Unusual Loop-Sequence Flexibility of the Proximal RNA Replication Element in EMCV

Jan Zoll,Willem J G Melchers,Paul Gielen,Marc M Hahn,Frank J M Van Kuppeveld,Hans A Heus

doi:10.1371/journal.pone.0024818

Jan Zoll, Willem J G Melchers + Show 4 more

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https://doi.org/10.1371/journal.pone.0024818

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Abstract

Picornaviruses contain stable RNA structures at the 5′ and 3′ ends of the RNA genome, OriL and OriR involved in viral RNA replication. The OriL RNA element found at the 5′ end of the enterovirus genome folds into a cloverleaf-like configuration. In vivo SELEX experiments revealed that functioning of the poliovirus cloverleaf depends on a specific structure in this RNA element. Little is known about the OriL of cardioviruses. Here, we investigated structural aspects and requirements of the apical loop of proximal stem-loop SL-A of mengovirus, a strain of EMCV. Using NMR spectroscopy, we showed that the mengovirus SL-A apical loop consists of an octaloop. In vivo SELEX experiments demonstrated that a large number of random sequences are tolerated in the apical octaloop that support virus replication. Mutants in which the SL-A loop size and the length of the upper part of the stem were varied showed that both stem-length and stability of the octaloop are important determinants for viral RNA replication and virus reproduction. Together, these data show that stem-loop A plays an important role in virus replication. The high degree of sequence flexibility and the lack of selective pressure on the octaloop argue against a role in sequence specific RNA-protein or RNA-RNA interactions in which octaloop nucleotides are involved.

Highlights

Cardiovirus, a genus of the family of Picornaviridae, is a group of small, nonenveloped RNA viruses
The apical loop of encephalomyocarditis virus (EMCV) SL-A is an octaloop As a first approach to gain more insight into the secondary structure of the cardiovirus 59UTR SLA, a multiple alignment was made of available RNA sequences in combination with in silico folding using MFold [14] (Figure 1)
Folding of SL-A showed a high degree of similarity in the secondary structures of this element within the entire cardiovirus genus

Summary

Introduction

Cardiovirus, a genus of the family of Picornaviridae, is a group of small, nonenveloped RNA viruses. Cardioviruses contain a singlestranded RNA genome of positive polarity of about 7500 nucleotides. The genomic RNA contains a single large open reading frame, preceded by a long 59-untranslated region (59 UTR) of approximately 750 nucleotides and followed by a short 39 UTR of approximately 125 nucleotides. The RNA is translated into a single large polyprotein, which is processed by a virusencoded protease to yield the individual structural capsid proteins and the nonstructural P2 and P3 region proteins, including the viral encoded RNA-dependent RNA polymerase (RdRp) 3Dpol and some relatively stable processing intermediates performing activities in viral RNA replication distinct from their final cleavage products [2]. A small viral peptide (VPg) is covalently attached via a tyrosine residue to the 59-side of both the plus and minus strand viral RNA

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