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Journal of Virology, October 2008, p. 9400-9408, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00427-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Poliovirus cis-Acting Replication Element-Dependent VPg Uridylylation Lowers the Km of the Initiating Nucleoside Triphosphate for Viral RNA Replication{triangledown}

Benjamin P. Steil1 and David J. Barton1,2*

Department of Microbiology,1 Program in Molecular Biology, University of Colorado Denver, School of Medicine, Aurora, Colorado 800452

Received 26 February 2008/ Accepted 12 July 2008


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ABSTRACT
 
Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5' terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5' end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpUOH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpUOH-independent manner, CRE-dependent VPgpUpUOH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the Km of UTP required for viral RNA replication and that CRE-dependent VPgpUpUOH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.


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INTRODUCTION
 
Concentrations of nucleoside triphosphates (NTPs) required for the initiation of viral RNA synthesis (NTPi) are typically greater than the concentrations required for the elongation of nascent RNA products (48). In mitochondria, this feature of RNA transcription machinery is used advantageously to regulate gene expression in response to ATP concentrations, thereby allowing regulation of mitochondrial gene expression as a function of the presence or absence of fermentable carbon sources (3, 4). In bacteria, ATP and GTP concentrations regulate the initiation of rRNA transcription, thereby regulating the ribosome formation and magnitudes of translation relative to available energy pools (15, 40). Prokaryotic and eukaryotic viral DNA-dependent and RNA-dependent RNA polymerases (RdRps) also require elevated concentrations of NTPi for the initiation of RNA synthesis (18, 20, 23, 24, 39, 45).

Relatively unique features of picornavirus RNA replication include the primer-dependent nature of the RdRp 3DPol (14) and the viral protein primers of RNA replication, VPg and VPgpUpUOH (37). Poliovirus (PV) RNA replication can be studied using defined reaction mixtures containing purified 3DPol; however, depending on the primers and templates used, 3DPol will copy both viral and nonviral templates (47). Furthermore, defined reaction mixtures containing purified 3DPol fail to replicate negative- and positive-strand RNAs in a normal asymmetric manner (1). In contrast, authentic VPg and VPgpUpUOH primer-dependent RNA replication can be studied using replication complexes formed in reaction mixtures containing cytoplasmic extracts from uninfected cells (9, 27). Basic features of PV RdRp, including elongation rates, are comparable when measured using either defined reaction mixtures containing purified 3DPol (49) or replication complexes formed during the translation of PV RNA in cell-free reaction mixtures (7).

A cis-acting replication element referred to as CRE is an RNA structure located within the PV 2CATPase open reading frame that functions as a template for the uridylylation of VPg, leading to the formation of VPgpUpUOH (26, 31, 36, 51). Two adenosine residues within a loop of the CRE RNA function as a template for the 3DPol-catalyzed addition of two uridine residues onto VPg in a slide-back mechanism, resulting in the synthesis of VPgpUpUOH (38). PV CRE-dependent VPg uridylylation can be achieved using purified 3DPol and CRE RNA templates in defined reaction mixtures (33, 35, 37, 38) or in replication complexes that synchronously and sequentially replicate PV RNA (28, 29). CRE-dependent VPg uridylylation was confirmed for other picornaviruses using cognate 3DPol and CRE RNA templates from human rhinovirus 2 (16), foot-and-mouth disease virus (31), coxsackievirus B3 (50), and human rhinovirus 14 (42). In addition to 3DPol and CRE, PV 5' cloverleaf RNA and viral protein 2CATPase activities are required for VPg uridylylation within RNA replication complexes (25).

In this investigation, we compared negative-strand RNA synthesis by PV RNA replicons with and without intact CRE RNA elements. The PV RNA replicons were compared in cell-free reaction mixtures which faithfully recapitulated all of the metabolic steps of viral mRNA translation and viral RNA replication found in the cytoplasm of infected cells (6, 9, 27). Significant advantages of this experimental system are the ability to study one cycle of sequential negative- and positive-strand RNA synthesis (7) and the ability to characterize biochemical defects in PV RNA replicons containing lethal mutations (8, 25, 30) that would preclude detailed phenotypic characterization in transfected cells or other experimental systems. Herein we report that CRE-dependent VPg uridylylation lowers the concentration of UTP required for PV negative-strand RNA synthesis. We discuss how CRE-dependent VPgpUpUOH synthesis helps overcome the rate-limiting step of RNA synthesis initiation.


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MATERIALS AND METHODS
 
PV cDNAs. (i) pRNA2 (referred to as wild-type [WT] CRE in this report). 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).

(ii) pDNVR27 (referred to as Ribo WT CRE in this report). 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).

(iii) pRibo KO CRE and pKO CRE. 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 S10 translation-replication reactions. 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) 10x 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. 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 x g for 15 min at 4°C and resuspended in [{alpha}-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. [{alpha}-32P]NTPs (1 µCi per 1-µl reaction mixture; 1.25 µM radiolabeled NTP) (MP Biomedicals) were included to radiolabel viral RNA and VPgpUpUOH. 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 x 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 VPgpUpUOH 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 VPgpUpUOH in the gel was detected by phosphorimaging.

RNase T1 digestion. 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 2x 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).


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RESULTS
 
[32P]UTP labeling conditions revealed a defect in CRE-independent negative-strand RNA synthesis. 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.


Figure 1
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  		FIG. 1. CRE mutation inhibited nascent negative-strand RNA synthesis within PIRCs. (A) Diagram of PV RNAs with either a WT or mutant (KO CRE) CRE. Eight silent mutations (PV nucleotides C4456U, A4459C, G4462A, C4465U, G4468A, A4469U, G4470C, and A4474G) were engineered to completely disrupt the CRE RNA structure and prevent any detectable VPgpUpUOH synthesis (29). PV RNA2 templates make only negative-strand RNA due to two nonviral 5' guanosine residues (10, 19). ORF, open reading frame. (B and C) PIRCs with PV RNA2 WT CRE (lanes 1 to 6) or PV RNA2 KO CRE (lanes 7 to 12) templates were incubated in [{alpha}-32P]UTP labeling reaction mix containing 1 mM ATP, 250 µM GTP, 250 µM CTP, 10 µM unlabeled UTP, and 1.25 µM [{alpha}-32P]UTP (40 µCi per 40-µl reaction mixture). RNA replication reaction mixtures were incubated from 4 to 20 min in the absence of guanidine (lanes 1 to 5 and 7 to 11, as indicated in panel C). A total of 2 mM of guanidine was included in two reaction mixtures (lanes 6 and 12). RNA products from the reaction mixtures were separated by electrophoresis in 1% agarose and detected by ethidium bromide and UV light (B) or by phosphorimaging (C). a, RNA ladder; b and c, PV RNA2 WT CRE and KO CRE template RNAs. Note mobility of 28S and 18S rRNAs relative to that of PV RNAs.


Figure 3
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  		FIG. 3. CRE-independent negative-strand RNA synthesis was sensitive to low concentrations of UTP. (A) VPg uridylylation. PIRCs with PV RNA2 WT CRE (lanes 1 and 2), PV RNA2 KO CRE (lanes 3 and 4), PV ribozyme KO CRE (lanes 5 and 6), or PV ribozyme WT CRE (lanes 7 and 8) RNA templates were incubated for 60 min in the absence (–) or presence (+) of guanidine in reaction mixtures containing [{alpha}-32P]UTP, 100 µM UTP, and 250 µM CTP. Reaction products were separated by electrophoresis, and VPgpUpUOH was detected as described in Materials and Methods (25, 29). (B) RNA synthesis in reaction mixtures containing 10 µM UTP (lanes 1 to 5) or 500 µM UTP (lanes 6 to 10), 250 µM CTP, and [{alpha}-32P]CTP. PIRCs with PV RNA2 WT CRE (lanes 1, 2, 6, and 7), PV RNA2 KO CRE (lanes 3 and 8), PV ribozyme KO CRE (lanes 4 and 9), or PV ribozyme WT CRE (lanes 5 and 10) RNA templates were incubated for 60 min in the absence (lanes 2 to 5 and 7 to 10) or presence (lanes 1 and 6) of guanidine. (C) RNA synthesis in reaction mixtures containing 10 µM CTP (lanes 1 to 5) or 250 µM CTP (lanes 6 to 10), 500 µM UTP, and [{alpha}-32P]GTP. PIRCs with PV RNA2 WT CRE (lanes 1, 2, 6, and 7), PV RNA2 KO CRE (lanes 3 and 8), PV ribozyme KO CRE (lanes 4 and 9), or PV ribozyme WT CRE (lanes 5 and 10) RNA templates were incubated for 60 min in the absence (lanes 2 to 5 and 7 to 10) or presence (lanes 1 and 6) of guanidine. Reaction products were separated by electrophoresis in 1% agarose, and radiolabeled RNAs were detected by phosphorimaging. GuHCl, guanidine HCl.

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 [{alpha}-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 3DPol 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 3DPol 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).

Low UTP concentrations did not support CRE-independent negative-strand RNA synthesis. 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.


Figure 2
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  		FIG. 2. Elevated UTP concentrations compensated for the defect in negative-strand RNA synthesis associated with CRE mutations. PIRCs with PV RNA2 KO CRE (lanes 1 to 6) or PV RNA2 WT CRE (lanes 7 to 12) templates were incubated for 60 min in the absence (–) or presence (+) of guanidine in reaction mixtures containing either [{alpha}-32P]UTP (A) or [{alpha}-32P]CTP (B). Concentrations of exogenous UTP and exogenous CTP in the reaction mixtures varied from 10 to 250 µM as indicated. A total of 1 mM ATP, 250 µM GTP, and 250 µM of either CTP (A) or UTP (B) were also present in the reaction mixtures. RNA products from the reaction mixtures were separated by electrophoresis in 1% agarose, and radiolabeled RNAs were detected by phosphorimaging. GuHCl, guanidine HCl.

CRE-dependent and CRE-independent RNA replication. 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 VPgpUpUOH (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). VPgpUpUOH 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 VPgpUpUOH synthesis (37), does not function as a template for VPgpUpUOH 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.

Apparent Km of UTP for CRE-independent and CRE-dependent negative-strand RNA synthesis. To measure the Km 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 Km measurements. Therefore, because the amounts of endogenous NTPs are unmeasurable, the Kms derived from these reaction mixtures are apparent Kms 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 Kms, 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 Vmax 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.


Figure 4
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  		FIG. 4. Apparent Km of UTP required for negative-strand RNA synthesis. PIRCs containing PV RNA2 WT CRE (lanes 1 to 9) or PV RNA2 KO CRE (lanes 10 to 18) templates were incubated for 60 min in the presence (lanes 9 and 18) or absence (lanes 1 to 8 and 10 to 17) of guanidine in reaction mixtures containing [{alpha}-32P]CTP, 100 µM CTP, and 0 to 500 µM UTP as indicated. RNA products from the reaction mixtures were separated by electrophoresis in 1% agarose and detected by ethidium bromide and UV light (A) or by phosphorimaging (B). Mobilities of PV RNA, RF RNA, 28S rRNAs, and 18S rRNAs are indicated. (C) Apparent Km of UTP. The amount of radiolabeled negative-strand RNA within RF bands in panel B was measured by phosphorimaging using Quantity One software with background from the lane 18 guanidine HCl control subtracted. Magnitude of negative-strand RNA synthesis (in arbitrary phosphorimager units) was plotted versus the concentration of UTP in each reaction mixture, and the Km values for WT (•) and KO CRE ({blacksquare}) PV RNAs were determined using GraphPad software. Nonlinear regression was used to fit our data using the Michaelis-Menten equation to define the relationship of product formation and substrate concentration, with Vmax and Km as the parameters, as follows: Y = (Vmax · X)/(Km + X), where X is the concentration of UTP. The Km values of UTP with standard errors for the respective RNAs are presented in the graph. Representative data are from two independent experiments.

RNase T1 digestion and gel electrophoresis of radiolabeled PV RNAs revealed the size of 3' poly(A) in positive-strand RNA templates and the size of 5' VPg-linked poly(U) products in negative-strand RNA products. VPg and VPgpUpUOH 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 (VPgpUpUOH priming) and CRE-independent (VPg priming) reaction conditions (Fig. 5). [{alpha}-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).


Figure 5
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  		FIG. 5. VPg-linked poly(U) from CRE-dependent and CRE-independent negative-strand RNA synthesis. (A) PV RF RNA fractionated by 1% agarose gel electrophoresis. PIRCs containing WT CRE RNA templates (lanes 1 to 2) or KO CRE RNA templates (lanes 3 to 4) were incubated in reaction mixtures containing 1 mM ATP, 250 µM GTP, 250 µM CTP, 10 µM UTP, [{alpha}-32P]UTP (3 µCi per 1-µl reaction mixture), and 2 mM guanidine HCl (GuHCL; lanes 2 and 4). Reaction products were separated by 1% agarose gel electrophoresis and detected by phosphorimaging. The mobility of PV RF RNA is indicated. (B) RNase T1 oligonucleotides (Oligos) in PV RNAs. WT CRE RNA templates (lane 1) and KO CRE RNA templates (lane 2) were synthesized by T7 RNA transcription in reaction mixtures containing [{alpha}-32P]ATP, digested with RNase T1, and separated by electrophoresis in 7 M urea-18% polyacrylamide (RNase T1; see Materials and Methods) (lanes 1 to 2). [{alpha}-32P]UTP-radiolabeled products of WT CRE (lanes 3 and 4) and KO CRE (lanes 5 and 6) RNA replication, from reaction mixtures with (lanes 4 and 6) and without (lanes 3 and 5) 2 mM guanidine, were digested with RNase T1 and separated by electrophoresis in 7 M urea-18% polyacrylamide (PV RNA replication and RNase T1; see Materials and Methods). Mobilities of specific T1 oligonucleotides and VPg-linked poly(U) are indicated.

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 [{alpha}-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 [{alpha}-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 VPgpUpUOH priming along the poly(A) tail of positive-strand RNA templates.


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DISCUSSION
 
PV 3DPol is strictly primer-dependent under all experimental circumstances (14, 37, 38), including those used in this investigation (28, 29). De novo (primer-independent) initiation of viral RNA synthesis is not observed under any of the experimental conditions used in this investigation. There are two potential primers for PV RNA replication, VPg and VPgpUpUOH (Fig. 6). VPgpUpUOH, the product of CRE-dependent VPg uridylylation, is the undisputed primer for the initiation of positive-strand RNA synthesis (28, 29, 41, 50). Disrupting the structure of CRE coordinately prevents both VPgpUpUOH synthesis and positive-strand RNA synthesis but does not prevent negative-strand RNA synthesis (Fig. 3). The uridine residues of VPgpUpUOH prime the initiation of positive-strand RNA synthesis via complementarity with the two adenosine residues at the 3' end of negative-strand RNA (41). If the two adenosine residues at the 3' end of negative-strand RNA are extended by two nonviral cytosine residues, as would occur for PV RNA2 templates, then VPgpUpUOH can no longer efficiently prime positive-strand RNA synthesis (note that RNA2 templates with WT CRE make VPgpUpUOH but do not function as a template for positive-strand RNA synthesis) (Fig. 3, lanes 2 and 7). Thus, in the case of positive-strand RNA synthesis, VPgpUpUOH must align with two adenosine residues at the very 3' end of negative-strand RNA templates. Removal of the two nonviral residues via ribozyme action allows for efficient VPgpUpUOH priming of positive-strand RNA synthesis (Fig. 3, lanes 5 and 10) (19).


Figure 6
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  		FIG. 6. Model of CRE-independent and CRE-dependent priming of negative-strand RNA synthesis. CRE-independent, VPg-primed, negative-strand RNA synthesis (top) exhibited a Km of 103 µM UTP, whereas CRE-dependent, VPgpUpUOH-primed, negative-strand RNA synthesis (bottom) had a Km of 12 µM UTP. CRE-dependent VPgpUpUOH must translocate from the CRE RNA template to the 3' poly(A) template to prime negative-strand RNA initiation as previously suggested by others (36).

Because VPgpUpUOH would also be complementary to adenosine residues at the 3' end of polyadenylated positive-strand templates, it has been proposed to function as a primer for negative-strand RNA synthesis (34) (Fig. 6). Yet when CRE-dependent VPg uridylylation is prevented with mutations that disrupt the CRE RNA structure, the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis directly (Fig. 3), independent of CRE and independent of detectable VPgpUpUOH intermediates (17, 28, 29) (Fig. 6). Thus, despite assertions by some that VPgpUpUOH is generally accepted as the primer for both negative- and positive-strand RNA synthesis (38), some uncertainty remains as to whether VPg, VPgpUpUOH, or both prime the initiation of negative-strand RNA synthesis (Fig. 6).

Compared to CRE-independent VPg-primed negative-strand RNA synthesis, CRE-dependent VPg uridylylation lowered the concentration of UTP required for efficient negative-strand RNA synthesis (Fig. 2 to 4), indicating that under such conditions at least a portion of negative-strand RNA was primed with VPgpUpUOH rather than VPg (Fig. 6). At low UTP concentrations (≤5 µM), the magnitude of negative-strand RNA synthesis by a WT template with an intact CRE was 95% greater than that with a KO CRE (Fig. 4, lanes 1 and 10). This suggested that at this low UTP concentration, 95% of negative-strand RNA synthesis was primed with CRE-dependent VPgpUpUOH and 5% was primed with VPg in a CRE-independent mechanism. At higher UTP concentrations (≥250 µM), the difference in magnitude of negative-strand RNA synthesis between templates with or without intact CRE structures was less dramatic, consistent with ~60% of negative-strands being primed by VPgpUpUOH and 40% being primed by VPg (Fig. 4, lanes 8 and 17). These data support the conclusion that VPgpUpUOH molecules derived from CRE-dependent VPg uridylylation may be the predominant primers for negative-strand RNA synthesis under normal conditions. An elegant pulse-chase experiment, wherein VPgpUpUOH molecules are chased into negative- and/or positive-strand RNA products, would provide more definitive evidence of VPgpUpUOH priming; however, such data have yet to be generated by us or others. Together, our data indicate that CRE, the template for VPgpUpUOH synthesis (33, 35), lowers the Km of UTP required for VPg uridylylation by 3DPol and subsequent VPgpUpUOH priming on the poly(A) template compared to VPg priming on the poly(A) template (Fig. 4 and 6). This is consistent with the low Km of UTP (~5 µM) reported for CRE-dependent VPg uridylylation (22). The fact that VPgpUpUOH oligonucleotides made from CRE RNA templates can lower the Km of the NTPi is not without precedent, as short oligonucleotide primers acting as the NTPi mimic can lower the Km of NTPs required for RNA synthesis by the hepatitis C virus RdRp (12), the bovine viral diarrhea virus RdRp (13), and the brome mosaic virus RdRp (21).

The average physiologic concentrations of ribonucleotides are 3,152 ± 1,698 µM for ATP, 468 ± 224 µM for GTP, 278 ± 242 µM for CTP, and 567 ± 460 µM for UTP (46). These mean values reflect what is generally found in normal human cells, human tumor cells, and human tissues (46); however, NTP concentrations vary from cell to cell due to physiological conditions and could vary within specific locations of a cell. Furthermore, NTP concentrations may change (decrease) during the time course of virus infection within cells. As shown in Fig. 4, CRE-dependent VPg uridylylation lessened the amount of exogenous UTP required for efficient negative-strand RNA synthesis from 103 µM to 12 µM. The total of 12 µM is comparable to the Km of NTPs required by PV 3DPol and other RNA polymerases for RNA elongation (5, 21, 32, 48). Thus, CRE-dependent VPg uridylylation may serve two important purposes: (i) providing VPgpUpUOH primers for both negative- and positive-strand RNA replication and (ii) overcoming the rate-limiting effects of NTPi concentrations, which in the absence of VPg uridylylation would otherwise restrict the initiation of RNA replication.

Our results support the conclusion that CRE-dependent VPg uridylylation, by virtue of VPgpUpUOH, lowers the Km of UTP required for the initiation of PV RNA replication. In this manner, PV RNA replication mechanisms circumvent the requirement of a high NTPi concentration, which typically constrains RNA synthesis by prokaryotic and viral RNA polymerases (3, 4, 15, 20, 48).


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ACKNOWLEDGMENTS
 
This work was supported by NIH grants AI42189 and T32 AI052066.


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FOOTNOTES
 
* Corresponding author. Mailing address: University of Colorado Denver, School of Medicine, Department of Microbiology MS8333, P.O. Box 6511, 12800 East 19th Ave., Aurora, CO 80045. Phone: (303) 724-4215. Fax: (303) 724-4226. E-mail: david.barton{at}uchsc.edu Back

{triangledown} Published ahead of print on 23 July 2008. Back


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Journal of Virology, October 2008, p. 9400-9408, Vol. 82, No. 19
0022-538X/08/$08.00+0     doi:10.1128/JVI.00427-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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  • Steil, B. P., Barton, D. J. (2009). Conversion of VPg into VPgpUpUOH before and during Poliovirus Negative-Strand RNA Synthesis. J. Virol. 83: 12660-12670 [Abstract] [Full Text]  

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