High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre)

doi:10.1073/pnas.0403079101

High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre)

^*Sealy Center for Structural Biology, and Departments of ^†Human Biological Chemistry and Genetics and ^§Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555; and ^¶University of Texas M. D. Anderson Cancer Center, Houston, TX 77030

Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved July 15, 2004 (received for review May 3, 2004)

Abstract

Picornaviruses constitute a medically important family of RNA viruses in which genome replication critically depends on a small RNA element, the cis-acting replication element (cre), that templates 3D^pol polymerase-catalyzed uridylylation of the protein primer for RNA synthesis, VPg. We report the solution structure of the 33-nt cre of human rhinovirus 14 under solution conditions optimal for uridylylation in vitro. The cre adopts a stem-loop conformation with an extended duplex stem supporting a novel 14-nt loop that derives stability from base-stacking interactions. Base-pair interactions are absent within the loop, and base substitutions within the loop that favor such interactions are detrimental to viral RNA replication. Conserved adenosines in the 5′ loop sequence that participate in a slide-back mechanism of VPg-pUpU synthesis are oriented to the inside of the loop but are available for base templating during uridylation. The structure explains why substitutions of the 3′ loop nucleotides have little impact on conformation of the critical 5′ loop bases and accounts for wide variation in the sequences of cres from different enteroviruses and rhinoviruses.

Footnotes

↵ ∥ To whom correspondence should be addressed. E-mail: smlemon{at}utmb.edu.
↵ ‡ V.T., Y.Y., and K.K. contributed equally to this work.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: cre, cis-acting replication element; HRV-14, human rhinovirus type 14; NTR, nontranslated region.
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 1T28), and the NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession no. BMRB-6115).