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

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. 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. His-154 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 the 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 Mg2+ and Mn2+ 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 the 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.

Eukaryotic mRNA is capped at the 5Ј end by a 7-methyl GMP moiety via an inverted 5Ј-5Ј-triphosphate linkage (m 7 GpppN-) (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)(3)(4)(5)(6). In eukaryotic cells and for many viruses, the cap structure is installed onto the 5Ј end of PolII transcripts through three enzymatic reactions (7,8). The 5Ј-␥-phosphate at the 5Ј end of the transcript is removed by RNA triphosphatase. Then Gp is transferred from GTP to the 5Ј end of the transcript by a guanylyltransferase, thus forming a trisphosphate linkage. During the transfer, Gp is covalently attached to the guanylyltrans-ferase 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 nucleotides (nt) 2 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 nonsegmented double-stranded RNA (dsRNA) 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 made by a Ϫ1 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 5Ј 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-acetyltransferase 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 m 7 Gp moiety of the cap structure was attached to His-154 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, which causes crowding of macromolecules in the aqueous solution (26), transfers m 7 Gp from mRNA to the 5Ј end of the L-A transcript, thus forming a cap structure at the 5Ј terminus (11) (Fig. 1A). The ␣and ␤-phosphates of the triphosphate linkage are derived from the 5Ј terminus of the L-A transcript and the ␥-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 * This work was supported by Grant BFU2010-15768 from the Spanish Ministry of Education and Science. 1  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 5Ј-diphosphate of the 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 the L-A transcript. Fungal totiviruses (including L-BC) closely related to L-A share His-154 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 5Ј 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 (Mat␣ 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 liters of YPAD (2% peptone, 1% yeast extract, 2% glucose, and 0.04% adenine) for 3 days at 28°C were harvested, washed once with deionized water, suspended in 1 volume/g of cells of buffer B (0.1 M Tris-HCl, pH 7.5, 1.4 M sorbitol, 1% 2-mercaptoethanol, and 2 mg/ml Zymolyase 20T (Seikagaku Corp.)), and incubated at 30°C for 45 min. All subsequent steps were carried out at 0 -4°C. 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 ϫ g, 20 min). To the supernatant, 0.25 volume of 25% PEG 4000 and 2.5 M NaCl was added. 30 min later, the suspension was centrifuged again (18,500 ϫ g 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 the 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 l of buffer A containing 20% (v/v) glycerol, and kept at Ϫ70°C before use.
Cap-snatching Reactions-The standard cap-snatching reaction in trans (25 l) contains 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 20 mM NaCl, 5 mM KCl, 0.1 mM EDTA, 0.5 mM m 7 GpppG as cap donor, 4.5 mg/ml bentonite, 20% PEG 4000, and 0.5 mM ATP, 32 P-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°C 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 m 7 GpppG and the 32 P-labeled cap acceptor were replaced by GTP and UTP, 0.5 mM each, and a 32 P-labeled 6-nt cap donor (3,000 -5,000 cpm). We used the 6-nt m 7 Gp*ppGGGCGp (labeled with 32 P, indicated by the asterisk) instead of m 7 GpppGGGCp (27) because of its ease in handling. The reaction was kept at 30°C for 1 h, and the products were analyzed as described above. The amount of L-BC virions used was 13 g of protein per reaction, approximately half of that of L-A (25 g), except that in Fig. 5, we used fewer L-BC particles (7 g of protein).
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 (-GGRHRGGAPIL-NQS). L-BC Gag with H156R mutation was generated by sitedirected mutagenesis. Strain 2927 (Mata ura3 trp1 his3 ski2-2, L-BC-o, 20 S RNA) transformed with the vector containing the wild type or mutant L-BC Gag was grown overnight at 28°C 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 l 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°C, the lysates (ϳ20 g of protein) were incubated in the reaction mixture for cap adduct formation as described above, and proteins were analyzed by SDS-PAGE.

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 the L-A virus. Due to the fragility and the low copy number of the 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 under "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 32 P-labeled 6-nt cap donor, L-BC Gag formed a cap adduct (Fig. 2B, right panel) as demonstrated previously (24).
His-156 of L-BC Gag Is Essential for Cap Adduct Formation-In L-A virus, Gag covalently binds to the m 7 Gp moiety of the cap structure through His-154. This residue corresponds to His-156 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 His-156 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, whereas H156R Gag failed to do so. Thus these results indicate that His-156 of L-BC Gag is essential for cap adduct formation. Although L-BC virus 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 5Ј End-In the 5Ј 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 [␣-32 P]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 was released as GTP (Fig. 3B). Because the 8-nt transcript has a single G at the 5Ј terminus, the result indicates that the L-BC transcript has diphosphate at its 5Ј 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 m 7 Gp* (labeled with 32 P, indicated by the asterisk) from a cap donor to an ongoing transcript in a CTPomitted transcription reaction resulting in the production of a 17-nt 5Ј 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). Although the 8-nt transcript was the major product in transcription, it did not incorporate the label efficiently. To confirm that the product was 5Ј-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 m 7 Gp*ppG and m 7 Gp*, respectively. Because S1 does not work on the triphosphate linkage of the cap structure and TAP cleaves anhydrous bonds between ␣and ␤-, and also ␤and ␥-phosphates of the triphosphate linkage, these results indicate that, like cap snatching in the L-A virus, the L-BC virus also can transfer the m 7 Gp moiety from the cap donor to the diphosphorylated 5Ј terminus of the transcripts, thus forming a cap structure at the 5Ј ends (Fig. 1A).
Cap Snatching in trans-L-A virus can transfer m 7 Gp from the cap analog m 7 GpppG to an externally added L-A transcript (cap acceptor) to form a cap structure at the 5Ј 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 m 7 GpppG. 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 the 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 (Fig. 3A), hereafter we used this L-A transcript as acceptor for cap snatching in trans with L-BC virus. We confirmed cap snatching in trans by the L-BC virus as shown in Fig. 6. The label located at the 5Ј-␣phosphate of the 16-nt L-A transcript (acceptor) can be removed by bacterial alkaline phosphatase (Fig. 6A, lane 2), whereas the label in the 17-nt product generated by the L-BC virus became resistant to the treatment due to its internalization (Fig. 6A, lane 4). Bacterial alkaline phosphatase is a phosphomonoesterase that hydrolyzes 5Ј-and 3Ј-phosphates from RNA and DNA. We isolated the bacterial alkaline phosphatase-resistant 17-nt transcript from the gel. S1 treatment of the 17-nt product generated m 7 GpppG, thus confirming the formation of cap structure at the 5Ј 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 ␣-phosphate of the triphosphate linkage in the cap structure.
Characterization of L-BC Cap Snatching in trans-Capsnatching activity of the L-BC virus has characteristics very similar to those of the L-A virus. Like in the case of L-A, the L-BC virus could utilize m 7 GpppG but not GpppG as the cap donor (Fig. 7B), indicating that N7 methylation is essential for  Lane Ϫ 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 nontreated sample. The deduced transfer reaction is shown on the bottom with the S1 and TAP cleavage sites.
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 snatching in trans required a primer for transcription, either a guanine nucleotide or the cap analog m 7 GpppG, resulting in the synthesis of 5Ј-XAAAAA-OH (X indicates primer). This led to the conclusion that the cap transfer process is activated by viral transcription. In accordance, a nonhydrolyzable ATP analog ATP␥S can replace ATP for cap snatching because 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, ATP␥S could replace ATP for the reaction (Fig. 7C). These results suggest that even the synthesis of the small fragment m 7 GpppGAA-OH by transcription is sufficient to activate L-BC cap snatching. The addition of EDTA inhibited the cap snatching of the L-BC virus (Fig. 7D), indicating that the reaction requires divalent cations. Mg 2ϩ and Mn 2ϩ could support both cap snatching (Fig. 8, A and B) and transcription (Fig. 8C) in L-A and L-BC viruses, although Mn 2ϩ is less active than Mg 2ϩ 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 2ϩ and Mn 2ϩ . 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 5Ј-diphosphorylated RNA but not trior monophosphorylated molecules as cap acceptors in the capsnatching reaction (27). Here we synthesized 16-nt L-A 5Ј-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 5Ј termini. Like L-A virus, L-BC admitted only 5Ј-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. This 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 the L-A transcript is not needed for L-A cap snatching. Likewise 4 UUU 6 of L-BC transcript is not necessary for  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 nuclease (S1) or TAP alone, or sequentially treated with TAP and S1, and analyzed on PEI-cellulose. The acceptor was also treated with S1 and analyzed on the same plate. None: nontreated sample. The deduced cap transfer reaction is shown on the bottom with S1 and TAP cleavage sites.
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 5Ј 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 5Ј termini of the viral transcripts but apparently no specific viral nucleotide sequences for cap acceptor activity. The role of the 5Ј-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 the 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 cappingdeficient 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 the L-BC virus is 1 or 2 orders of magnitude lower than that of L-A, the effects of the 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 the L-A virus. L-BC virus synthesizes transcripts with diphosphate at their 5Ј termini and transfers m 7 Gp from mRNA to the 5Ј 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 added transcript (cap snatching in trans), indicating that the cap-snatching site is externally accessible. The cap-snatching site of the 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  five-fold symmetric axes. The trenches are positioned in the Gag asymmetric dimer close to the two-fold and three-fold axes, far from the pores. There are no structural studies on the 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 five-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 5Ј end with the cap-snatching site while anchored at the 3Ј end in the pore. L-BC cap snatching requires ATP, and its analog ATP␥S 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 2ϩ or Mn 2ϩ . 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 the L-BC virus uses N7 methylated cap molecules but not nonmethylated ones as cap donors. Like L-A virus, L-BC requires 5Ј-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 5Ј-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 3Ј end followed by decapping at the 5Ј end by the Dcp1-Dcp2 decapping enzyme to generate an RNA body bearing 5Ј-monophosphate (34,35). Then decapped RNA is digested by the SKI1/XRN1 5Ј-exonuclease. Alternatively, the deadenylated RNA is digested by the 3Ј-exonuclease activity of the exosome. It is not clear how RNAs with 5Ј-diphosphate are produced in the cytoplasm and how stable they are. The scavenger decapping enzyme removes m 7 Gp from dinucleotide-or oligonucleotide-capped molecules generated by the exosome, leaving the 3Ј fragment with 5Ј-diphosphate (36,37). The CTL1 gene product has RNA triphosphatase activity (38). It localizes in the cytoplasm, but its physiological role is unknown. We have also observed that ski1⌬ cells accumulate L-A transcripts, most of which are 5Ј-monophosphorylated (19), thus indicating the presence of RNA diphosphatase activity in the cytoplasm. If this activity operates efficiently enough to convert the 5Ј-diphosphorylated viral transcripts, once released from the virions, to 5Ј-monophosphorylated ones, then it might suppress cross capping in vivo. FIGURE 9. A diphosphate status at the 5Ј end is required for cap acceptor activity in L-BC cap snatching. A, 16-nt L-A 5Ј-terminal fragments bearing tri (ppp-), di (pp-), and mono (p-) phosphate at their 5Ј ends were synthesized by SP6 RNA polymerase using GTP, GDP, and GMP, respectively, as primers, and their cap acceptor activity in the L-BC cap-snatching reaction was tested. B, effects of nucleotide sequence on cap acceptor activity. A 16-nt L-A 5Ј-terminal fragment having the WT sequence or the A3U mutation was synthesized by SP6 RNA polymerase using GDP as a primer, and its cap acceptor activity in the L-A or L-BC cap-snatching reaction was tested. The A3U mutation is indicated by the dot. In L-A virus, His-154 of Gag forms a phosphoamide bond with m 7 Gp. His-154 is located at the tip of a loop (residues 144 -163) that is part of the upper rim of the trench. Upon m 7 GDP binding, the rim moves inwardly and forms a closed conformation (39). In the trench, Tyr-150, Asp-152, Tyr-452, and Tyr-538 are located close to the bound m 7 GDP, 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 a closed conformation during the canonical mRNA capping reaction (40,41). This enzyme forms a Gpenzyme intermediate with GTP and transfers Gp to the diphosphorylated 5Ј termini of PolII transcripts. Therefore, the cap-snatching reaction of L-A resembles that of guanylyltransferase. Because His-156 of L-BC Gag is essential for cap adduct formation, it is likely that L-BC Gag also forms an intermediate with m 7 Gp through His-156 and transfers m 7 Gp to the 5Ј 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 5Ј end, resulting in the formation of an activated A(5Ј)pp(5Ј)DNA/ RNA intermediate (42,43). Then a terminal DNA deoxyribose (RNA ribose) 3Ј-OH attacks the 5Ј ␣-phosphate of A(5Ј)pp(5Ј)DNA/RNA to produce a 3Ј,5Ј-phosphodiester at the splice junction. In the nonconventional capping reaction of vesicular stomatitis virus, however, L protein covalently binds the 5Ј-monophosphorylated pre-mRNA through His and transfers the bound pre-mRNA to GDP (44). Members of the alphavirus-like superfamily methylate GTP first and then appear to form an m 7 Gp-enzyme intermediate through His and transfer m 7 Gp to the 5Ј-diphosphate end of the viral transcript during the capping reaction (45). Recently, it has been demonstrated that the RNA ligase RtcB from Pyrococcus horikoshii or Escherichia coli forms a Gp-enzyme intermediate through His with GTP and transfers Gp to a polynucleotide 3Ј-phosphate to form polynucleotide-(3Ј)pp(5Ј)G (46,47). Then attack of an RNA 5Ј-OH on the -N(3Ј)pp(5Ј)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 3Ј-phosphate terminus to form RNA(3Ј)pp(5Ј)A (48). Then the terminal RNA ribose 2Ј-OH attacks the 3Ј-phosphate of RNA(3Ј)pp(5Ј)A to generate RNA 2Ј,3Ј-cyclic phosphate. Interestingly, this enzyme can also transfer Ap to an RNA 5Ј-phosphate terminus, resulting in the formation of A(5Ј)pp(5Ј)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-trimer 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, 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 the 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 contribute to the understanding of how these simplest dsRNA viruses with only two encoded proteins can live and propagate in their eukaryotic host.