5′ cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis

Abstract

A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.

Requirements for West Nile Virus (–)- and (+)-Strand Subgenomic RNA Synthesis in Vitro by the Viral RNA-dependent RNA Polymerase Expressed in Escherichia coli

“…From this study, the requirement for WNV (Ϫ)-strand RNA synthesis also follows the trend previously established for other flaviviruses (41, 54 -56) and for poliovirus with regard to the requirement for interaction between the 5Ј and 3Ј ends (57)(58)(59). We examined the role of 5Ј-cap as well as CYC motifs for synthesis of WNV RNA of not only (Ϫ)-strand but also of (ϩ)-strand polarity.…”

Cis-acting RNA elements at the 5′ end of Sindbis virus genome RNA regulate minus- and plus-strand RNA synthesis

“…In conclusion, we have shown that the 51-nt CSE is not essential for SIN RNA replication, but it does enhance its efficiency+ Additionally, we have demonstrated that the 59 UTR of the SIN genome is an essential component of the promoter for both plus-and minusstrand RNA synthesis and that the 59 requirements for each of these processes differ+ Our data demonstrate that interaction between the 59 and 39 ends of the alphavirus genome is required for efficient initiation of minus-strand RNA synthesis+ These data, together with the recent reports for dengue virus and poliovirus (You & Padmanabhan, 1999;Barton et al+, 2001;Herold & Andino, 2001), suggest that communication between I. Frolov, R. Hardy, and C.M. Rice host mRNA translation (Gallie, 1998;Imataka et al+, 1998) and RNA virus replication+…”

“…To further define the role of the 59 end of the alphavirus genome in RNA replication, we employed complementary in vivo and in vitro approaches+ These methods allowed us to distinguish between defects in plus-and minus-strand RNA synthesis while separating RNA synthetic events from translation+ Our data begin to define the elements required at the 59 end of the genome for initiation of RNA synthesis by the viral replicase+ These findings and previously published data suggest a model for initiation of minus-strand synthesis that may be broadly applicable to plus sense RNA viruses (Barton et al+, 2001;Herold & Andino, 2001)+…”

“…interactions+ For poliovirus, two recent reports have shown that the host poly(C) binding protein, PCBP2, and the viral polymerase precursor, 3CD, bind to a 59 cloverleaf structure and interact with poly(A) binding protein (PABP) bound to 39 poly(A)+ This 59-39 interaction, mediated by a protein-protein bridge, is required for efficient initiation of poliovirus minus-strand RNA synthesis (Barton et al+, 2001;Herold & Andino, 2001)+ With this in mind, we propose a model for the initiation of SIN minus-strand RNA synthesis based on the requirement for host translational machinery+ Capdependent translation initiation requires the formation of a cap-binding complex that consists of initiation factor eIF4F bound to the cap structure of mRNAs (Merrick & Hershey, 1996)+ The eIF4F complex contains eIF4E, eIF4A, and eIF4G subunits+ The eIF4G subunit interacts with PABP bringing the 59 and 39 end of mRNAs together during the initiation of translation (Gallie, 1998;Imataka et al+, 1998)+ We hypothesize that the SIN replicase interacts directly or indirectly with the 59 end of the genome, and then is brought into position for initiation at the 39 end of the genome by the circularization of the template mediated by components of the translational machinery (Fig+ 9)+ In support of this model, SIN minus-strand RNA synthesis is sensitive to the length of the poly(A) tract and the presence of PABP (R+W+ Hardy, R+C+ Deo, S+K+ Burley, & C+M+ Rice, unpubl+ data)+ This mechanism of minus-strand initiation would serve multiple purposes for the virus; it would ensure that the template RNA had both 39 and 59 ends, it could potentially abrogate translation from the template RNA, thus clearing it of ribosomes and facilitating replication, and it may play a role in the shut-off of vertebrate host cell translation, a process that exhibits a strong positive correlation with the level of viral RNA replication+ This is just one of many possible models and further work is needed to define specific RNA elements required for minus-strand initiation and the host and viral factors that recognize them+ A replication model involving the interaction of the 59 and 39 ends of the genome may also help to explain the extraordinarily high level of recombination of alphavirus RNAs+ Even RNAs incapable of replication can efficiently recombine in infected cells+ Recombination has been detected between an RNA lacking the 59 end of the genome and an RNA lacking the 39 end of the viral genome (Raju et al+, 1995)+ These pairs of defective RNAs may be brought together by initiation complexes and recombine using a copy-choice mechanism to produce viable recombinant virus+…”

Structural Features of a Picornavirus Polymerase Involved in the Polyadenylation of Viral RNA

“…2; see Fig. S2 in the supplemental material) (19,21,24). Mutant viral RNAs were divided into four groups on the basis of the structural and functional characteristics of particular 3D pol amino acids: mutations of glycine 64, an amino acid involved in the fidelity of viral RNA replication ( Fig.…”