Abstract
Footnotes
†Present address: School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading, UK.Poliovirus, the prototypic member of the family Picornaviridae, is a model system for the replication of economically important relatives, e.g. rhinovirus, foot-and-mouth disease virus, as well as other positive-strand RNA viruses. The genome, a 7·5 kb positive-sense RNA molecule, is covalently linked at the 5' end to a 22 amino acid virus-encoded peptide (VPg) (Lee et al., 1977; Nomoto et al., 1977). The genome encodes a single polyprotein, post-translationally cleaved by virus-encoded proteases into structural and non-structural components that, in conjunction with cellular proteins, replicate and package the newly synthesized genome into progeny virus particles. VPg is covalently attached to the 5' end of the genome because the peptide is the protein primer for genome replication (Paul et al., 1998). To fulfil this function VPg must be modified by the addition of two uridylate (U) residues to a conserved tyrosine residue, a process designated uridylylation, thereby allowing complementary base-pairing to the A nucleotides at the 3' end of the positive- and negative-sense genomes (Andino et al., 1999).
RNA structures within the non-coding regions (NCR) at the 5' and 3' ends of the genome are known to play important roles in translation and replication of the viral genome (reviewed in Andino et al., 1999). The interactions of viral and cellular proteins with these RNA structures are critical for the coordination of genome translation, replication and subsequent encapsidation. For example, the 5' cloverleaf structure is required in cis for negative-strand synthesis, suggesting the genome may circularize during replication (Barton et al., 2001; Lyons et al., 2001). Further studies have demonstrated that the interaction of the poly(C) binding protein (PCBP), bound to the 5' cloverleaf structure, with poly(A) binding protein (PABP), bound to the 3' poly(A) tail, is important in this circularization and is essential for genome replication (Herold & Andino, 2001).
We have previously reported the identification and characterization of an additional small RNA structure (designated 2CCRE), located approximately centrally in the genome in the region encoding the 2C protein, that is absolutely required for replication (Goodfellow et al., 2000). Analysis of this structure, and related elements in other picornaviruses, has demonstrated that the functional domain is the terminal 14 nt single-stranded loop, which must be presented on a base-paired RNA stem (Goodfellow et al., 2003; Paul et al., 2000; Yang et al., 2002). We and others have demonstrated that 2CCRE functions as the template for the formation of the peptide primer VPgpUpU with the A1 position of a highly conserved CA1A2A3CA motif acting as the templating nucleotide (Goodfellow et al., 2003; Paul et al., 2000; Rieder et al., 2000; Yang et al., 2002). The addition of the second U nucleotide probably occurs via a slide-back mechanism as recently described (Gerber et al., 2001).
In our previous analysis of 2CCRE function, using a two-step RNase protection assay, we were unable to detect negative-sense genomes after transfection of replicon RNA containing a defective 2CCRE (Goodfellow et al., 2000). However, the sensitivity of this in vivo assay was unlikely to detect the first round of genome replication during which the input RNA is copied into a negative-sense genome forming a doubled-stranded RNA replicative form (RF; see Fig. 1C). Therefore, the formal possibility that 2CCRE was required for positive- but not negative-strand initiation could not be excluded. Recent studies from D. J. Barton and colleagues imply that there may be differences in the initiation of opposing genome strands (Barton et al., 2001; Lyons et al., 2001) which prompted us to more precisely define the role of 2CCRE-mediated uridylylated VPg in poliovirus replication. We have studied the replication of 2CCRE-defective replicons using in vitro translation and replication reactions (IVTR) containing HeLa S10 extracts prepared as previously described (Molla et al., 1991). The HeLa S10 and Xenopus oocyte (Gamarnik & Andino, 1996) replication systems are currently the only reproducible methods of detecting the first round of genome replication. Efficient formation of positive-sense genomes in IVTR reactions requires an authentic (i.e. identical to viral RNA) 5' end as the two G nucleotides added to the 5' end of in vitro-synthesized RNA transcripts generated using T7 RNA polymerase block positive-sense genome synthesis (Barton et al., 1996, 1999; Herold & Andino, 2000). To overcome this block, the efficient removal of the additional nucleotides is required; this is achieved using a cis-acting hammerhead ribozyme (Herold & Andino, 2000).
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Pre-initiation replication complexes, programmed with 1 µg of in vitro-transcribed RNA derived from wild-type poliovirus type 3 (PV3) replicons (pT7Rep3 and pT7Rep3+R) or from 2CCRE-defective replicons (pT7Rep3/SL3 and pT7Rep3/SL3+R), with (indicated by the +R suffix) or without a hammerhead ribozyme at their 5' ends (Fig. 1A and Goodfellow et al., 2000), were formed in the presence of 2 mM guanidine hydrochloride to allow translation, but prevent replication (as described by Barton et al., 1995). Genome replication was subsequently monitored using [32P]UTP to label newly synthesized RNA in a guanidine hydrochloride-free environment, as previously described (Barton et al., 1995). RNA from pT7Rep3/SL3 contains eight synonymous mutations that disrupt 2CCRE structure and prevent detectable VPgpUpU formation in an in vitro uridylylation assay (Goodfellow et al., 2000). We have never succeeded in selecting revertant viruses when these mutations are present in a full-length genome, confirming a completely null phenotype (Goodfellow et al., 2000). In Fig. 1(B), lanes 1 and 2, we demonstrate for the first time that PV3-derived replicons replicate in IVTR reactions and that, as for PV1, additional nucleotides at the 5' end of the transcript prevent efficient replication (compare the formation of SS form). The 2CCRE-defective replicons Rep3/SL3 and Rep3/SL3+R both failed to produce positive-sense SS progeny, though the double-stranded RF was readily detectable in IVTR reactions primed with these RNAs (compare lanes 3 and 4 in Fig. 1B). Correct function of the hammerhead ribozyme present at the 5' end of transcripts derived from pT7Rep3+R and pT7Rep3/SL3+R was verified by monitoring the release of the ribozyme-containing RNA fragment from the full-length RNA transcript (data not shown). The formation of RF in extracts programmed with the 2CCRE-defective replicon (Rep3/SL3+R) RNA requires the formation of negative-strand RNA. This implies that the 2CCRE, and therefore 2CCRE-mediated uridylylation of VPg, is not required for the initiation of negative-sense genome synthesis.
Priming of positive-sense genome synthesis occurs on the AA dinucleotide present at the 3' end of the negative-sense genome. To confirm that positive-sense RNA was not produced by Rep3/SL3+R due to lack of the nucleotidepeptide primer (VPgpUpU), we directly monitored the formation of VPgpUpU in IVTR reactions (as described by Lyons et al., 2001). VPgpUpU was readily detected in IVTR reactions programmed with wild-type PV3 replicon RNA (Rep3+R) but was absent in reactions programmed with the 2CCRE-defective replicon (Rep3/SL3+R; Fig. 2, lanes 1 and 2 respectively). The labelled VPg observed in IVTR reactions was confirmed as VPgpUpU by comparison with in vitro uridylylated VPg (data not shown and Paul et al., 2000).
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We have previously demonstrated that genomes containing a defective 2CCRE cannot be rescued by co-infection with a helper virus, implying that 2CCRE functions only in cis under these conditions (Goodfellow et al., 2000). To address whether the VPgpUpU primer formed from a helper genome in IVTR reactions could function in trans, we studied the ability of luciferase-encoding replicons with functional 2CCRE structures (Rep3-L and Rep3-L+R) to restore the replication of a 2CCRE-defective replicon (Rep3/SL3+R) by co-translation and replication in IVTR reactions (Fig. 3). Pre-initiation replication complexes were formed (as described above) containing 2CCRE-defective replicon (Rep3/SL3+R; Fig. 3, lane 1), wild-type replicons (Rep3-L and Rep3-L+R; lanes 2 and 3) or an equal amount of 2CCRE-defective replicon with wild-type replicon RNA (Fig. 3, lanes 4 and 5). RNA from the 2CCRE-defective replicon (Rep3/SL3+R) is 750 nt smaller than the helper replicons (Rep3-L and Rep3-L+R) because of the difference in the size of luciferase and chloramphenicol acetyltransferase (CAT) reporter genes present in the replicons, and so can be distinguished by gel electrophoresis.
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In contrast to our previous cell-based assays (Goodfellow et al., 2000), functional 2CCRE in the luciferase-encoding replicons could rescue 2CCRE-defective replicons in trans. An authentic 5' end on the helper genome increased, but was not absolutely necessary for, the trans-rescue of the smaller CAT-encoding 2CCRE-defective replicon (Fig. 3, lanes 4 and 5). The apparent difference between our previous results obtained in vivo (Goodfellow et al., 2000) and those generated in vitro are likely to be due to the lack of compartmentalization in IVTR reactions, which allows for enhanced mixing of replication complexes. The large amounts of RNA used to prime the IVTR also means that the available levels of VPgpUpU are likely to be higher than those in intact cells. Tiley et al. (2003) have recently reported the trans-rescue of an foot-and-mouth disease virus cre mutant in vivo using a helper virus with reduced replication.
E. Wimmer and colleagues have previously reported the formation of VPgpUpU and VPgpoly(U) in vitro using poly(A) RNA as a template (Paul et al., 1998, 2003). The efficiency of this in vitro reaction is very poor compared with VPgpUpU formation using 2CCRE as a template, and works optimally with manganese as a cofactor (Paul et al., 2000, 2003), conditions that are known to reduce the specificity of polymerase-template recognition (Arnold et al., 1999). Paul et al.(2000) suggest that the 2CCRE is the in vivo template for uridylylation of VPg, and that 2CCRE-mediated uridylylated VPg translocates to the 3' poly(A) tail of the virus to prime the initiation of the negative-sense genome. Our results demonstrate that 2CCRE is not required for negative-strand synthesis. Furthermore, the double-stranded RF (Fig. 1C) is generated in the absence of detectable VPgpUpU. We therefore propose a new model in which 2CCRE functions to form a pool of uridylylated VPg (VPgpUpU) which is retained in the replication complex until positive-strand initiation begins. We further suggest that the 3' poly(A) tail is likely to be the template for the uridylylation of VPg, and probably acts to form VPgpoly(U). Although apparently relatively inefficient in vitro (Paul et al., 1998), the yield of VPgpoly(U) could be influenced by the length of the 3' poly(A) tail [which has been shown by Herold & Andino (2001) to have a marked effect on the efficiency of genome replication], or by the adjacent 3' NCR sequences of the virus. Our previous analysis of VPg uridylylation using an RNA transcript encompassing the poliovirus 3' NCR with a poly(A) tail of 9 nt indicated that the majority of the product formed in this reaction is VPgpoly(U) and not VPgpUpU (Goodfellow et al., 2003). These reactions were done in the presence of manganese; more recent studies using magnesium as a cofactor which increases the specificity of polymerasetemplate interaction (Arnold et al., 1999) have suggested that the structured 3' NCR specifically recruits 3Dpol, thereby increasing the efficiency of VPgpoly(U) formation from the poly(A) tail (I. G. Goodfellow and others, unpublished). Numerous other studies also support a sequence-specific interaction of the 3' NCR and the virus polymerase. It has been reported that poliovirus 3Dpol exhibits 5-fold greater affinity for RNA substrates containing the virus 3' NCR (Oberste & Flanegan, 1988), an interaction also observed in the cardioviruses, in which a requirement for both the structured elements of the 3' NCR and the poly(A) tail has been shown (Cui & Porter, 1995; Cui et al., 1993). Revertants of viruses containing debilitating mutations in the 3' NCR have been mapped to residues in 3Dpol, further supporting a direct and specific interaction of the polymerase and the 3' NCR (Duque & Palmenberg, 2001; Meredith et al., 1999). Additional studies will be needed to define the 3' sequences that could function in the formation of VPgpoly(U), and to confirm the role of this nucleotidepeptide primer in negative-strand synthesis.
It is interesting to note that several other products appear to be uridylylated in the IVTR reactions (Fig. 2). We speculate that these labelled products are uridylylated VPg-containing precursor proteins, though this interpretation will need verification. There are recent data to suggest that 2CCRE can function in vitro to uridylylate VPg-containing precursors (Pathak et al., 2002). Given the defect in the 2CCRE present in the RNA derived from the replicon pT7Rep3/SL3+R, it is likely that the radiolabelled products observed are derived from poly(A)-templated VPg uridylylation. The identity of the VPg-containing precursor(s) uridylylated in vivo during poliovirus replication is the subject of ongoing analysis.
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Received 30 January 2003; accepted 18 June 2003.