Mechanism of Formation of Reovirus mRNA V-terminal Blocked and Methylated Sequence, m7GpppGmpC

Blocked and methylated 5’ termini of reovirus mRNA are formed by viral cores at an early stage of transcription. Cores incubated in a complete transcription reaction mixture for 30 s, or in a mixture lacking UTP and ATP for a longer time, synthesize the “cap” structure, m’GpppG”pC. The dinucleotide ppGpC functions as substrate for a core-associated guanylyltransferase and is converted to GpppGpC by addition of pG from GTP. For optimal conversion, both the diphosphate terminus and phosphodiester bond are required. pGpC is not a substrate, but pppGpC is utilized after removal of the y-phosphate by a core nucleotide phosphohydrolase. Methyltransferases also present in cores transfer methyl groups sequentially from S-adenosylmethionine (AdoMet) to the W-position of the 5’-terminal guanosine followed by the 2’-OH of the penultimate guanosine. GpppGpC is hydrolyzed by cores in the presence of pyrophosphate to ppGpC, the predominant 5’.terminal structure of reovirus mRNA made in the absence of S-adenosylmethionine. NT-methylation prevents pyrophosphorolysis of m’GpppGpC, which may explain the increased proportion of blocked, methylated

Blocked and methylated 5' termini of reovirus mRNA are formed by viral cores at an early stage of transcription.
Cores incubated in a complete transcription reaction mixture for 30 s, or in a mixture lacking UTP and ATP for a longer time, synthesize the "cap" structure, m'GpppG"pC.
The dinucleotide ppGpC functions as substrate for a core-associated guanylyltransferase and is converted to GpppGpC by addition of pG from GTP. For optimal conversion, both the diphosphate terminus and phosphodiester bond are required. pGpC is not a substrate, but pppGpC is utilized after removal of the y-phosphate by a core nucleotide phosphohydrolase.
Methyltransferases also present in cores transfer methyl groups sequentially from S-adenosylmethionine (AdoMet) to the W-position of the 5'-terminal guanosine followed by the 2'-OH of the penultimate guanosine. GpppGpC is hydrolyzed by cores in the presence of pyrophosphate to ppGpC, the predominant 5'.terminal structure of reovirus mRNA made in the absence of S-adenosylmethionine.
NT-methylation prevents pyrophosphorolysis of m'GpppGpC, which may explain the increased proportion of blocked, methylated 5' termini in viral mRNA synthesized in the presence of S-adenosylmethionine.
On the basis of these findings, the following reaction series is proposed for the synthesis of reovirus mRNA caps. In the series, AdoHcy is the abbreviation for S-adenosylhomocysteine.
The widespread distribution of caps in eukaryotic mRNAs is consistent with an important role in translation. Reovirus and vesicular stomatitis virus-capped mRNAs are translated more efficiently in wheat germ extracts than the unmethylated viral mRNAs (22). Furthermore, chemical removal of 5'-terminal 7-methylguanosine from rabbit globin mRNA results in a reduced ability to direct cell-free protein synthesis (23), and in vaccinia mRNA, synthetic poly (A) and poly(G) (42,43). In order to determine if m'GpppG" . . . is synthesized as an initial or a final step in reovirus mRNA formation, nascent RNA products of reovirus cores were examined for the presence of modified 5' termini.
RNA synthesis was initiated by addition of magnesium acetate to standard reaction mixtures containing chymotrypsindigested, washed reovirus cores and [cx-~*P]GTP and [Me-3H]/ AdoMet as radioactive precursors. After incubation for the indicated intervals at 37", reaction mixtures were extracted with phenol and the products were analyzed by filtration on calibrated columns of Sephadex G-75 (Fig. 1A). As shown in Fig. 1. B, and C, RNA of size sufficient to be excluded from the gel became apparent between 30 s and 1 min of incubation and subsequently increased in amount (Fig. 1D). The szP/3H ratio was 0.3 in the products of a 1-min incubation as compared to 0.54 after 2 min, consistent with the presence of 5'-terminal [3H]methyl groups in the incomplete, internally ?'-labeled chains. Although no large RNA was synthesized in a 30-s reaction, the gel elution profile included 'H-labeled material in the position of an oligonucleotide marker of 8 chain length 12 to 18 nucleotides (Fig. 1B). The [3H]methyllabeled material which presumably corresponds to blocked and methylated nascent products was pooled as indicated and digested with PI nuclease to produce 5'-mononucleotides and unhydrolyzed caps (19, 32). The digestion products were further incubated with bacterial alkaline phosphatase and analyzed by paper chromatography. Fig. 2 (panel I) shows that component I, which corresponds to oligonucleotides of average chain length (about 15 nucleotides) (44), contains all the 3H radioactivity in the cap structure, m'GpppGm. Component II, which is shorter in length than component I and elutes close to the position of GTP and AdoMet, consists of a mixture of oligonucleotides containing either the dimethylated cap, m'GpppG" (93%) or the monomethylated cap, m'GpppG (7%) (Fig. 2, panel ZZ). The results demonstrate that reovirus nascent mRNA of chain length 15 nucleotides or less already have blocked and methylated 5' termini. Some of the shorter nascent chains contain an incompletely methylated cap, m'GpppG, suggesting that methylation of the terminal guanosine precedes that of the penultimate residue.
In order to determine how early in their synthesis nascent chains are blocked and methylated, the products of a reaction mixture containing unwashed cores, [Me-SH]AdoMet, GTP, and CTP but no UTP and ATP were analyzed. Under these conditions, chain elongation is arrested at the sites of pU and  A, an aliquot (20 ~1) was analyzed by paper electrophoresis.
B, the phosphatase-resistant material in another aliquot that migrated in the position of pA was extracted from the paper and divided into three fractions. The first was directly analyzed by chromatography (solid line), and the second fraction was digested with P, nuclease before paper chromatography in isobutyric acid/O. to '*P radioactivity in the purified m'G$ppG" was about twice that in the m'G$ppG, in agreement with the proposed structure. Thus, the structures in peaks I, II, and III are G;ppG, m7G$ppG, and m'G$ppG" derived from G$ppGpC, m'G;ppGpC, and m7G$ppGmpC, respectively.
In addition to GcppGpC and m7G6ppGmpC synthesized from ppGpC, low levels of G$p6G, and m7G$p6G were also detected in the phosphatase-treated products (in Fractions 11 to 12 and Fraction 8, respectively, of Fig. 5A). They were identified by paper chromatography with authentic marker samples as in Fig. 5B (data not shown). These compounds presumably were formed in a limited reaction involving condensation of 2 molecules of pp;G. The quantities of the different 5'-terminal structures synthesized by reovirus cores in the presence of AdoMet are summarized in Table I. Conversion of ppGpC to GpppGpC appears to be the most efficient reaction and is at least 20-fold more effective than pppG condensation for the formation of the blocked structure, GpppG.
N'-methylation of the terminal guanosine in GpppGpC (or GpppG) was incomplete in these partial reaction mixtures, and only about 10% of the products were methylated. The second methylation, i.e. 2'-0-methylguanosine formation, of the blocked trinucleotide was even more limited (<l%), as observed for short nascent chains synthesized in an incomplete reaction mixture (Fig. 3).
Previously it was found that the initiation of transcription by purified cytoplasmic polyhedrosis virus was almost completely dependent upon the presence of AdoMet in the incubation mixture (41). This was in contrast to viral mRNA synthesis by reovirus cores which was not increased by addition of AdoMet (18). Similarly, the formation by reovirus cores of 32P-labeled blocked structures from ppGpC and [W"P]GTP was not increased by AdoMet (Fig. 6). Furthermore, the inhibitor of methylation, AdoHcy, did not reduce the rate of G;ppGpC formation ( Fig. 6).
Reovirus mRNA synthesis in vitro depends upon the presence of Mg'+, and cap formation shows the same requirement. Under standard reaction conditions for reovirus core-mediated cap synthesis from ppGpC and [w~~P]GTP, optimal incorporation of 32pG into GfippGpC occurred at 2 mM Mg'+ (950 pmol/h/mg of cores). No incorporation was observed in the absence of Mg", and increasing the concentration of the cation to 4, 6, 8, and 12 mM decreased cap synthesis by 4%, lo%, 13%, and 20%, respectively.
Modification of pppGpC for Synthesis of Blocked, Methylated Structures-The formation of [3H]methyl-labeled blocked structures was also studied with [Me-3H]AdoMet, unlabeled GTP, and pppGpC (Table II). Like ppGpC, pppGpC was cdnverted to a mixture of the capped structures, Values shown were calculated from the data in Fig. 5, A  Although the yield of methylated, blocked compounds with pppGpC was 70% of that obtained with ppGpC, the distribution of mono-and dimethylated compounds produced from each was almost identical. These data indicate that the y-phosphate of pppGpC is removed by the core-associated phosphohydrolase (13)(14)(15), and the resulting ppGpC used for capping and methylation. Consistent with this suggestion, it was found that pppGpC was converted to ppGpC at a rate of about 9 nmol/mg of cores/h at 45"; under similar incubation conditions, 0.2 nmol of caps in mRNA were formed per mg of reovirus cores/h. Thus, the core-associated phosphohydrolase apparently is required to modify 5'-terminal pppGpC in nascent chains before blocked structures are synthesized.
Substrate Utilization for Blocked Structure Synthesis-A variety of different substrates were tested as precursors for the synthesis of blocked structures by reovirus cores. As shown in Table III thesis. Incubation conditions with ppGpC and [a-"P]GTP and analysis of GrjpfiGpC products were as described under "Experimental Procedures" and in Fig. 6, except that 0.5 mM AdoHcy was present i n the incubation mixture together with the indicated amount of sodium pyrophosphate. bond formation before transfer of the 5'-terminal pG in the synthesis of blocked structures (Fig. 4) > pppG + ppGpC Guanylyltransferase GpppGpC + PP, internal nucleotides in nascent mRNA and recognizes only blocked structures consisting of 2 guanosine residues linked 5'-5' through 3 phosphates. Such a strict substrate recognition may be a property characteristic of the reovirus-associated enzyme. The NT-G-methylase solubilized and purified from vaccinia virus methylates GpppGp(Gp),G and GpppAp-(Ap),A, producing m'GpppGp(Gp),G and m'GpppAp(Ap),A in the absence of viral genome DNA (42, 43). The purified vaccinia methylase does not require metal ions for activity. In contrast, the reovirus core-associated N'-G-methylase, one of the several activities in the transcriptional complex, apparently requires Mg '+ ion for activity (Table V) (Table III). The proposed reaction series for its synthesis includes ppGpC and pppGpC as intermediates.
Since 2 mM pyrophosphate almost completely inhibits reaction 3 in the series, i.e. the formation of GpppGpC from ppGpC plus GTP (Fig. 7), it was of interest to test for the accumulation of ppGpC as an intermediate when cores were incubated with [LY-~~P]GTP, [Y]CTP, and 2 mM pyrophosphate. As shown in Fig. 9A, electrophoretic analysis of a reaction mixture incubated under these conditions included several radioactive compounds including material in the position of ppGpC. However, because of the presence of residual radioactivity including [%]CDP, it was difficult to determine directly if ppGpC was present. In order to assay for '"C-labeled, phosphodiester bond-containing compounds: pGpC, ppGpC, and pppGpC, samples were eluted from the electrophoretogram. An aliquot of each eluate was exhaustively digested with alkaline phosphatase and re-analyzed by electrophoresis for the presence of '"C-labeled GpC (Fig. 9B). A small amount of radioactivity was obtained from the fractions corresponding to the positions of GpC and pGpC, but the bulk of the phosphatase-resistant "C radioactivity was recovered in fractions corresponding to the position of marker ppGpC. The calculated amount of "C-labeled ppGpC plus pGpC synthesized in the presence of 2 mM pyrophosphate was 160 pmol as compared to the value of 280 pmol of GpppGpC synthesized under similar conditions but without added pyrophosphate (Table III, Experiment 2).
In order to identify further the major '*C-labeled product, another aliquot of the radioactive material from the region of ppGpC (Fig. 9A, bracket) was analyzed by Dowex 1 chromatography with marker compounds. Again, a peak of "P-labeled material with coincident "C radioactivity eluted in the position of ppGpC (Fig. 9C). After phosphatase digestion, the Incubation conditions and analysis procedures were the same as described in the legend for Table IV. GpppG, 10 nmol, was used in each assay as substrate. MgZ' [SH]Methyl incorporated rnM pm01 2 16.9 0 1.3 0.2 rn~ EDTA 0.9 double-labeled material was converted to ["C]GpC plus "P-labeled inorganic phosphate (Fig. 1OA). In addition, treatment of the double-labeled ppGpC with P, nuclease yielded '"C-labeled pC and 3ZP-labeled ppG and pG, the latter possibly due to contaminating pyrophosphatase (Fig. 10B).

DISCUSSION
Reovirions consist of an outer protein shell enclosing an inner core which contains the segmented, double-stranded RNA genome. One strand ("minus") of each of the 10 duplex segments is transcribed by a core-associated RNA polymerase. Transcription is probably end to end since the resulting single-stranded ("plus") products function as precursors of double-stranded RNA as well as messenger for viral protein synthesis (44). Furthermore, the 5'.terminal sequences of the mRNA and the corresponding strand in genome RNA are the same: m'G(5')ppp (5' for 6 h at 45". After incubation, the mixture was analyzed by paper electrophoresis. B, adjacent fractions in A were combined in pairs and extracted to obtain the radioactive compounds. Aliquots (10% of each extract) were digested in 100 al of 10 mr+r Tris/HCl buffer with 0.5 unit of alkaline phosphatase at 37" for 2 h, and analyzed again by paper electrophoresis for ["C ]GpC with authentic marker GpC. The counts per min profile shows the yield of ["C]GpC from the fractions in A. C, an aliquot (50%) of the material from the region indicated by the bracket in A was applied to a column (0.6 x 25 cm) of Dowex l-X8 resin equilibrated with 0. 1 M HCl, and the chromatography was performed with a 0 to 0.4 M NaCl linear gradient in 0.01 M HCl (total = 400 ml). Fractions (2 ml) were collected and 0.1 ml of each was counted in Aquasol scintillant.