INTRODUCTION

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Bacteriophage 6, infecting the plant-pathogenic bacterium Pseudomonas syringae, is one of the best-characterized double-stranded RNA (dsRNA) viruses (15, 16). Its genome consists of three linear dsRNA segments: small (S), medium (M), and large (L). These are brought into the host cell inside a transcriptionally active virus core particle. In the course of transcription, plus-sense single-stranded RNAs (ssRNAs) s⁺, m⁺, and l⁺ are produced and extruded into the cytoplasm, where they are translated by the cellular protein synthesis machinery. Early 6 proteins P1, P2, P4, and P7 assemble into the polymerase complex, or procapsid (PC). This is followed by packaging of s⁺, m⁺, and l⁺ into the PC and their subsequent replication to form S, M, and L, respectively. We have previously demonstrated that purified P2 protein, the Pol subunit of the 6 PC, can act as a replicase and transcriptase in vitro (11, 13). Although it accepts RNA templates of both 6 and heterologous origin, 6 Pol shows a clear preference for templates containing 6-specific plus-strand initiation signals at their 3' ends. This suggests that the enzyme may play an active role in selection between plus- and minus-strand initiation signals, thus favoring the synthesis of plus strands during transcription in vivo (11).

6 has long been the only known species in the family Cystoviridae (20). Recently, the family has been updated with several newly isolated bacteriophages (17). Like 6, they contain tripartite dsRNA genomes and a lipid membrane as a structural element. Genomes of two of these bacteriophages, 8 and 13, have been sequenced completely, thus enabling their phylogenic analysis (8, 18). 13 shows limited sequence homology with 6, whereas 8 differs from 6 and 13 dramatically. At the amino acid level, the polymerase complex proteins P1, P2, P4, and P7 are homologous between 6 and 13. However, the only protein that shows detectable similarity across all three bacteriophages is the P2 polymerase, referred to here as Pol (Fig. 1).

View larger version (56K): [in this window] [in a new window]   FIG. 1.   Comparision of three recombinant Pol proteins (P2 proteins) from 6, 8, and 13. The protein sequences can be accessed at http://www.ncbi.nlm.nih.gov (phi6P2, AAA32355; phi8P2 AAF63300; phi13P2, AAG00444). Amino acid sequence alignment is based on structure alignment with ClustalW (http://www.ebi.ac.uk/clustalw/) and ESPript 1.9 (http://www-pgm1.ipbs.fr:8080/ESPript/). Strictly conserved residues are highlighted, and similar residues are boxed. Numbers correspond to the 6 Pol sequence. Secondary-structure elements are from the high-resolution crystal structure of 6 Pol (3), courtesy of S. J. Butcher.

Replication and transcription initiation sites vary considerably between 6, 8, and 13 (Fig. 2). It is obvious, however, that the 3' ends of both minus and plus strands, used in the initiation of plus- and minus-strand syntheses, respectively, are pyrimidine rich, with an invariable C at the 3'-most position of all minus strands. Within each bacteriophage genome, the 3'-proximal sequences of l, m, and s are generally more conserved than those of l⁺, m⁺, and s⁺. 13 is the best example of this asymmetry. Interestingly, the 3' end of l does not match the m/s consensus at one (6 and 13) or several (8) positions. In the case of 6, this single-nucleotide substitution is known to be the reason for the lower transcription efficiency of L compared to M or S (6, 11).

View larger version (73K): [in this window] [in a new window]   FIG. 2.   Terminal homologies among dsRNA genomic segments S, M, and L in 6, 8, and 13. Twenty nucleotides (each) of the left- and right-hand termini are shown. Boxed plus and minus signs represent the rest of the plus- and minus-strand sequences, respectively. Nucleotides conserved within each genome are highlighted. Positions of transcription (left-hand) and replication (right-hand) initiation sites are indicated.

Since 6 Pol has been suggested to control the bacteriophage RNA metabolism through selective initiation at the transcriptional promoter, the question arises whether a similar regulatory function could also be assigned to the polymerases of 8 and 13. To this end, we purified the two recombinant polymerase subunits and assayed their activities in vitro. Like their 6 counterpart, both polymerases catalyze RNA-dependent RNA synthesis in vitro. However, they differ from each other and from 6 Pol in several biochemical properties. Most interestingly, each enzyme has a distinct template specificity, with a clear bias for ssRNAs containing 3' ends similar to those found in bacteriophage-specific minus strands. These data suggest a possible scenario in which polymerase subunit specificity has coevolved with the minus-strand 3'-proximal sequence to ensure efficient transcription initiation. On the other hand, the results with genomic RNAs indicate that some additional factors, such as the melting temperature of the initiation site, are likely to be involved in the regulation of RNA synthesis from dsRNA templates.

	MATERIALS AND METHODS

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Plasmids and strains. 6 polymerase was produced using Escherichia coli strain BL21(DE3/pEM2) (13). The 8 Pol expression strain was prepared as follows. The p2 gene of 8 was first PCR amplified from plasmid pLM2424 (8) using the primer pair p2phi8up and p2phi8down primer pair (Table 1) and Pfu DNA polymerase (Stratagene). The PCR fragment was digested with NdeI and BamHI (underlined sites in primer sequences) and ligated with the similarly cut vector pMG60, a derivative of pMG59 (7). E. coli BL21 was then transformed with the resultant plasmid pHY1 to produce BL21(pHY1). To construct the 13 Pol expression strain, the p2 gene of 13 was amplified from pLM2200 (18) using primers p2phi13up and p2phi13down. The PCR fragment was inserted into pET32b(+) (Novagen) at NdeI-HindIII sites to produce plasmid pHY2. This was introduced into BL21(DE3) (Novagen), yielding BL21(DE3/pHY2).

Expression and purification of recombinant polymerase subunits. Recombinant polymerases from 8 and 13 were expressed by the procedure described for 6 Pol (4, 13). Briefly, starter cultures were grown in Luria-Bertani medium containing 150 mg of ampicillin/ml at 37°C to an optical density at 540 nm (OD₅₄₀) of 0.6. Cultures were then diluted 50-fold, and incubation was continued until the OD₅₄₀ reached 1.0. Expression of the recombinant polymerase was induced with 1 mM isopropyl--D-thiogalactopyranoside (IPTG). After addition of IPTG, bacterial cultures were shaken for 15 h at 23°C [for BL21(pHY1)] or 18 h at 19°C [for BL21(DE3/pHY2)]. Protein purification was carried out at +4°C. Throughout purification, the pH value was 7.4 for 8 Pol and 8.0 for 13 Pol. Bacterial pellets were resuspended in 35 ml of 100 mM NaCl-50 mM Tris-HCl-1 mM EDTA. Suspensions were passed three times through a precooled French pressure cell at ~105 MPa. Phenylmethylsulfonyl fluoride was added to 1 mM after the first passage. Lysates were cleared at 120,000 × g for 2.5 h. 6 Pol and 13 Pol were purified successively on Cibacron Blue 3GA agarose (Sigma), heparin agarose (Sigma), and a HiTrap Q Sepharose column (Pharmacia) as described elsewhere (13). For the 8 Pol purification, Reactive Brown 10 agarose (Sigma) and a Superdex 75 gel filtration column (Pharmacia) were used. Protein concentrations were determined by absorbance at 280 nm in 6 M guanidine hydrochloride based on an optical density (OD) of 1.21 OU for 1 mg of 8 Pol/ml and an OD of 1.37 for 1 mg of 13 Pol/ml (5). Purified proteins were stored at +4°C.

Preparation of RNA substrates. ssRNA substrates were produced by in vitro transcription with T7 RNA polymerase as described previously (13, 14). Templates for the transcription were prepared either by cutting recombinant plasmid DNA with restriction endonucleases or by PCR amplification with Pfu DNA polymerase. For PCR, oligonucleotide T7-1, containing the T7 polymerase promoter sequence, was used as an upstream primer. Oligonucleotides 3'end to 3'end4 were used as downstream primers for amplification of the s⁺ fragment from pEM15 (11), whereas pT7-3'end to pT7-3'end14 served as downstream primers to amplify the luciferase gene from pT7luc (9). Genomic dsRNA was extracted from purified bacteriophage particles with phenol-chloroform, precipitated with ethanol, and dissolved in sterile water.

Polymerase activity assay. Polymerase activity was assayed in 10-µl reaction mixtures. In the initial experiments, we used the conditions reported previously for the 6 polymerase (11, 13). These were further optimized to 50 mM HEPES-KOH (pH 7.4 to 7.8; see Results for details), 20 mM ammonium acetate (NH₄OAc), 6% (wt/vol) polyethylene glycol 4000, 5 mM MgCl₂, 2 mM MnCl₂, 0.1 mM EDTA, 0.1% Triton X-100, 1 mM (each) ATP and GTP, 0.2 mM (each) CTP and UTP, 0.8 U of RNasin/µl, and 0.25 mCi of [-³²P]UTP (Amersham; 3,000 Ci/mmol)/ml. The final concentration of RNA substrates was 50 to 200 µg/ml. Reactions were started by adding one of the three polymerases up to 0.02 to 0.04 mg/ml, and reaction mixtures were further incubated at 30°C for 1 h. Reaction products were separated by standard agarose gel electrophoresis (13). Gels were dried and exposed to Fuji Super RX film or analyzed with a Fuji BAS1500 phosphorimager.

	RESULTS

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Expression and purification of recombinant Pol proteins. To construct plasmids for 8 Pol and 13 Pol expression, full-length polymerase genes were cloned under the control of inducible promoters and strong Shine-Dalgarno sequences. Soluble polymerases from bacteriophages 6, 8, and 13 were expressed by incubating the corresponding E. coli strains at 16 to 23°C in the presence of IPTG. Expression at higher temperatures (28 to 37°C) led to substantial increases in overall polymerase production, but most of the protein was insoluble. The 6 Pol purification protocol, employing a combination of Cibacron Blue 3GA agarose and heparin agarose and an anion-exchange column, gave satisfactory results for 13 Pol (Fig. 3A). However, 8 Pol failed to bind to the blue agarose, leading us to test other dye affinity resins (Sigma kit no. RDL-9). Reactive Brown 10 agarose bound 8 Pol well, without retarding most of the contaminating proteins (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1 mM EDTA). Bound 8 Pol was eluted with 50 mM Tris-HCl (pH 7.4)-500 mM NaCl- 1 mM EDTA and was further purified to near-homogeneity using a Superdex 75 gel filtration column equilibrated with 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 0.1 mM EDTA (Fig. 3A). The relative electrophoretic mobilities of the three purified enzymes correlated with their calculated molecular masses (Table 2): 13 Pol is larger, and 8 Pol is smaller, than 6 Pol. The yields of purified proteins in milligrams per liter of bacterial culture are given in Table 2.

View larger version (37K): [in this window] [in a new window]   FIG. 3.   Purified polymerase subunits catalyze RNA replication in vitro. (A) SDS-PAGE analysis of purified recombinant Pol proteins from 6, 8, and 13. Lane Mk, protein marker. Molecular masses (in kilodaltons) are given on the right. (B) Agarose electrophoresis of reaction mixtures containing s+13 RNA and one of the three purified polymerases: 6 Pol, 8 Pol, or 13 Pol. Lane C, control without polymerase. The upper panel shows an ethidium bromide-stained gel, with the ssRNA (ss) and dsRNA (ds) marked on the right; the lower panel is the corresponding autoradiogram.

View this table: [in this window] [in a new window]   TABLE 2.   Properties of the Pol subunits from 6, 8, and 13

Effects of reaction conditions on the polymerase activity. The above assay was utilized to study the effects of several parameters on the activities of the three polymerases. All three enzymes synthesized dsRNA over a wide pH range, with the Tris-HCl pH optima increasing in the order 13 Pol-6 Pol-8 Pol. Interestingly, the enzyme activity in HEPES-KOH was always higher than that in Tris-HCl at the same pH (Fig. 4A, D, and G).

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Effects of pH (A, D, and G) and of ammonium acetate (B, E, and H) and Mn2+ (C, F, and I) concentrations on the activities of the three polymerases. Reaction mixtures containing s+13 RNA and 6 Pol (A, B, and C), 8 Pol (D, E, and F) or 13 Pol (G, H, and I) were incubated at 30°C for 1 h and analyzed by agarose gel electrophoresis. Radioactivity in the bands of the newly produced dsRNA was quantified with a phosphorimager. Tris-HCl (Tris) or HEPES-KOH (HEPES) buffers were used in panelsA, D, and G, whereas in the other panels pH was buffered with HEPES-KOH (pH 7.8 for 6 Pol and 8 Pol, and pH 7.4 for 13 Pol). Graphs are normalized so that the highest observed value within each panel is set to 1.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Effects of temperature on replication. Replication mixtures containing s+13 RNA and 6 Pol (A), 8 Pol (B), or 13 Pol (C) were incubated at 20 to 60°C, as indicated. Aliquots were withdrawn at different time points and analyzed by agarose gel-electrophoresis followed by phosphorimager analysis. The highest observed value within each panel is set to 1.

8 and 13 polymerases catalyze dsRNA transcription in vitro. In addition to its replication activity, purified 6 Pol can catalyze dsRNA transcription in vitro employing a semiconservative (strand displacement) mechanism (11). To test the transcriptional activities of 8 Pol and 13 Pol, the two polymerases, along with 6 Pol, were assayed with genomic dsRNAs extracted from bacteriophage particles. In all three cases, radioactive products that migrated at the position of the input dsRNA species were found, consistent with the idea of semiconservative transcription (Fig. 6). However, the product patterns varied significantly depending on the polymerase and the dsRNA source.

View larger version (50K): [in this window] [in a new window]   FIG. 6.   Polymerase subunits from 6, 8, and 13 catalyze dsRNA transcription in vitro. Reaction mixtures contained 6 Pol (lanes 1 to 3), 8 Pol (lanes 4 to 6) or 13 Pol (lanes 7 to 9) and dsRNA extracted from 6 (lanes 1, 4, and 7), 8 (lanes 2, 5, and 8) or 13 (lanes 3, 6, and 9). The upper panel shows an ethidium bromide-stained gel, and the lower panel is the corresponding autoradiogram. The positions of dsRNA segments L, M, and S are marked on the right.

Effect of the template 3' end on the efficiency of RNA synthesis. To further explore the template preferences of 6 Pol, 8 Pol, and 13 Pol, we compared replication efficiencies with five variants of s⁺ ssRNA (6 s⁺ segment with an extensive internal deletion [11]). One of the variants, s⁺(), had the natural 6 terminus CUCUCUCUCU3' (also present in the 13 m⁺; see Fig. 2); the other four templates contained different one-nucleotide additions: A3', C3', G3', and U3'. For all three polymerases, replication efficiency increased considerably when C3' was used as a terminal template nucleotide. G3' was the second-best addition (Fig. 7, s⁺). The effects of the other two terminal bases were polymerase dependent. Both A3' and U3' reduced 6 Pol replication, whereas for 13 Pol they were neutral or somewhat stimulatory. 8 Pol was substantially inhibited by A3' and was activated by U3'. A similar result was also obtained for the set of five firefly luciferase mRNAs: luc(), containing CCCAAGCUUA3', and its one-nucleotide-longer derivatives luc(A3'), luc(C3'), luc(G3'), and luc(U3'). However, in this case, U3' enhanced 8 Pol replication by an order of magnitude (Fig. 7, luc).

View larger version (12K): [in this window] [in a new window]   FIG. 7.   Effect of the 3'-terminal nucleotide of the template on replication efficiency. Two sets of ssRNA templates, luc and s+, were assayed with 6 Pol, 8 Pol, or 13 Pol at 30°C for 1 h. Each of the sets contained five RNA species with different 3' ends (N3'): either without modifications () or extended with one additional 3'-terminal nucleotide (U, G, C or A). Reaction products were separated by agarose gel-electrophoresis, and radioactivity in the dsRNA product bands was quantified with a phosphorimager. The graphs are normalized so that the highest value within each panel is set to 1.

View larger version (11K): [in this window] [in a new window]   FIG. 8.   Replication efficiency of luc ssRNAs containing different 3'-terminal extensions. Reaction mixtures containing one of the seven luciferase ssRNA templates with the different 3' extensions and 6 Pol, 8 Pol, or 13 Pol were incubated at 30°C for 1 h and analyzed as for Fig. 7. The highest value within each panel is set to 1.

	DISCUSSION

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Previous studies have shown that the polymerase subunit of bacteriophage 6 can catalyze RNA synthesis without the assistance of other proteins (11, 13). In addition, the high-resolution crystal structures of the 6 Pol apoenzyme, as well as its template and NTP complexes, have recently been solved (3). We now demonstrate RNA-dependent RNA polymerization activity of two polymerase subunits isolated from the 6-related bacteriophages 8 and 13.

The three polymerases are homologous to each other, with an overall amino acid identity of 11% and a mean similarity of 41% (Fig. 1). Pairwise sequence alignments reveal ~50 and 20% identity of 6 Pol to 13 Pol and 8 Pol, respectively (8, 18). All three proteins contain the (G/S)DD motif (Fig. 1, turn between 15 and 16) critical for catalysis in all RNA-dependent RNA polymerases (10). Also strictly conserved is the positively charged RRRTA sequence (11), which has been shown to interact with the phosphate groups of the incoming NTP substrate in 6 Pol (3). We also notice the conservation in the C-terminal domain (23 to 24), implicated in stabilizing the initiation complex via stacking interactions with the first two nucleotides of the nascent strand (3). More-reliable molecular comparisons, however, require knowledge of the atomic structures for 8 Pol and 13 Pol. Crystallization experiments are under way.

The polymerases are shown to catalyze replication and transcription in vitro using, respectively, ssRNA and dsRNA templates (Fig. 3 and 6). Replication is at least 1 order of magnitude more efficient than transcription for all three enzymes (data not shown), in agreement with earlier data on 6 Pol (11).

The three enzymes can be distinguished biochemically. In Tris-HCl buffer, 6 Pol is the most active at pH 8.9, with 13 Pol having a lower and 8 Pol a higher pH optimum (Fig. 4A, D, G). This distribution is mirrored by the predicted isoelectric points (Table 2) and is likely to be a function of protein surface charge at different pH values. When Mn²⁺ is added to a reaction mixture containing an excess of Mg²⁺, it increases the activities of all three enzymes, albeit to different extents (Fig. 4C, F, and I). Manganese has been reported to affect the activities of several RNA-dependent RNA polymerases, including those from 6 (11, 13), hepatitis C virus (1, 21), brome mosaic virus (19), bacteriophage Q (2), and some other viruses. Recent structural data for 6 Pol suggest that Mn²⁺ could serve to stabilize the polymerase molecule in a compact initiatory conformation (3). Intriguingly, 8 Pol and 13 Pol are stimulated by Mn²⁺ to a lesser extent than 6 Pol and, at the same time, show a higher thermostability (Fig. 5).

Most importantly, the purified enzymes have different template specificities, for both ssRNAs and dsRNAs (Fig. 6 to 8). Several generalizations are appropriate in this respect. Both 6 Pol and 13 Pol prefer ssRNA templates with the 3'-terminal cytosine; CC3' and CCC3' are even better initiation signals (Fig. 7 and 8). While 8 Pol is also increasingly stimulated with C3', CC3', and CCC3', it initiates more efficiently at the termini containing a 3'-proximal uridine(s), such as GAAAAUUUC 3', GUUUUUUCC 3', UC 3', or U 3'. This is further corroborated by the observation that s⁺13 RNA (ending in ... CCCC 3') is a better template for 6 Pol and 13 Pol than for 8 Pol (Fig. 3B). Although 13 Pol shows a somewhat higher affinity for the U-containing 3'-terminal extensions than 6 Pol (Fig. 7 and 8), the two polymerases are obviously closer to each other than to 8 Pol in terms of their template specificities, exactly as one would expect from the sequence comparison.

The polymerases from the Cystoviridae do not require a primer to commence RNA synthesis (12, 13). As shown for 6 Pol, the polymerase forms a quaternary initiation complex where the template 3' terminus is engaged in Watson-Crick base pairing with two cognate NTPs (3). The NTPs are additionally stabilized by stacking interactions to each other and to a special C-terminal "platform" in the polymerase. Since purine bases possess a very high stacking propensity, it is not surprising that the cystoviral polymerases show a bias for pyrimidine-rich template termini. In fact, many other RNA- and DNA-dependent RNA polymerases also initiate de novo and prefer purine nucleotides at the 5' end of the nascent RNA, thus indicating that all these enzymes might utilize protein-NTP stacking to facilitate the initiation step.

There is a clear correlation between the terminal preferences of the polymerase subunits and the transcription initiation sequences from the corresponding bacteriophages (Fig. 2). Indeed, all three 8 minus strands end with UC 3', which is shifted to (C/U)C 3' in 13 and (C/A)C 3' in 6, consistent with the Pol specificity profiles. As the transcription in dsRNA viruses is asymmetric, we suggest that the polymerase subunit and the plus sense initiation sites have become mutually fit as a result of coevolution. The replication promoters seem to experience a lower evolutionary pressure, consistent with weaker conservation of the right-hand termini in 8 and 13 (Fig. 2). Indeed, both 6 Pol and 13 Pol prefer the minus-strand 3'-terminal sequence to that of the plus strands (compare GUUUUUUCC 3' and UCUCUCUCU 3' in Fig. 8).

In addition to the 3'-terminal preferences of the polymerase subunits, plus-strand synthesis in the Cystoviridae is also likely to be controlled by the melting temperature of the dsRNA termini. The GC content of the transcription initiation sites is noticeably lower than that of the replication promoters in all three bacteriophages (Fig. 2), which should facilitate access of the polymerase to the 3' termini of the minus strands. This is consistent with the fact that the template tunnel inside the polymerase molecule can accommodate only ssRNA, not dsRNA (3). The efficiency of the dsRNA terminal unzipping could explain, for example, why minus strands of 6 and 13 do not end in CCC 3', although it is preferred by 6 Pol and 13 Pol for ssRNA (Fig. 8). Furthermore, different accessibilities of initiation sites in a single-stranded form might explain the results shown in Fig. 6, where 8 Pol utilized the 13 S segment more efficiently than homologous templates, and 13 Pol preferred 13 L to M and S (Fig. 6).

In summary, biochemical comparison of three Pol subunits from Cystoviridae reveals, in addition to the expected similarity, a substantial degree of functional divergence. Further experiments with the purified polymerases will address the molecular basis for the difference in template specificity and will provide more-detailed insights into the regulation of the viral RNA metabolism.