and 7-UU-8 sequences (Fig. 3C). Therefore, 4-AAA-6 of L-A transcript is not needed for L-A cap snatching. Likewise 4-UUU-6 of L-BC transcript is not necessary for L-BC cap snatching. We modified the second or third A of the L-A sequence to U by site-directed mutagenesis. SP6 RNA polymerase, however, made very few transcripts with the A2U mutation because the polymerase requires GA at the 5’ end of the transcript as part of the promoter. When RNA with the A3U mutation was tested, L-A and L-BC viruses used this RNA as cap acceptor (Fig. 9B). Therefore, L-A and L-BC viruses require diphosphate at the 5’ termini of the viral transcripts but apparently no specific viral nucleotide sequences for cap acceptor activity. The role of the 5’ terminal GA sequence in the activity, however, remains to be clarified.

In many strains, L-A and L-BC viruses cohabit in the same cells. Because these viruses can use either viral transcript as cap acceptor in the in vitro cap-snatching reactions, this raises the possibility that their cohabitation may be beneficial for their mutual cap acquisition in vivo. M1, a satellite RNA of L-A virus, requires L-A-encoded proteins for encapsidation and replication. M1 encodes the K1 killer toxin and its immunity (32). Efficient expression of the toxin requires capping of M1 transcripts (11). Cells carrying M1 maintained by capping-deficient L-A Gag (H154R) showed a negative phenotype in killer assay (11) and the presence or absence of L-BC viruses apparently did not affect this phenotype (Fig. 10). These results suggest that M1 (or L-A) and L-BC preferentially install the cap structure on their ongoing transcript by cap snatching in cis in the cell. Alternatively, because the copy number of L-BC virus is one or two orders of magnitude lower than that of L-A, the effects of L-BC virus on M1 capping may be subtle and thus we may need to develop a more sensitive capping assay DISCUSSION

In this work we have demonstrated that yeast L-BC virus can perform cap snatching to furnish its transcript with a cap structure, and that the capping reaction is very similar to that found in L-A virus. L-BC virus synthesizes transcripts with diphosphate at their 5’ termini and transfers m7Gp from mRNA to the 5’ terminus of the transcript. L-BC virus can form a cap structure not only on an ongoing transcript (cap snatching in cis) but also on an externally

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added transcript (cap snatching in trans), indicating that the cap-snatching site is externally accessible. The cap-snatching site of L-A virus is located in a trench on the outer surface of Gag (33). L-A transcripts are made inside the virion and presumably released to the cytoplasm through one of pores located at its icosahedral 5-fold symmetric axes. The trenches are positioned in the Gag asymmetric dimer close to the 2-fold and 3-fold axes, far from the pores. There are no structural studies on L-BC virus. Considering the similarities between L-A and L-BC viruses, however, it is likely that the cap-snatching site of L-BC is also located on the surface of Gag far from its 5-fold axes. This could explain why L-BC virus preferentially capped a 15-nt transcript over a shorter 8-nt transcript in cap snatching in cis. The 8-nt transcript might be too short to interact at the 5’ end with the cap-snatching site while anchored at the 3’ end in the pore. L-BC cap snatching requires ATP and its analog γ-S-ATP can replace ATP for the activity. This suggests that as in the case of L-A, L-BC cap snatching is also activated by transcription. L-BC cap snatching requires a divalent cation Mg++ or Mn++. Both transcription and cap-adduct formation require the same cations. Therefore, these cations have dual roles in cap snatching: one through transcription activation, and the other a direct involvement in the cap transfer reaction itself.

The capping reaction of L-BC virus uses N7 methylated cap molecules but not non-methylated ones as cap donors. Like L-A virus, L-BC requires 5’ terminal diphosphate for cap acceptor activity. Interestingly, L-A and L-BC viruses can admit either viral transcript as cap acceptor in vitro. It raises the possibility that their cohabitation in the same cell is beneficial for their cap acquisition. Furthermore, because host RNA with 5’ terminal diphosphate is rare in the cytoplasm, it also suggests that this requirement alone is sufficient to discriminate host RNA as cap acceptor in cap snatching. mRNA degradation in eukaryotes usually begins with the shortening of the poly A tail at the 3’ end, followed by decapping at the 5’ end by the Dcp1-Dcp2 decapping enzyme to generate an RNA body bearing 5’ monophosphate (34, 35). Then decapped RNA is digested by the SKI1/XRN1 5’ exonuclease. Alternatively the deadenylated RNA is digested by the 3’ exonuclease activity of the exosome. It is not clear how RNAs with 5’ diphosphate are produced in the cytoplasm and how stable they

are. The scavenger decapping enzyme removes m7Gp from dinucleotide- or oligonucleotide-capped molecules generated by the exosome, leaving the 3’ fragment with 5’ diphosphate (36, 37). The CTL1 gene product has RNA triphosphatase activity (38). It localizes in the cytoplasm, but its physiological role is unknown. We also have observed that ski1Δ cells accumulate L-A transcripts, most of which are 5’ monophosphorylated (19) thus indicating the presence of RNA diphosphatase activity in the cytoplasm. If this activity operates efficiently enough to convert the 5’ diphosphorylated viral transcripts, once released from the virions, to 5’ monophosphorylated ones, then it might suppress cross capping in vivo.

In L-A virus, His154 of Gag forms a phophoamide bond with m7Gp. His154 is located at the tip of a loop (residues 144-163) that is part of the upper rim of the trench. Upon m7GDP binding the rim moves inwardly and forms a closed conformation (39). In the trench, Tyr150, Asp152, Tyr452 and Tyr538 are located close to the bound m7GDP, suggesting their involvement in cap recognition. Mutagenesis studies indicate that these residues are crucial for cap-adduct formation (39). Guanylyltransferase also contains a trench that can adopt either an open or closed conformation during the canonical mRNA capping reaction (40, 41). This enzyme forms a Gp-enzyme intermediate with GTP and transfers Gp to the diphosphorylated 5’ termini of PolII transcripts. Therefore, the cap snatching reaction of L-A resembles that of guanylyltransferase. Because His156 of L-BC Gag is essential for cap-adduct formation, it is likely that L-BC Gag also forms an intermediate with m7Gp through His156 and transfers m7Gp to the 5’ terminus of its transcript. Guanylyltransferase relies on Lys but not on His to form a Gp-enzyme intermediate. DNA and T4 RNA ligases also use Lys to form an AMP (NAD)-enzyme intermediate and transfer the Ap moiety to a DNA/RNA 5’ end resulting in the formation of an activated A(5’)pp(5’)DNA/RNA intermediate (42, 43). Then a terminal DNA deoxyribose (RNA ribose) 3’-OH attacks the 5’ α-phophate of A(5’)pp(5’)DNA/RNA to produce a 3’, 5’-phosphodiester at the splice junction. In the non-conventional capping reaction of vesicular stomatitis virus, however, L protein covalently binds the 5’ monophosphorylated pre-mRNA through His and transfers the bound pre-mRNA to GDP (44). Members of the alphavirus-like superfamily methylate GTP first, then appear to

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form an m7Gp-enzyme intermediate through His and transfer m7Gp to the 5’ diphosphate end of the viral transcript during the capping reaction (45). Recently it has been demonstrated that the RNA ligase RtcB from Pyrococcus horikoshii forms a Gp-enzyme intermediate through His with GTP and transfers Gp to a polynucleotide 3’-phosphate to form polynucleotide-(3’)pp(5’)G (46, 47). Then attack of an RNA 5’-OH on the –N(3’)pp(5’)G end completes the ligation reaction. RNA cyclase RtcA from E. coli reacts with ATP to form an Ap-enzyme intermediate through His and transfers Ap to an RNA 3’-phosphate terminus to form RNA(3’)pp(5’)A (48). Then the terminal RNA ribose 2’-OH attacks the 3’-phosphate of RNA(3’)pp(5’)A to generate RNA 2’, 3’-cyclic phosphate. Interestingly, this enzyme can also transfer Ap to an RNA 5’-phosphate terminus resulting in the formation of A(5’)pp(5’)RNA, reminiscent of the intermediates produced by DNA and T4 RNA ligases, although RtcA cannot promote the ligation reaction (49). Therefore, many enzymes use not only Lys but also His to form (poly)nucleotide-enzyme intermediates for chemical transformations at polynucleotide termini.

Transcription of Influenza virus occurs in the nucleus in the form of a ribonucleoprotein

complex consisting of viral RNA, RNA-dependent RNA polymerase and NP proteins. The polymerase is a hetero-trimmer composed of PA, PB1 and PB2 subunits. The polymerase is accessible to pre-mRNA in the nucleus: PB2 binds to the cap structure of pre-mRNA and PA cleaves the bound RNA endonucleolytically 10-13 nt downstream of the cap, and finally PB1 uses the capped fragment as a primer to transcribe the viral RNA. By contrast, transcription and replication of L-A and L-BC totiviruses occur inside the virion. Once assembled, the Pol domain of Gag-Pol is confined inside the virion and is not accessible to mRNA in the cytoplasm. In this context it is quite reasonable that the cap-snatching site of totivirus is located at the cytoplasmic side of the capsid protein Gag. This raises another interesting question of how L-A and L-BC viruses can activate cap snatching at the surface of the virion by transcription that occurs inside the virion. The answer to this will not only provide insights on virus structure and function but also contributes to the understanding of how these simplest dsRNA viruses with only two encoded proteins can live and propagate in their eukaryotic host.

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FOOTNOTES *This work was supported by Grant BFU2010-15768 from The Spanish Ministry of Education and Science. We wish to thank “Fundación Ramón Areces” for financial support of our Institute. 1

The abbreviations used are: dsRNA, double-stranded RNA; nt, nucleotides; BAP, bacterial alkaline phosphatase; TAP, tobacco acid pyrophosphatase; TLC, thin layer chromatography; PEI, polyethyleneimine.

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LEGENDS TO FIGURES

FIGURE 1. A. Schematic diagram of cap-snatching mechanism in L-A and L-BC viruses. Gag decaps mRNA and forms an intermediate with m7Gp through His (H154 for L-A and H156 for L-BC). Then m7Gp is transferred to the diphosphorylated 5’ end of the emerging viral transcript to form a 5’-5’ triphosphate linkage (cap snatching in cis). m7Gp also can be transferred to an externally added viral transcript (cap snatching in trans). B. Comparison of selected L-A sequences with those of other fungal totiviruses. H154 and crucial residues for cap recognition are boxed and indicated by the arrow and vertical bars, respectively. GenBank accession numbers are: L-A (AAA50320.1), TaV1 (ADQ54105.1), XdL1A (NC_020903.1), XdL1B (AFH09413.1), L-BC (NP 042580.1) and SgVL (AGG68772.1)

FIGURE 2. L-BC Gag has cap-adduct forming activity. A. RNAs extracted from L-A and L-BC virus preparations were separated on an agarose gel and visualized by ethdium bromide staining (left panel, EtBr). λ HindIII markers (λ) and some of their sizes in Kb are also shown. Autoradiograms of the Northern blots hybridized with L-A- and L-BC-specific probes are shown in the middle (L-A probe) and on the right panel (L-BC probe). The L-A probe recognizes the positive strand of L-A from nt 1323-1786. The L-BC probe hybridizes the positive strand of L-BC from nt 63-502. B. L-A and L-BC virions were incubated with the 6-nt cap donor and then their proteins were analyzed by SDS-PAGE. Left panel (Coomassie) shows Coomassie brilliant blue staining of the gel with protein markers (M) whose sizes in kDa are indicated on the left. An autoradiogram of the same gel is shown on the right panel (Cap adduct). Gag and Gag-Pol of L-A and L-BC viruses are marked on the right of the panels. C. Cell lysates from two independent transformants with a plasmid expressing WT (lanes 2 and 3) or H156R mutant (lanes 4 and 5) L-BC Gag were incubated with the 6-nt cap donor and then analyzed by SDS-PAGE. Coomassie brilliant blue staining of the gel (Coomassie) and an autoradiogram of the same gel (Cap-adduct) are shown. A lysate from untransformed cells is run in parallel as control (lane 1). M, protein markers. The position of L-BC Gag is indicated.

FIGURE 3. L-BC transcript has diphosphate at the 5’ end. A. Transcripts made by L-A or L-BC viruses in CTP-omitted reactions were separated on a 15% acrylamide/8 M urea gel. An autoradiogram of the gel is shown. The sizes of transcripts are indicated. B. A gel-purified 8-nt L-BC transcript labeled with [α-32P]GTP in a CTP-omitted reaction was treated with S1 nuclease and analyzed on PEI-cellulose (S1). None indicates the non-treated 8-nt transcript. The positions of non-labeled guanine nucleotides are indicated on the right. C. The 5’ terminal sequences from L-BC and L-A positive strands. Cs relevant to the transcripts observed in A are marked by the arrows and arrowhead. Identical nucleotides between the first 8-nt L-A and L-BC sequences are underlined. Note that the 8-nt L-BC transcript has a single G at the 5’ terminus.

FIGURE 4. L-BC cap snatching in cis. A. L-A or L-BC virions were incubated with the cap donor m7Gp*ppGGGCGp (* indicates the position of 32p) in a CTP-omitted reaction and the capped products were separated on a denaturing acrylamide gel. An autoradiogram of the gel is shown. The positions of the donor and the capped products are indicated. Lane (-) indicates the cap donor alone without incubation as control. B. The 16-nt L-BC capped product was isolated from the gel, treated with S1 nuclease (S1) or tobacco acid pyrophosphatase (TAP) and analyzed on PEI-cellulose. The cap donor was also analyzed in parallel. None indicates non-treated sample. The deduced transfer reaction is shown on the bottom with the S1 and TAP cleavage sites. FIGURE 5. Cap snatching in trans. A cap acceptor, 16-nt L-A transcript (lanes 2 and 3) or 8-nt L-BC transcript (lanes 5 and 6) was incubated with L-A (lanes 2 and 5) or L-BC (lanes 3 and 6) virions along with the cap donor m7GpppG. After incubation, capped products were separated in a denaturing acrylamide gel and visualized by autoradiography. The L-A (lane 1) or L-BC (lane 4) transcript without incubation was run in parallel as control. The acceptors (Acceptor), capped products (Capped) and their nucleotide sizes are indicated. The low capping efficiency by L-BC is due to a lower amount of L-BC virions used, as described in the Experimental Procedures.

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FIGURE 6. Proof of L-BC cap snatching in trans. A. A 16-nt L-A transcript (Acceptor) 5’-labeled with [α-32P]GTP was incubated with L-BC virions in the presence of the cap donor m7GpppG. The product (lanes 3 and 4) or the acceptor (lanes 1 and 2) was non-treated (lanes 1 and 3) or treated with bacterial alkaline phosphatase (BAP, lanes 2 and 4) and separated on a denaturing acrylamide gel. An autoradiogram of the gel is shown. B. The bacterial alkaline phosphatase-resistant product shown in A, lane 4, was isolated from the gel, treated with S1 or TAP alone, or sequentially with TAP and S1, and analyzed on PEI-cellulose. The acceptor was also treated with S1 and analyzed on the same plate. None: non-treated sample. The deduced cap transfer reaction is shown on the bottom with S1 and TAP cleavage sites. FIGURE 7. Requirements for L-BC cap snatching in trans. A. Time course of L-BC cap snatching. The incubation times are indicated below the panel. m7GpppG and the 16-nt L-A transcript were used as cap donor and acceptor, respectively. B. The cap-snatching reaction was carried out in the presence of 0.5 mM m7GpppG or GpppG or in their absence (-) as indicated. The 16-nt L-A transcript was used as cap acceptor. The presence (+) or absence (-) of L-BC virions (L-BC) in the reaction is also indicated. C. The cap-snatching reaction was carried out in the presence of 0.5 mM ATP (+) or γ-S-ATP or in their absence (-) as indicated. 0.5 mM m7GpppG was used as cap donor. The presence (+) or absence (-) of 5 mM MgCl2 in the reaction is also shown. D. ATP requirements. The cap-snatching reaction was carried out in the presence of one of nucleotide triphosphates (0.5 mM) or in their absence (-). In the rightest lane, 10 mM EDTA was also added to the reaction. FIGURE 8. Effects of divalent cations on cap snatching and cap-adduct formation. A and B. Effects of divalent cations (5 mM) on L-BC (A) or L-A (B) cap snatching. C. Effects of divalent cations (5 mM) on L-A or L-BC transcription. The transcripts were analyzed on an agarose gel and visualized by ethidium bromide staining (upper panel). An autoradiogram of the gel is also shown in the lower panel. D. Cap-adduct formation of L-BC Gag in the presence of 5 mM divalent cations or in their absence (-). L-BC virions were incubated with the labeled 6-nt cap donor in the presence or absence of divalent cations. Then the protein was separated by SDS-PAGE and visualized by Coomassie brilliant blue staining (Coomassie). An autoradiogram of the gel (Cap-adduct) is shown on the right. M: protein mobility markers (kDa). FIGURE 9. A diphosphate status at the 5’ end is required for cap-acceptor activity in L-BC cap snatching. A. 16-nt L-A 5’ terminal fragments bearing tri(ppp-) di(pp-) and mono(p-)phosphate at their 5’ ends were synthesized by SP6 RNA polymerase using GTP, GDP and GMP, respectively, as primers and tested their cap-acceptor activity in L-BC cap-snatching reaction. B. Effects of nucleotide sequence on cap-acceptor activity. A 16-nt L-A 5’ terminal fragment having the WT sequence or the A3U mutation was synthesized by SP6 RNA polymerase using GDP as a primer and tested its cap acceptor activity in the L-A or L-BC cap-snatching reaction. The A3U mutation is indicated by the dot. FIGURE 10. Effects of L-BC virus on M1 killer phenotype. A. Diagram of L-A expression plasmid. Gag (closed dot) and Gag-Pol (closed dot-closed square) are expressed from the constitutive PGK1 promoter (PGK1). Thick line represents the L-A cDNA sequence and thin lines are vector sequences. The position of H154 in Gag is indicated by the vertical bar. B. Wild type (WT) or mutated (H154R) virions were expressed from the vectors in L-BC or L-BC-o strains. Satellite M1 RNA can be encapsidated into virions produced by L-A proteins expressed from the plasmids (29). Mutated (H154R) virions can also maintain M1 RNA, although M1 transcripts produced by the mutant virions lack the cap structure at the 5’ ends and thus express the secreted killer toxin poorly (11). C. Killer toxin assay. Colonies separated by the vertical line harbor M1 satellite RNA maintained by a vector expressing either WT (left) or H154R (right) L-A Gag. Colonies harboring L-BC virus (L-BC) or no virus (L-BC-o) are shown below or above the horizontal line. The expression of the killer toxin is visualized by clear halos resulting from killing the toxin-sensitive lawn cells.

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Tsutomu Fujimura and Rosa EstebanCap Snatching in Yeast L-BC Double-Stranded RNA Totivirus

published online July 3, 2013J. Biol. Chem. 

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