Poliovirus cis-Acting Replication Element-Dependent VPg Uridylylation Lowers the K_m of the Initiating Nucleoside Triphosphate for Viral RNA Replication

pRNA2 was kindly provided by James B. Flanegan (University of Florida College of Medicine, Gainesville, FL). pRNA2 encodes a PV subgenomic RNA replicon containing an in-frame deletion of nucleotides (nt) 1175 to 2956 within the capsid genes (11). T7 transcription of MluI-linearized pRNA2 cDNA produces PV RNA2 replicon RNA with two nonviral guanosine residues at its 5′ terminus that prevent positive-strand RNA synthesis (10).

pDNVR27 is identical to pRNA2 except for a 5′-terminal hammerhead ribozyme, such that T7 transcription and ribozyme cleavage produce a PV RNA2 replicon possessing an authentic PV 5′ terminus (29).

pRibo knockout (KO) mutant CRE and pKO CRE are identical to pRNA2 and pDNVR27, respectively, except for eight silent mutations that disrupt the CRE RNA structure. These plasmids were described previously as pDNVR26 and pDNVR28 (29).

HeLa cell S10 extracts (S10) and HeLa cell translation initiation factors were prepared as previously described (9). Reaction mixtures contained 50% (by volume) S10, 20% (by volume) translation initiation factors, 10% (by volume) 10× nucleotide reaction mix (10 mM ATP, 2.5 mM GTP, and 2.5 mM CTP; 600 mM KCH₃CO₂; 300 mM creatine phosphate; 4 mg per ml of creatine kinase; and 155 mM HEPES-KOH [pH 7.4]), and T7 transcripts of PV replicon RNA at 45 μg/ml. Preinitiation RNA replication complexes (PIRCs) formed during 3 h of PV RNA translation at 34°C in the presence of 2 mM guanidine HCl as previously described (7). Exogenous UTP was intentionally omitted from the translation-replication reaction (when PIRCs were forming in the presence of 2 mM guanidine) to reduce the amounts of residual UTP in isolated PIRCs.

Translation of PV replicon mRNA was monitored by including [³⁵S]methionine (1.2 mCi/ml) (Amersham) in HeLa S10 translation-replication reaction mixtures. Magnitudes of protein synthesis were measured by the incorporation of [³⁵S]methionine into acid-precipitable material and/or gel electrophoresis. [³⁵S]methionine-labeled proteins were solubilized in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer (2% SDS [Sigma], 62.5 mM Tris-HCl [pH 6.8], 0.5% 2-mercaptoethanol, 0.1% bromophenol blue, 20% glycerol). The samples were heated at 100°C for 5 min and separated by gel electrophoresis in SDS 9 to 18% polyacrylamide gels. [³⁵S]methionine-labeled proteins were detected by phosphorimaging (Bio-Rad).

PV RNA replication and VPg uridylylation were assayed using PIRCs containing PV RNAs as described previously (29). PIRCs were isolated from HeLa S10 translation-replication reaction mixtures by centrifugation at 17,000 × g for 15 min at 4°C and resuspended in [α-³²P]NTP-labeling reaction mix containing 27 mM HEPES-KOH [pH 7.4], 60 mM KCH₃CO₂, 2.3 mM Mg(CH₃CO₂)₂, 2.6 mM dithiothreitol, 2.3 mM KCl, 50 μg/ml puromycin, 1 mM ATP, 250 μM GTP, and the indicated concentrations of CTP and UTP, with or without guanidine HCl, as indicated in the figure legends. [α-³²P]NTPs (1 μCi per 1-μl reaction mixture; 1.25 μM radiolabeled NTP) (MP Biomedicals) were included to radiolabel viral RNA and VPgpUpU_OH. Reaction mixtures were incubated at 37°C using continuous labeling for the indicated periods of time as described in figure legends.

Replication complexes containing radiolabeled viral RNAs were reisolated by centrifugation at 17,000 × g for 15 min at 4°C. This allowed unincorporated radiolabel and other contaminating materials in the supernatant fractions to be discarded. Pelleted replication complexes containing radiolabeled PV RNA products of RNA replication were solubilized in 0.5% SDS buffer (0.5% SDS, 10 mM Tris HCl [pH 7.5],1 mM EDTA, 100 mM NaCl). Radiolabeled RNA products were phenol-chloroform extracted, ethanol precipitated, and separated by gel electrophoresis on a nondenaturing 1% agarose-Tris-borate-EDTA (TBE) gel. Radiolabeled RNAs within the gels were detected and quantified by phosphorimaging.

Radiolabeled VPgpUpU_OH was solubilized in Tris-Tricine sample buffer (3% SDS, 62.5 mM Tris-hydrochloride [pH 6.8], 5% β-mercaptoethanol, 10% glycerol, and 0.1% bromophenol blue) and fractionated by electrophoresis (70 mA of constant current for 15 min followed by 17 mA of constant current for 12 h) in a 0.75-mm-thick polyacrylamide (a 29:1 ratio of acrylamide to bis-acrylamide)-Tris-Tricine gel system (a 4% polyacrylamide stacking gel [4% polyacrylamide, 1.0 M Tris, pH 8.45, and 0.1% SDS] and a 12% polyacrylamide separating gel [1.0 M Tris, pH 8.45, and 0.1% SDS]) by using a Tris-Tricine running buffer (0.1 M Tris-Tricine [pH 8.25], 0.1% SDS). Gels were fixed in 50% trichloroacetic acid and then dried. Radiolabeled VPgpUpU_OH in the gel was detected by phosphorimaging.

Radiolabeled RNAs were resuspended in 10 μl methylmercury hydroxide (MeHgOH) sample buffer (50 mM boric acid, 5 mM sodium borate, and 10 mM sodium sulfate). MeHgOH was not added to the sample until a 1-μl portion of the sample was placed in 5 ml of trichloroacetic acid solution (428 mM trichloroacetic acid, 44.8 mM sodium pyrophosphate) to acid precipitate the labeled RNA products, which were then collected on nitrocellulose filters and quantified by scintillation counting. One microliter of 0.5 M MeHgOH was then added to the remaining 9 μl of the RNA sample and incubated at room temperature for 10 min to denature the double-stranded RNAs. The MeHgOH in the samples was then inactivated by adding 1 μl 1.4 M β-mercaptoethanol. One microliter of RNase T1 (300 U/μl) was then added to each reaction mixture and they were incubated at 37°C for 30 min. When indicated, proteinase K (200 μg/ml) was added to reaction mixtures, which were incubated at 37°C for another 30 min to remove VPg from the VPg-linked poly(U) products of RNA replication. Reaction mixtures were terminated by the addition of an equal volume of 2× urea sample buffer (18 M urea, 8.9 mM Tris base, 8.9 mM boric acid [pH 8.3], 0.2 mM EDTA, 20% [wt/vol] sucrose, 0.05% [wt/vol] bromophenol blue, 0.05% xylene cyanol). Radiolabeled T1 oligonucleotides in the urea sample buffer were denatured at 100°C for 3 min and separated by electrophoresis in 7 M urea-18% polyacrylamide gels in TBE buffer (89 mM Tris base, 89 mM boric acid, pH 8.3, 2 mM EDTA) for 5 h at 25 W. Radiolabeled RNAs within the gels were detected and quantified by phosphorimaging (Bio-Rad).

Mutations were used to disrupt the integrity of the CRE RNA structure within a PV RNA replicon without affecting the amino acid sequence in this region of the open reading frame (Fig. 1A). As shown previously (29; see Fig. 3), these mutations completely abrogated CRE-dependent VPg uridylylation within PIRCs.

To examine nascent negative-strand RNA synthesis, we incubated PIRCs containing defined PV RNA templates (PV RNA, WT CRE, or KO CRE) (Fig. 1A) for increasing periods of time in [α-³²P]UTP labeling reaction mix containing 1 mM ATP, 250 μM GTP, 250 μM CTP, and 10 μM UTP. RNA products from the reaction mixtures were separated by electrophoresis and detected by ethidium bromide staining (Fig. 1B) and by phosphorimaging (Fig. 1C). Template RNA along with residual 28S and 18S rRNAs was detected by ethidium bromide staining and UV light (Fig. 1B), providing evidence that relatively equal amounts of reaction product were recovered and loaded in each lane. Nascent negative-strand RNAs that were less than full-length were evident in reaction mixtures containing the WT RNA replicon after 8, 12, and 16 min of incubation, with full-length negative strands accumulating within replicative form (RF) RNA by 20 min of incubation (Fig. 1C, lanes 1 to 5). RF RNA consists of full-length negative-strand RNA hybridized to positive-strand RNA templates. Nascent negative-strand RNAs that were less than full-length remained hybridized to PV RNA templates and migrated between the single-stranded PV RNA templates and the mature double-stranded RF RNA in the agarose gel (Fig. 1C, lanes 2 to 4). The burst of RF RNA accumulation observed between the 16- and 20-min time points was not unexpected because this corresponds to the time it takes for 3D^Pol to elongate across the templates under the conditions of the experiment. RF RNA has more UMP residues per molecule than nascent negative-strand RNAs, and the accumulating RF RNA also migrates more discretely in the agarose gel than nascent negative-strand RNAs of variable length. The apparent rate of negative-strand RNA elongation with this relatively low UTP concentration was ∼285 nt per minute, comparable to that previously reported (7). Maximal 3D^Pol elongation rates of ∼1,200 nt per minute are achieved when normal concentrations of each NTP are used in reactions (≥200 μM) (7, 49). Guanidine HCl (2 mM) inhibited PV negative-strand RNA synthesis (Fig. 1C, lane 6); however, guanidine did not affect the incorporation of radiolabel into ribosomal RNAs (Fig. 1C, lanes 6 to 12). The reason(s) for incorporation of radiolabel into rRNAs is unknown to the authors. Mutations that disrupted the CRE RNA structure potently inhibited negative-strand RNA synthesis (Fig. 1C, lanes 7 to 11). This result was not congruent with previously published data using high-specific-activity [³²P]CTP labeling conditions (1 mM ATP, 250 μM GTP, 250 μM UTP, and 10 μM CTP), where CRE was found to be unnecessary for negative-strand RNA synthesis (28, 29).

Because the result shown in Fig. 1 was discordant with previous data using [³²P]CTP labeling conditions (29), we examined the effects of various UTP and CTP concentrations on negative-strand RNA synthesis (Fig. 2). When increasing concentrations of unlabeled UTP from 10 to 250 μM were used to dilute the specific activity of [³²P]UTP in the labeling reaction mixture, the incorporation of [³²P]UMP into negative-strand RNA within RF RNA on WT templates decreased (Fig. 2A, lanes 7, 9, and 11). Notably, robust amounts of [³²P]UMP were incorporated into the negative strand of RF RNA in reactions containing the WT template at the highest specific activity of [³²P]UTP (10 μM UTP) (Fig. 2A, lane 7). In contrast, only small amounts of [³²P]UMP were incorporated into RF RNA when PIRCs containing KO CRE templates were incubated in labeling reaction mixtures containing 10 μM UTP (Fig. 2A, lane 1). Significantly, when higher concentrations of UTP were included in the labeling reaction, the KO CRE RNA functioned efficiently as a template for negative-strand RNA synthesis (Fig. 2A, compare amounts of RF RNA in lanes 3 and 5 with amounts of RF RNA from wild-type RNA templates in lanes 9 and 11). A very small amount of positive-strand RNA was made from the PV RNA template despite the presence of two nonviral G residues on the template RNA used in this experiment (Fig. 2A, lanes 9 and 11), whereas no positive-strand RNA was made from the KO CRE template (Fig. 2A, lanes 1 to 6). In contrast to the results using low concentrations of UTP described above, and consistent with our previous report (29), our results showed that low concentrations of CTP did not diminish the magnitude of negative-strand RNA synthesis from KO CRE templates (Fig. 2B, lane 1). The data in Fig. 1 and 2 indicate that negative-strand RNA synthesis on KO CRE templates was uniquely sensitive to low UTP concentrations.

In order to examine the coordinate effects of CRE mutations and alternate NTP concentrations on both negative- and positive-strand RNA replication, we used templates competent for negative-strand RNA synthesis alone or competent for both negative- and positive-strand RNA replication (Fig. 3). As previously reported (10, 19, 29), two nonviral G residues at the 5′ end of PV RNA2 templates prevent positive-strand RNA synthesis (Fig. 3B and C, lanes 2 and 7), whereas a ribozyme-processed version of PV RNA2 functions as a template for both negative- and positive-strand RNA replication (Fig. 3B and C, lanes 5 and 10). When CRE was intact in these templates, VPg uridylylation was readily detected by the incorporation of [³²P]UMP into VPgpUpU_OH (Fig. 3A, lanes 1 and 7). Guanidine HCl (2 mM), as previously reported (29), inhibited CRE-dependent VPg uridylylation within PIRCs (Fig. 3A, lanes 2 and 8). Likewise, mutations that completely disrupted the CRE RNA structure prevented any detectable VPg uridylylation (Fig. 3A, lanes 3 and 5). VPgpUpU_OH was not detected in reaction mixtures containing CRE KO templates, despite the presence of 3′ poly(A) sequences on the PV RNA templates (Fig. 3A, lanes 3 and 5). Thus, as shown here (Fig. 3A, lanes 3 and 5) and elsewhere (28, 29), the 3′ poly(A) tail of PV RNA, which was originally considered a potential template for VPgpUpU_OH synthesis (37), does not function as a template for VPgpUpU_OH synthesis within PIRCs.

RNA replication was measured by [³²P]CTP incorporation in reaction mixtures containing 10 μM UTP (Fig. 3B, lanes 1 to 5) or 500 μM UTP (Fig. 3B, lanes 6 to 10). In addition, RNA replication was measured by [³²P]GTP incorporation in reaction mixtures containing 10 μM CTP (Fig. 3C, lanes 1 to 5) or 250 μM CTP (Fig. 3C, lanes 6 to 10). These conditions maintained a constant specific activity of radiolabel (in contrast to the changing specific activity conditions shown in Fig. 2), allowing for a quantitative comparison of negative-strand RNA synthesis under each reaction condition. KO CRE mutations that prevented VPg uridylylation prevented positive-strand RNA synthesis but did not prevent negative-strand RNA synthesis (Fig. 3B, compare lanes 9 and 10; Fig. 3C, compare lanes 4 and 5 to lanes 9 and 10); however, when UTP concentrations were low, CRE-independent, VPg-primed, negative-strand RNA synthesis was notably less than negative-strand RNA synthesis by templates with intact CRE elements (Fig. 3B, compare RF RNAs in lanes 3 and 4 with RF RNAs in lanes 2 and 5). The relative magnitudes of negative-strand RNA synthesis were not different in reaction mixtures containing either 10 μM or 250 μM CTP (Fig. 3C). Thus, the amounts of CRE-independent negative-strand RNA synthesis were reduced in reaction mixtures containing low UTP concentrations but were not reduced in reaction mixtures containing low CTP or GTP concentrations. Therefore, CRE-dependent VPg uridylylation appeared to be required for at least a portion of negative-strand RNA synthesis, especially at low UTP concentrations.

To measure the K_m of UTP required for negative-strand RNA synthesis, we incubated WT and KO CRE PIRCs in labeling reaction mixtures containing increasing concentrations of exogenous UTP and measured the incorporation of [³²P]CTP into negative-strand RNA (Fig. 4). Ethidium bromide staining and UV light revealed the mobilities of PV RNA, 28S rRNA, and 18S rRNA and demonstrated relatively equal sample recovery between experimental samples (Fig. 4A). As previously established, guanidine HCl inhibited negative-strand RNA synthesis (Fig. 4B, lanes 9 and 18). Notably, negative-strand RNA was synthesized in the absence of exogenous UTP (Fig. 4B, lanes 1 and 10). The synthesis of negative-strand RNA in the absence of exogenous UTP indicated that residual amounts of endogenous UTP from cytoplasmic extracts used to make PIRCs were sufficient to support full-length negative-strand RNA synthesis during the 60-min incubation period (Fig. 4B, lanes 1 and 10). Small but unmeasurable amounts of residual NTPs from the cytoplasmic extracts used to form PIRCs confound precise K_m measurements. Therefore, because the amounts of endogenous NTPs are unmeasurable, the K_ms derived from these reaction mixtures are apparent K_ms based on the amounts of exogenous NTPs added to the reaction mixtures. Half-maximal amounts of negative-strand RNA were made in reaction mixtures containing 12 μM UTP when CRE was intact (Fig. 4B, lanes 1 to 8, and 4C), whereas half-maximal amounts of negative-strand RNA were made in reaction mixtures containing 103 μM UTP when CRE was disrupted by mutations (Fig. 4B, lanes 10 to 17, and 4C). For the determination of K_ms, the values on the y axis should represent initial velocities. This would require that the rates of negative-strand RNA synthesis be linear for the 60-min duration of the reaction. We find that negative-strand RNA replication continuously initiates at the same rate for 60 min or longer after the removal of guanidine from the population of RNA replication complexes within the reactions (B. P. Steil and D. J. Barton, unpublished data). Consistent with modest deficits in the magnitude of negative-strand RNA synthesis for KO CRE RNA, the V_max of negative-strand RNA synthesis was 2.3-fold lower for KO CRE RNA than for WT CRE RNA (Fig. 4B and C). Thus, CRE-dependent VPg uridylylation dramatically lowered the concentration of UTP required for efficient negative-strand RNA synthesis.

VPg and VPgpUpU_OH are predicted to prime the initiation of RNA replication along the 3′ poly(A) tail of PV RNA templates, leading to the synthesis of VPg-linked poly(U) at the 5′ end of negative-strand RNA (43, 52). RNase T1 cleaves on the 3′ side of single-stranded RNA at guanosine residues and is useful in the determination of the length of homopolymeric RNA sequences in viral RNA (2). Therefore, we used RNase T1 and urea polyacrylamide gel electrophoresis to determine whether VPg-linked poly(U) was present in the radiolabeled negative-strand RNA products synthesized under CRE-dependent (VPgpUpU_OH priming) and CRE-independent (VPg priming) reaction conditions (Fig. 5). [α-³²P]UTP was incorporated into PV negative-strand RNA, as it was synthesized within PV RNA replication complexes containing WT RNA and CRE KO RNA templates (Fig. 5A, lanes 1 and 3). Smaller amounts of radiolabel were incorporated into RF RNA products in reaction mixtures containing the CRE KO templates due to the limiting UTP (10 μM) in the reaction mixtures (Fig. 5A), as expected (Fig. 4). Guanidine prevented the incorporation of radiolabel into RF RNA (Fig. 5A, lanes 2 and 4).

Because the pattern of T1 oligonucleotides in PV positive-strand RNA is different from the pattern of T1 oligonucleotides in negative-strand RNA, RNase T1 can be used to distinguish PV positive-strand RNA from negative-strand RNA (7). When [α-³²P]ATP-labeled PV positive-strand RNA templates were digested with RNase T1 and the RNA fragments were separated by electrophoresis in 7 M urea polyacrylamide gels, the predicted T1 oligonucleotides were evident (Fig. 5B, lanes 1 and 2). The largest T1 oligonucleotides within the positive strand of PV RNA include a 37-mer, a 36-mer, and a 31-mer within the heteropolymeric portion of PV RNA as well as the 3′ terminal poly(A)₈₄ tail (Fig. 5B, lanes 1 and 2). When [α-³²P]UTP-labeled PV negative-strand RNA products were digested with RNase T1 and the RNA fragments were separated by electrophoresis into 7 M urea polyacrylamide gels, the predicted negative-strand-specific T1 oligonucleotides were evident (Fig. 5B, lane 3). Radiolabeled VPg-linked poly(U) T1 oligonucleotides migrating more slowly than poly(A)₈₄ sequences were evident in the reaction products (Fig. 5B, lanes 3 and 5). The amounts of T1 oligonucleotides from CRE-independent negative-strand RNA synthesis were smaller than those from the WT RNA control (Fig. 5B, lanes 1 and 3). Nonetheless, VPg-linked poly(U) was present in the RF RNA from both WT and CRE KO templates (Fig. 5B, lanes 1 and 3). These data confirm that PV RF RNA from CRE-dependent and CRE-independent reaction products contained poly(U) products, consistent with VPg and VPgpUpU_OH priming along the poly(A) tail of positive-strand RNA templates.