1 Division of Virology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 5JR, UK
2 University of California San Francisco, Genentech Hall, San Francisco, CA 94143-2280, USA
Correspondence
David Evans
david.evans{at}vir.gla.ac.uk
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ABSTRACT |
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Present address: School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading, UK.
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MAIN TEXT |
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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. 1
C). 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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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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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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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Arnold, J. J., Ghosh, S. K. & Cameron, C. E. (1999). Poliovirus RNA-dependent RNA polymerase (3Dpol). Divalent cation modulation of primer, template, and nucleotide selection. J Biol Chem 274, 3706037069.
Barton, D. J., Black, E. P. & Flanegan, J. B. (1995). Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA. J Virol 69, 55165527.[Abstract]
Barton, D. J., Morasco, B. J. & Flanegan, J. B. (1996). Assays for poliovirus polymerase, 3Dpol, and authentic RNA replication in HeLa S10 extracts. Methods Enzymol 275, 3557.[Medline]
Barton, D. J., Morasco, B. J. & Flanegan, J. B. (1999). Translating ribosomes inhibit poliovirus negative-strand RNA synthesis. J Virol 73, 1010410112.
Barton, D. J., O'Donnell, B. J. & Flanegan, J. B. (2001). 5' cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J 20, 14391448.
Cui, T. & Porter, A. G. (1995). Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3'-noncoding region of the viral RNA. Nucleic Acids Res 23, 377382.[Abstract]
Cui, T., Sankar, S. & Porter, A. G. (1993). Binding of encephalomyocarditis virus RNA polymerase to the 3'-noncoding region of the viral RNA is specific and requires the 3'-poly(A) tail. J Biol Chem 268, 2609326098.
Duque, H. & Palmenberg, A. C. (2001). Phenotypic characterization of three phylogenetically conserved stem-loop motifs in the mengovirus 3' untranslated region. J Virol 75, 31113120.
Gamarnik, A. V. & Andino, R. (1996). Replication of poliovirus in Xenopus oocytes requires two human factors. EMBO J 15, 59885998.[Abstract]
Gerber, K., Wimmer, E. & Paul, A. V. (2001). Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: identification of a cis-replicating element in the coding sequence of 2Apro. J Virol 75, 1097910990.
Goodfellow, I. G., Chaudhry, Y., Richardson, A., Meredith, J. M., Almond, J. W., Barclay, W. S. & Evans, D. J. (2000). Identification of a cis-acting replication element within the poliovirus coding region. J Virol 74, 45904600.
Goodfellow, I. G., Kerrigan, D. & Evans, D. J. (2003). Structure and function of the poliovirus cis-acting replication element (CRE). RNA 9, 124137.
Herold, J. & Andino, R. (2000). Poliovirus requires a precise 5' end for efficient positive-strand RNA synthesis. J Virol 74, 63946400.
Herold, J. & Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol Cell 7, 581591.[Medline]
Lee, Y. F., Nomoto, A., Detjen, B. M. & Wimmer, E. (1977). The genome-linked protein of picornaviruses. I. A protein covalently linked to poliovirus genome RNA. Proc Natl Acad Sci U S A 74, 5963.[Abstract]
Lyons, T., Murray, K. E., Roberts, A. W. & Barton, D. J. (2001). Poliovirus 5'-terminal cloverleaf RNA is required in cis for VPg uridylylation and the initiation of negative-strand RNA synthesis. J Virol 75, 1069610708.
Meredith, J. M., Rohll, J. B., Almond, J. W. & Evans, D. J. (1999). Similar interactions of the poliovirus and rhinovirus 3D polymerase with the 3' untranslated region of rhinovirus 14. J Virol 73, 99529958.
Molla, A., Paul, A. V. & Wimmer, E. (1991). Cell-free, de novo synthesis of poliovirus. Science 254, 16471651.[Medline]
Nomoto, A., Kitamura, N., Golini, F. & Wimmer, E. (1977). The 5' terminal structures of poliovirus RNA and poliovirus mRNA differ only in the genome-linked protein VPg. Proc Natl Acad Sci U S A 77, 53455349.
Oberste, M. S. & Flanegan, J. B. (1988). Measurement of poliovirus RNA polymerase binding to poliovirion and nonviral RNAs using a filter-binding assay. Nucleic Acids Res 16, 1033910352.[Abstract]
Pathak, H. B., Paul, A. V. & Cameron, C. E. (2002). A 3B-containing precursor serves as both the VPg donor and 3D stimulatory factor in the oril-template uridylylation reaction. Europicamerica 2002 (Cape Cod, 1319 May 2002), F11. Edited by B. Baxt, E. Ehrenfeld, M. Grubman, P. W. Mason & E. Wimmer.
Paul, A. V., van Boom, J. H., Filippov, D. & Wimmer, E. (1998). Protein-primed RNA synthesis by purified poliovirus RNA polymerase. Nature 393, 280284.[CrossRef][Medline]
Paul, A. V., Rieder, E., Kim, D. W., van Boom, J. H. & Wimmer, E. (2000). Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg. J Virol 74, 1035910370.
Paul, A. V., Peters, J., Mugavero, J., Yin, J., Van Boom, J. H. & Wimmer, E. (2003). Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus. J Virol 77, 891904.[CrossRef][Medline]
Rieder, E., Paul, A. V., Kim, D. W., van Boom, J. H. & Wimmer, E. (2000). Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation. J Virol 74, 1037110380.
Tiley, L., King, A. M. Q. & Belsham, G. J. (2003). The foot-and-mouth disease virus cis-acting replication element (cre) can be complemented in trans within infected cells. J Virol 77, 22432246.
Yang, Y., Rijnbrand, R., McKnight, K. L., Wimmer, E., Paul, A., Martin, A. & Lemon, S. M. (2002). Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14. J Virol 76, 74857494.
Received 30 January 2003;
accepted 18 June 2003.