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Journal of Virology, August 2002, p. 7485-7494, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7485-7494.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Sequence Requirements for Viral RNA Replication and VPg Uridylylation Directed by the Internal cis-Acting Replication Element (cre) of Human Rhinovirus Type 14

Yan Yang,1 Rene Rijnbrand,1 Kevin L. McKnight,1,{dagger} Eckard Wimmer,2 Aniko Paul,2 Annette Martin,1,3 and Stanley M. Lemon1*

Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1019,1 Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794,2 Unité de Génétique Moléculaire des Virus Respiratoires, Institut Pasteur, 75724 Paris Cedex 15, France3

Received 24 January 2002/ Accepted 30 April 2002

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998). The nucleotide sequence of the apical loop of this internal cis-acting replication element (cre) was critical for RNA synthesis, while secondary RNA structure, but not primary sequence, was shown to be important within the duplex stem. Similar cres have since been identified in other picornaviral genomes. These RNA segments appear to serve as template for the uridylylation of the genome-linked protein, VPg, providing the VPg-pUpU primer required for viral RNA transcription (A. V. Paul et al., J. Virol. 74:10359-10370, 2000). Here, we show that the minimal functional HRV-14 cre resides within a 33-nucleotide (nt) RNA segment that is predicted to form a simple stem-loop with a 14-nt loop sequence. An extensive mutational analysis involving every possible base substitution at each position within the loop segment defined the sequence that is required within this loop for efficient replication of subgenomic HRV-14 replicon RNAs. These results indicate that three consecutive adenosine residues (nt 2367 to 2369) within the 5' half of this loop are critically important for cre function and suggest that a common RNNNAARNNNNNNR loop motif exists among the cre sequences of enteroviruses and rhinoviruses. We found a direct, positive correlation between the capacity of mutated cres to support RNA replication and their ability to function as template in an in vitro VPg uridylylation reaction, suggesting that these functions are intimately linked. These data thus define more precisely the sequence and structural requirements of the HRV-14 cre and provide additional support for a model in which the role of the cre in RNA replication is to act as template for VPg uridylylation.

* Corresponding author. Mailing address: Department of Microbiology & Immunology, The University of Texas Medical Branch, Galveston, TX 77755-1019. Phone: (409) 772-4793. Fax: (409) 772-9598. E-mail: smlemon{at}utmb.edu.

{dagger} Present address: Eli Lilly and Company, Indianapolis, IN 46285.

Journal of Virology, August 2002, p. 7485-7494, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7485-7494.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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